U.S. patent application number 14/438607 was filed with the patent office on 2015-09-03 for renal cell populations and uses thereof.
The applicant listed for this patent is REGENMEDTX, LLC. Invention is credited to Joydeep Basu, Andrew Bruce, Teresa Burnette, Kelly Guthrie, Dominic Justewicz, Russell W. Kelley, John W. Ludlow.
Application Number | 20150246073 14/438607 |
Document ID | / |
Family ID | 49517782 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246073 |
Kind Code |
A1 |
Basu; Joydeep ; et
al. |
September 3, 2015 |
RENAL CELL POPULATIONS AND USES THEREOF
Abstract
The present invention concerns enriched heterogeneous mammalian
renal cell populations characterized by biomarkers, and methods of
making and using the same.
Inventors: |
Basu; Joydeep;
(Winston-Salem, NC) ; Guthrie; Kelly;
(Winston-Salem, NC) ; Justewicz; Dominic;
(Winston-Salem, NC) ; Burnette; Teresa;
(Winston-Salem, NC) ; Bruce; Andrew;
(Winston-Salem, NC) ; Kelley; Russell W.;
(Winston-Salem, NC) ; Ludlow; John W.;
(Winston-Salem, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENMEDTX, LLC |
Winston-Salem |
NC |
US |
|
|
Family ID: |
49517782 |
Appl. No.: |
14/438607 |
Filed: |
October 24, 2013 |
PCT Filed: |
October 24, 2013 |
PCT NO: |
PCT/US13/66707 |
371 Date: |
April 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61718150 |
Oct 24, 2012 |
|
|
|
61876616 |
Sep 11, 2013 |
|
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Current U.S.
Class: |
424/93.7 ;
435/325; 435/7.1; 435/7.21 |
Current CPC
Class: |
A61K 35/22 20130101;
G01N 33/56966 20130101; C12N 5/0686 20130101; C12N 5/0081 20130101;
C12N 2500/02 20130101; C12N 2501/58 20130101; G01N 2333/9108
20130101; G01N 2333/4742 20130101; C12N 2501/599 20130101; A61P
43/00 20180101; A61P 13/12 20180101; G01N 2333/705 20130101 |
International
Class: |
A61K 35/22 20060101
A61K035/22; G01N 33/569 20060101 G01N033/569; C12N 5/071 20060101
C12N005/071 |
Claims
1. An enriched heterogeneous mammalian renal cell population that
express the biomarkers GGT-1 and a cytokeratin.
2. The cell population according to claim 1 wherein the biomarkers
are detected by a monoclonal or polyclonal antibody.
3. The cell population according to claim 1 wherein greater than
18% of the cells express GGT-1.
4. The cell population according to claim 1 wherein the cytokeratin
is selected from CK8, CK18, CK19 and combinations thereof.
5. The cell population according to claim 4 wherein the level of
expression of the cytokeratin is greater than 80%.
6. The cell population according to claim 1 wherein the cytokeratin
is detected by polyclonal antibody.
7. The cell population according to claim 1 wherein the cell
population expresses AQP.
8. The cell population according to claim 1 wherein less than 40%
of the cells express AQP2.
9. The cell population according to claim 7 wherein the AQP2 is
detected by monoclonal antibody.
10. The cell population according to claim 1 wherein the mammal is
human.
11. A method of identifying a heterogeneous renal cell population
suitable for implantation and/oreliciting a regenerative response
said method comprising the steps Isolating cells from a mammalian
kidney sample Exposing said isolated cells to one or more labeled
detection moiety, wherein each labeled detection moiety is directed
to a different biomarker and is labeled with a different label
Determining the percentage of cells that express each of said
different biomarker.
12. The method of claim 11 wherein the biomarker is selected from
AQP1, AQP2, AQP4, Calbindin, Calponin, CD117, CD133, CD146, CD24,
CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK40 to 67, CK7,
CK8, CK8, CK18, CK19, combinations of CK8, CK18 and CK19, Connexin
43, Cubilin, CXCR4 (Fusin), DBA, E-cadherin (CD324), EPO
(erythropoeitin), GGT1, GLEPP1 (glomerular epithelial protein 1),
Haptoglobulin, Itgb1 (Integrin .beta.1), KIM-1/TIM-1 (kidney injury
molecule-1/T-cell immunoglobulin and mucin-containing molecule),
MAP-2 (microtubule-associated protein 2), Megalin, N-cadherin,
Nephrin, NKCC (Na-K-Cl-cotransporters), OAT-1 (organic anion
transporter 1), Osteopontin, Pan-cadherin, PCLP1 (podocalyxin-like
1 molecule), Podocin, SMA (smooth muscle alpha-actin),
Synaptopodin, THP (tamm-horsfall protein), Vimentin, .alpha.GST-1
(alpha glutathione S-transferase) and combinations thereof.
13. The method of claim 11 wherein the labeled detection moiety is
an antibody.
14. A method of treating kidney disease in a mammal comprising
administering a heterogeneous renal cell population to the patient,
wherein the cell population expresses two or more biomarkers
selected from the group consisting of GGT-1, AQP2 and a
cytokeratin.
15. A method of treating kidney disease in a mammal comprising
administering a heterogeneous renal cell population to the patient,
wherein cells within the cell population have levels of expression
for GGT-1 and CK18 are greater than 18% and 80%, respectively.
16. A method of treating kidney disease in a mammal comprising
administering a heterogeneous renal cell population to the patient,
wherein cells within the cell population express GGT-1, AQP2 and
CK18 at levels of expression in Table 12.4.
17. The method according to claim 15 or 16 wherein the cell
population is cultured on a matrix.
18. The method according to claim 15 or 16 wherein the cell
population is suspended in a gelatin-based biomaterial.
19. The method according to claim 15 or 16 wherein the mammal is
human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 61/718,150, entitled "Isolated
Regenerative Renal Cells and Uses Thereof", filed 24 Oct. 2012, and
61/876,616, entitled "Renal Cell Populations and Uses Thereof"
filed 11 Sep. 2013.
TECHNICAL FIELD
[0002] The present disclosure generally relates to an enriched
heterogeneous mammalian kidney-derived cell population, methods of
identifying the cell population, methods for their use in the
preparation of a regenerative medicine therapy, and methods for
treating kidney disease by administration of the enriched
heterogeneous kidney-derived cell population to a mammalian subject
are provided herein.
BACKGROUND OF THE INVENTION
[0003] Collagen and gelatin-based biomaterials have been
successfully employed for a variety of tissue engineering
applications (Rohanizadeh et al. J Mater Sci Mater Med 2008; 19:
1173-1182; Takemoto et al. Tissue Eng Part A 2008; 14: 1629-1638;
Young et al. J Control Release 2005; 109: 256-274). Both of these
macromolecules are characterized by excellent biocompatibility and
low antigenicity (Cenni et al. J Biomater Sci Polym Ed 2000; 11:
685-699; Lee et al. Int J Pharm 2001; 221: 1-22; Waksman et al. J
Immunol 1949; 63: 427-433); however, since gelatin is obtained by
the hydrolysis of collagen, it has certain advantages over the
latter: (a) it is readily available and easy to use; (b) offers
options relative to molecular weight and bloom (i.e. control over
physical properties); and (c) is more flexible towards chemical
modification and more straightforward to manufacture. Moreover,
from a biological standpoint, gelatin maintains cytocompatibility
and cell adherence properties similar to collagen Engvall et al.
Int J Cancer 1977; 20: 1-5; Kim et al. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 2009; 108: e94-100).
[0004] Various methods have been reported for the crosslinking of
these macromolecules for the purpose of delaying their
biodegradation to prolong their in vivo residence (in tissue
engineering applications) or tailoring their drug releasing
capacity (when used as drug carriers). Numerous methods have been
published for chemical or photochemical crosslinking of collagen or
gelatin (Adhirajan et al. J Microencapsul 2007; 24: 647-659; Chang
et al. Macromol Biosci 2007; 7: 500-507; Gagnieu et al. Biomed
Mater Eng 2007; 17: 9-18; Kimura et al. J Biomater Sci Polym Ed
2010; 21: 463-476; Ma et al. J Biomed Mater Res A 2004; 71:
334-342; Vandelli et al. Int J Pharm 2001; 215: 175-184; Vandelli
et al. J Control Release 2004; 96: 67-84). The majority of these
procedures are targeted to reduce the susceptibility of these
biomaterials to enzymatic degradation and to extend their in viva
residence time (Chang et al. supra 2007; Ma et al. supra 2004).
Other crosslinking methods are typically employed to yield gelatin
or collagen-based biomaterials suitable as slow release drug,
protein or nucleic acid carriers (Kimura supra 2010; Vandelli supra
2004; Kommareddy et al. Nanomedicine 2007; 3: 32-42; Sehgal et al.
Expert Opin Drug Deliv 2009; 6: 687-695; Sutter et al. J Control
Release 2007; 119: 301-312). A widely used crosslinking agent class
for collagen and gelatin as well as other tissue
engineering-compatible systems is the carbodilmides (Adhirajan
supra 2007; Olde Damink et al. Biomaterials 1996; 17: 765-773;
Pieper et al. Biomaterials 2000; 21: 581-593; Comwell et al. Clin
Podiatr Med Surg 2009; 26: 507-523). These molecules are known as
zero-length crosslinkers and act by mediating the formation of
amide bonds between carboxyl and primary amine functionalities
present on the species to be crosslinked. In addition,
carbodiimides are less cytotoxic compared to other common
crosslinking agent (e.g. glutaraldehyde) (Lai et al. J Mater Sci
Mater Med 2010; 21: 1899-1911). Glutaraldehylde is used as a
crosslinker in Cultispherbeads. Burg U.S. Pat. No. 6,991,652
describes tissue engineering composites containing
three-dimensional support constructs for cells that may be
delivered to a subject.
[0005] Regenerative medicine technologies provide next-generation
therapeutic options for chronic kidney disease (CKD). Presnell et
al. WO/2010/056328 and Ilagan et al. PCT/US2011/036347 describe
isolated bioactive renal cells, including tubular and
erythropoietin (EPO)-producing kidney cell populations, and methods
of isolating and culturing the same, as well as methods of treating
a subject in need with the cell populations.
[0006] There is a need for therapeutic formulations that are
suitable for delivery of active agents, such as for example,
bioactive cells in tissue engineering and regenerative medicine
applications, to subjects in need.
[0007] The kidney is a complex organ that performs many functions
to keep the blood clean and chemically balanced. In addition to
removing wastes, the kidneys release three important hormones:
[0008] erythropoietin, or EPO, which stimulates the bone marrow to
make red blood cells
[0009] renin, which regulates blood pressure; and
[0010] calcitriol, the active form of vitamin D, which helps
maintain calcium for bones and for normal chemical balance in the
body.
[0011] To perform these functions the kidney comprises numerous
different cell types. However, not all renal cells are required for
a regenerative response and identifying the combination of cells
useful in eliciting a regenerative response has been the subject of
investigation. Thus, there remains a need for methods that identify
a heterogeneous renal cell population, i.e. bioactive cells, that
finds use in the therapeutic formulations disclosed herein.
SUMMARY OF THE INVENTION
[0012] Disclosed herein is a heterogeneous mammalian renal cell
population from mammalian kidney tissue. Methods for the isolation
and purification of the mammalian kidney-derived cell population
are provided. A unique population of mammalian kidney-derived cells
is characterized by phenotypic characteristics, for example,
biomarker phenotype. Biomarker expression phenotype is retained
after multiple passages of the mammalian kidney-derived cell
population in culture and is suitable for use in the preparation of
a regenerative therapy.
[0013] Described herein are a select population of human renal cell
population characterized by specific biomarkers and their use.
[0014] The select population of human renal cells allows the use a
smaller numbers of cells that provide a regenerative stimulus. This
smaller number is advantageous because it lowers the possibility of
adverse immunological events as well as provide a regenerative
stimulus. The selected renal cell population does not require a
large proportion of stem cells to be effective as a regenerative
stimulus. The selected renal cell population may be recovered from
a diseased kidney.
[0015] In one aspect, there is provided methods for indentifying
and/or characterizing a heterogeneous renal cell population. In one
embodiment, the heterogeneous renal cell population is
characterized by its phenotypic expression of biomarkers. In
certain embodiments, the renal cells are identified with one or
more reagents that allow detection of the biomarkers on/in the
heterogeneous renal cell population. Detection of the biomarkers
can be carried out by any suitable method, for example, those based
on immunofluorescent microscopy, flow cytometry, fiber-optic
scanning cytometry, or laser scanning cytometry. In one embodiment,
a method of identifying a heterogeneous renal cell population
suitable for implantation and/or eliciting a regenerative response,
said method comprising the steps:
[0016] Isolating cells from a mammalian kidney sample;
[0017] Exposing said isolated cells to one or more labeled
detection moiety, wherein each labeled detection moiety is directed
to a different biomarker and is labeled with a different label;
[0018] Determining the percentage of cells that express each of
said biomarker.
[0019] In one embodiment, the cell population is an SRC cell
population that expresses two or more biomarkers listed in Tables
12.2 and 12.3. In one embodiment, the biomarkers have levels of
expression as provided in Table 12.4. In an embodiment the SRC cell
population have levels of expression for GGT-1 and CK18 greater
than 18% and 80%, respectively.
[0020] In one aspect, there is provided injectable, therapeutic
formulations containing active agents, e.g., bioactive cells. In
one embodiment, the injectable formulation comprises bioactive
cells and a temperature-sensitive cell-stabilizing biomaterial. In
another embodiment, the a temperature-sensitive cell-stabilizing
biomaterial maintains (i) a substantially solid state at about
8.degree. C. or below and/or (ii) a substantially liquid state at
ambient temperature or above. In one other embodiment, the
bioactive cells comprise renal cells, as described herein. In
another embodiment, the bioactive cells are substantially uniformly
dispersed throughout the volume of the cell-stabilizing
biomaterial. In other embodiments, the biomaterial has a
solid-to-liquid transitional state between about 8.degree. C. and
about ambient temperature or above. In one embodiment, the
substantially solid state is a gel state. In another embodiment,
the cell-stabilizing biomaterial comprises a hydrogel. In one other
embodiment, the hydrogel comprises gelatin. In other embodiments,
the gelatin is present in the formulation at about 0.5% to about 1%
(w/v). In one embodiment, the gelatin is present in the formulation
at about 0.75% (w/v). In another embodiment, the formulation
further includes a cell viability agent. In one other embodiment,
the cell viability agent comprises an agent selected from the group
consisting of an antioxidant, an oxygen carrier, an
immunomodulatory factor, a cell recruitment factor, a cell
attachment factor, an anti-inflammatory agent, an
immunosuppressant, an angiogenic factor, and a wound healing
factor. In some embodiments, the cell viability agent is an
antioxidant. In one embodiment, the antioxidant is
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. In another
embodiment, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid is present at about 50 .mu.M to about 150 .mu.M. In one other
embodiment, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid is present at about 100 .mu.M. In some embodiments, the cell
viability agent is an oxygen carrier. In one embodiment, the oxygen
carrier is a perfluorocarbon. In other embodiments, the cell
viability agent is an immunomodulatory agent. In one embodiment,
the cell viability agent is an immunosuppressant.
[0021] In another aspect, there is provided injectable, therapeutic
formulations containing bioactive renal cells. In one embodiment,
the formulation comprises bioactive renal cells, about 0.75% (w/v)
gelatin, and about 100 .mu.M
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, wherein the
formulation has (i) a substantially solid state at about 8.degree.
C. or below, and (ii) a substantially liquid state at ambient
temperature or above. In another embodiment, the bioactive renal
cells are substantially uniformly dispersed throughout the volume
of the cell-stabilizing biomaterial. In one other embodiment, the
biomaterial comprises a solid-to-liquid transitional state between
about 8.degree. C. and about ambient temperature. In other
embodiments, the substantially solid state is a gel state. In some
embodiments, the formulation further includes a cell viability
agent. In yet another embodiment, the cell viability agent
comprises an agent selected from the group consisting of an
antioxidant, an oxygen carrier, an immunomodulatory factor, a cell
recruitment factor, a cell attachment factor, an anti-inflammatory
agent, an angiogenic factor, and a wound healing factor. In one
embodiment, the cell viability agent is an oxygen carrier. In
another embodiment, the oxygen carrier is a perfluorocarbon. In one
other embodiment, the cell viability agent is an immunomodulatory
agent. In other embodiments, the cell viability agent is an
immunosuppressant.
[0022] In one other aspect, the present disclosure provides a
formulation described herein that further includes biocompatible
beads. In one embodiment, the biocompatible beads comprise a
biomaterial. In another embodiment, the beads are crosslinked. In
one other embodiment, the crosslinked beads have a reduced
susceptibility to enzymatic degradation as compared to
non-crosslinked biocompatible beads. In other embodiments, the
crosslinked beads are carbodiimide-crosslinked beads. In one
embodiment, the carbodiimide is selected from the group consisting
of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC), DCC-N,N'-dicyclohexylcarbodiimide (DCC), and
N,N'-Diisopropylcarbodiimide (DIPC). In another embodiment, the
carbodiimide is 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDC). In one other embodiment, the crosslinked beads
comprise a reduced number of free primary amines as compared to
non-crosslinked beads. In other embodiments, the number of free
primary amines is detectable spectrophotometrically at about 355
nm. In some embodiments, the beads are seeded with the bioactive
cells. In one embodiment, the bioactive cells are renal cells. In
another embodiment, the formulation further comprises additional
biocompatible beads that comprise a temperature-sensitive
biomaterial that maintains (i) a substantially solid state at
ambient temperature or below, and (ii) a substantially liquid state
at about 37.degree. C. or above. In one other embodiment, the
biomaterial of the beads comprises a solid-to-liquid transitional
state between ambient temperature and about 37.degree. C. In other
embodiments, the substantially solid state is a gel state. In one
embodiment, the biomaterial of the beads comprises a hydrogel. In
another embodiment, the hydrogel comprises gelatin. In one other
embodiment, the beads comprise gelatin at about 5% (w/v) to about
10% (w/v). In some embodiments, the additional biocompatible beads
are spacer beads. In other embodiments, the spacer beads are not
seeded with bioactive cells.
[0023] In another aspect, the formulations of the present
disclosure contain products secreted by a renal cell population. In
one embodiment, the formulations comprise products secreted by a
renal cell population and/or bioactive cells. In one other
embodiment, the bioactive cells are renal cells. In another
embodiment, the products comprise one or more of paracrine factors,
endocrine factors, and juxtacrine factors. In one other embodiment,
the products comprise vesicles. In other embodiments, the vesicles
comprise microvesicles. In one embodiment, the vesicles comprise
exosomes. In another embodiment, the vesicles comprise a secreted
product selected from the group consisting of paracrine factors,
endocrine factors, juxtacrine factors, and RNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a flow diagram of the overall NKA manufacturing
process described herein.
[0025] FIG. 2 A-D is a flow diagram providing further details of
the process depicted in FIG. 1 and described herein.
[0026] FIG. 3 is a graph that exemplifies the variations in culture
duration and cell yields from six patients.
[0027] FIG. 4 is a picture of the SRC banding in a 7% OptiPrep.RTM.
density gradient. Reference is made to Example 5.
[0028] FIG. 5 is a bar graph depicting the expression of renal cell
markers in human SRC populations. Reference is made to Example
12.
[0029] FIG. 6 is a bar graph depicting the enzymatic activity of
human SRC. Reference is made to Example 12.
[0030] FIG. 7A-D depicts NKA injection in the kidney: (a) needle
inserted into the kidney cortex, (b) NKA delivery, (c) multiple
delivery points in the kidney, and (d) final implant of the NKA
(exemplary).
DETAILED DESCRIPTION OF THE INVENTION
[0031] Disclosed herein are therapeutic formulations for active
agents, such as bioactive cells, as well as methods of preparing
the same and methods of treating a subject in need with the
formulations. The bioactive cell formulations may be suitable for
heterogenous mixtures or fractions of bioactive renal cells (BRCs).
The bioactive renal cells may be isolated renal cells including
tubular and erythropoietin (EPO)-producing kidney cells. The BRC
cell populations may include enriched tubular and EPO-producing
cell populations. The BRCs may be derived from or are themselves
renal cell fractions from healthy individuals. In addition, there
is provided renal cell fractions obtained from an unhealthy
individual that may lack certain cellular components when compared
to the corresponding renal cell fractions of a healthy individual,
yet still retain therapeutic properties. The present disclosure
also provides therapeutically-active cell populations lacking
cellular components compared to a healthy individual, which cell
populations can be, in one embodiment, isolated and expanded from
autologous sources in various disease states.
[0032] Although bioactive cell formulations are described herein,
the present disclosure contemplates formulations containing a
variety of other active agents. Other suitable active agents
include, without limitation, cellular aggregates, acellular
biomaterials, secreted products from bioactive cells, large and
small molecule therapeutics, as well as combinations thereof. For
example, one type of bioactive cells may be combined with
biomaterial-based microcarriers with or without therapeutic
molecules or another type of bioactive cells, unattached cells may
be combined with acellular particles.
1. DEFINITIONS
[0033] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Principles of Tissue Engineering, 3.sup.rd Ed. (Edited by R Lanza,
R Langer, & J Vacanti), 2007 provides one skilled in the art
with a general guide to many of the terms used in the present
application. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described.
[0034] The term "cell population" as used herein refers to a number
of cells obtained by isolation directly from a suitable tissue
source, usually from a mammal. The isolated cell population may be
subsequently cultured in vitro. Those of ordinary skill in the art
will appreciate that various methods for isolating and culturing
cell populations for use with the present invention and various
numbers of cells in a cell population that are suitable for use in
the present invention. A cell population may be an unfractionated,
heterogeneous cell population derived from an organ or tissue,
e.g., the kidney. For example, a heterogeneous cell population may
be isolated from a tissue biopsy or from whole organ tissue.
Alternatively, the heterogeneous cell population may be derived
from in vitro cultures of mammalian cells, established from tissue
biopsies or whole organ tissue. An unfractionated heterogeneous
cell population may also be referred to as a non-enriched cell
population. In one embodiment, the cell populations contain
bioactive cells.
[0035] The term "native organ" shall mean the organ of a living
subject. The subject may be healthy or un-healthy. An unhealthy
subject may have a disease associated with that particular
organ.
[0036] The term "native kidney" shall mean the kidney of a living
subject. The subject may be healthy or un-healthy. An unhealthy
subject may have a kidney disease.
[0037] The term "regenerative effect" shall mean an effect which
provides a benefit to a native organ, such as the kidney. The
effect may include, without limitation, a reduction in the degree
of injury to a native organ or an improvement in, restoration of,
or stabilization of a native organ function. Renal injury may be in
the form of fibrosis, inflammation, glomerular hypertrophy, etc.
and related to a disease associated with the native organ in the
subject.
[0038] The term "admixture" as used herein refers to a combination
of two or more isolated, enriched cell populations derived from an
unfractionated, heterogeneous cell population. According to certain
embodiments, the cell populations are renal cell populations.
[0039] An "enriched" cell population or preparation refers to a
cell population derived from a starting organ cell population
(e.g., an unfractionated, heterogeneous cell population) that
contains a greater percentage of a specific cell type than the
percentage of that cell type in the starting population. For
example, a starting kidney cell population can be enriched for a
first, a second, a third, a fourth, a fifth, and so on, cell
population of interest. As used herein, the terms "cell
population", "cell preparation" and "cell prototype" are used
interchangeably.
[0040] In one aspect, the term "enriched" cell population as used
herein refers to a cell population derived from a starting organ
cell population (e.g., a cell suspension from a kidney biopsy or
cultured mammalian kidney cells) that contains a percentage of
cells capable of producing EPO that is greater than the percentage
of cells capable of producing EPO in the starting population. For
example, the term "B4" is a cell population derived from a starting
kidney cell population that contains a greater percentage of
EPO-producing cells, glomerular cells, and vascular cells as
compared to the starting population. The cell populations may be
enriched for one or more cell types and depleted of one or more
other cell types. For example, an enriched EPO-producing cell
population may be enriched for interstitial fibroblasts and
depleted of tubular cells and collecting duct epithelial cells
relative to the interstitial fibroblasts and tubular cells in a
non-enriched cell population, i.e. the starting cell population
from which the enriched cell population is derived. In all
embodiments citing EPO-enriched or "B4" populations, the enriched
cell populations are heterogeneous populations of cells containing
cells that can produce EPO in an oxygen-regulated manner, as
demonstrated by oxygen-tunable EPO expression from the endogenous
native EPO gene.
[0041] In another aspect, an enriched renal cell population, which
contains a greater percentage of a specific cell type, e.g.,
vascular, glomerular, or endocrine cells, than the percentage of
that cell type in the starting population, may also lack or be
deficient in one or more specific cell types, e.g., vascular,
glomerular, or endocrine cells, as compared to a starting kidney
cell population derived from a healthy individual or subject. For
example, the term "B4'," or B4 prime," in one aspect, is a cell
population derived from a starting kidney cell population that
lacks or is deficient in one or more cell types, e.g., vascular,
glomerular or endocrine, depending on the disease state of the
starting specimen, as compared to a healthy individual. In one
embodiment, the B4' cell population is derived from a subject
having chronic kidney disease. In one embodiment, the B4' cell
population is derived from a subject having focal segmental
glomerulosclerosis (FSGS). In another embodiment, the B4' cell
population is derived from a subject having autoimmune
glomerulonephritis. In another aspect, B4' is a cell population
derived from a starting cell population including all cell types,
e.g., vascular, glomerular, or endocrine cells, which is later
depleted of or made deficient in one or more cell types, e.g.,
vascular, glomerular, or endocrine cells. In yet another aspect,
B4' is a cell population derived from a starting cell population
including all cell types, e.g., vascular, glomerular, or endocrine
cells, in which one or more specific cell types e.g., vascular,
glomerular, or endocrine cells, is later enriched. For example, in
one embodiment, a B4' cell population may be enriched for vascular
cells but depleted of glomerular and/or endocrine cells. In another
embodiment, a B4' cell population may be enriched for glomerular
cells but depleted of vascular and/or endocrine cells. In another
embodiment, a B4' cell population may be enriched for endocrine
cells but depleted of vascular and/or glomerular cells. In another
embodiment, a B4' cell population may be enriched for vascular and
endocrine cells but depleted of glomerular cells. In preferred
embodiments, the B4' cell population, alone or admixed with another
enriched cell population, e.g., B2 and/or 83, retains therapeutic
properties. A B4' cell population, for example, is described herein
in the Examples, e.g., Examples 11-13.
[0042] In another aspect, an enriched cell population may also
refer to a cell population derived from a starting kidney cell
population as discussed above that contains a percentage of cells
expressing one or more vascular, glomerular and proximal tubular
markers with some EPO-producing cells that is greater than the
percentage of cells expressing one or more vascular, glomerular and
proximal tubular markers with some EPO-producing cells in the
starting population. For example, the term "B3" refers to a cell
population derived from a starting kidney cell population that
contains a greater percentage of proximal tubular cells as well as
vascular and glomerular cells as compared to the starting
population. In one embodiment, the B3 cell population contains a
greater percentage of proximal tubular cells as compared to the
starting population but a lesser percentage of proximal tubular
cells as compared to the B2 cell population. In another embodiment,
the B3 cell population contains a greater percentage of vascular
and glomerular cells markers with some EPO-producing cells as
compared to the starting population but a lesser percentage of
vascular and glomerular cells markers with some EPO-producing cells
as compared to the B4 cell population.
[0043] In another aspect, an enriched cell population may also
refer to a cell population derived from a starting kidney cell
population as discussed above that contains a percentage of cells
expressing one or more tubular cell markers that is greater than
the percentage of cells expressing one or more tubular cell markers
in the starting population. For example, the term "B2" refers to a
cell population derived from a starting kidney cell population that
contains a greater percentage of tubular cells as compared to the
starting population. In addition, a cell population enriched for
cells that express one or more tubular cell markers (or "B2") may
contain some epithelial cells from the collecting duct system.
Although the cell population enriched for cells that express one or
more tubular cell markers (or "B2") is relatively depleted of
EPO-producing cells, glomerular cells, and vascular cells, the
enriched population may contain a smaller percentage of these cells
(EPO-producing, glomerular, and vascular) in comparison to the
starting population. In general, a heterogeneous cell population is
depleted of one or more cell types such that the depleted cell
population contains a lesser proportion of the cell type(s)
relative to the proportion of the cell type(s) contained in the
heterogeneous cell population prior to depletion. The cell types
that may be depleted are any type of kidney cell. For example, in
certain embodiments, the cell types that may be depleted include
cells with large granularity of the collecting duct and tubular
system having a density of <about 1.045 g/ml, referred to as
"B1". In certain other embodiments, the cell types that may be
depleted include debris and small cells of low granularity and
viability having a density of >about 1.095 g/ml, referred to as
"B5". In some embodiments, the cell population enriched for tubular
cells is relatively depleted of all of the following: "B1", "B5",
oxygen-tunable EPO-expressing cells, glomerular cells, and vascular
cells.
[0044] The term "hypoxic" culture conditions as used herein refers
to culture conditions in which cells are subjected to a reduction
in available oxygen levels in the culture system relative to
standard culture conditions in which cells are cultured at
atmospheric oxygen levels (about 21%). Non-hypoxic conditions are
referred to herein as normal or normoxic culture conditions.
[0045] The term "oxygen-tunable" as used herein refers to the
ability of cells to modulate gene expression (up or down) based on
the amount of oxygen available to the cells. "Hypoxia-inducible"
refers to the upregulation of gene expression in response to a
reduction in oxygen tension (regardless of the pre-induction or
starting oxygen tension).
[0046] The term "biomaterial" as used herein refers to a natural or
synthetic biocompatible material that is suitable for introduction
into living tissue. A natural biomaterial is a material that is
made by or originates from a living system. Synthetic biomaterials
are materials which are not made by or do not originate from a
living system. The biomaterials disclosed herein may be a
combination of natural and synthetic biocompatible materials. As
used herein, biomaterials include, for example, polymeric matrices
and scaffolds. Those of ordinary skill in the art will appreciate
that the biomaterial(s) may be configured in various forms, for
example, as porous foam, gels, liquids, beads, solids, and may
comprise one or more natural or synthetic biocompatible materials.
In one embodiment, the biomaterial is the liquid form of a solution
that is capable of becoming a hydrogel.
[0047] The term "modified release" or the equivalent terms
"controlled release", "delayed release", or "slow release" refer to
formulations that release an active agent, such as bioactive cells,
over time or at more than one point in time following
administration to an individual. Modified release of an active
agent, which can occur over a range of desired times, e.g.,
minutes, hours, days, weeks, or longer, depending upon the
formulation, is in contrast to standard formulations in which
substantially the entire dosage unit is available immediately after
administration. For tissue engineering and regenerative medicine
applications, preferred modified release formulations provide for
the release of an active agent at multiple time points following
local administration (e.g., administration of an active agent
directly to a solid organ). For example, a modified release
formulation of bioactive cells would provide an initial release of
cells immediately at the time of administration and a later, second
release of cells at a later time. The time delay for the second
release of an active agent may be minutes, hours, or days after the
initial administration. In general, the period of time for delay of
release corresponds to the period of time that it takes for a
biomaterial carrier of the active agent to lose it structural
integrity. The delayed release of an active agent begins as such
integrity begins to degrade and is completed by the time integrity
fails completely. Those of ordinary skill in the art will
appreciate other suitable mechanisms of release.
[0048] The term "anemia" as used herein refers to a deficit in red
blood cell number and/or hemoglobin levels due to inadequate
production of functional EPO protein by the EPO-producing cells of
a subject, and/or inadequate release of EPO protein into systemic
circulation, and/or the inability of erythroblasts in the bone
marrow to respond to EPO protein. A subject with anemia is unable
to maintain erythroid homeostasis. In general, anemia can occur
with a decline or loss of kidney function (e.g., chronic renal
failure), anemia associated with relative EPO deficiency, anemia
associated with congestive heart failure, anemia associated with
myelo-suppressive therapy such as chemotherapy or anti-viral
therapy (e.g., AZT), anemia associated with non-myeloid cancers,
anemia associated with viral infections such as HIV, and anemia of
chronic diseases such as autoimmune diseases (e.g., rheumatoid
arthritis), liver disease, and multi-organ system failure.
[0049] The term "EPO-deficiency" refers to any condition or
disorder that is treatable with an erythropoietin receptor agonist
(e.g., recombinant EPO or EPO analogs), including anemia.
[0050] The term "organ-related disease" as used herein refers to
disorders associated with any stage or degree of acute or chronic
organ failure that results in a loss of the organ's ability to
perform its function.
[0051] The term "kidney disease" as used herein refers to disorders
associated with any stage or degree of acute or chronic renal
failure that results in a loss of the kidney's ability to perform
the function of blood filtration and elimination of excess fluid,
electrolytes, and wastes from the blood. Kidney disease also
includes endocrine dysfunctions such as anemia
(erythropoietin-deficiency), and mineral imbalance (Vitamin D
deficiency). Kidney disease may originate in the kidney or may be
secondary to a variety of conditions, including (but not limited
to) heart failure, hypertension, diabetes, autoimmune disease, or
liver disease. Kidney disease may be a condition of chronic renal
failure that develops after an acute injury to the kidney. For
example, injury to the kidney by ischemia and/or exposure to
toxicants may cause acute renal failure; incomplete recovery after
acute kidney injury may lead to the development of chronic renal
failure.
[0052] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures for kidney disease,
anemia, EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency wherein the object is to reverse, prevent or
slow down (lessen) the targeted disorder. Those in need of
treatment include those already having a kidney disease, anemia,
EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency as well as those prone to having a kidney
disease, anemia, EPO deficiency, tubular transport deficiency, or
glomerular filtration deficiency or those in whom the kidney
disease, anemia, EPO deficiency, tubular transport deficiency, or
glomerular filtration deficiency is to be prevented. The term
"treatment" as used herein includes the stabilization and/or
improvement of kidney function.
[0053] The term "in vivo contacting" as used herein refers to
direct contact in vivo between products secreted by an enriched
population of cells and a native organ. For example, products
secreted by an enriched population of renal cells (or an admixture
or construct containing renal cells/renal cell fractions) may in
vivo contact a native kidney. The direct in vivo contacting may be
paracrine, endocrine, or juxtacrine in nature. The products
secreted may be a heterogeneous population of different products
described herein.
[0054] The term "ribonucleic acid" or "RNA" as used herein refers
to a chain of nucleotide units where each unit is made up of a
nitrogenous base, a ribose sugar, and a phosphate. The RNA may be
in single or double stranded form. The RNA may be part of, within,
or associated with a vesicle. The vesicle may be an exosome. RNA
includes, without limitation, mRNAs, rRNA, small RNAs, snRNAs,
snoRNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), and
noncoding RNAs. The RNA is preferably human RNA.
[0055] The term "construct" refers to one or more cell populations
deposited on or in a surface of a scaffold or matrix made up of one
or more synthetic or naturally-occurring biocompatible materials.
The one or more cell populations may be coated with, deposited on,
embedded in, attached to, seeded, or entrapped in a biomaterial
made up of one or more synthetic or naturally-occurring
biocompatible biomaterials, polymers, proteins, or peptides. The
one or more cell populations may be combined with a biomaterial or
scaffold or matrix in vitro or in vivo. In general, the one or more
biocompatible materials used to form the scaffold/biomaterial is
selected to direct, facilitate, or permit the formation of
multicellular, three-dimensional, organization of at least one of
the cell populations deposited thereon. The one or more
biomaterials used to generate the construct may also be selected to
direct, facilitate, or permit dispersion and/or integration of the
construct or cellular components of the construct with the
endogenous host tissue, or to direct, facilitate, or permit the
survival, engraftment, tolerance, or functional performance of the
construct or cellular components of the construct.
[0056] The term "marker" or "biomarker" refers generally to a DNA,
RNA, protein, carbohydrate, or glycolipid-based molecular marker,
the expression or presence of which in a cultured cell population
can be detected by standard methods (or methods disclosed herein)
and is consistent with one or more cells in the cultured cell
population being a particular type of cell. Such biomarkers
include, but are not limited to, the genes set forth in Tables X
and Y. The marker may be a polypeptide expressed by the cell or an
identifiable physical location on a chromosome, such as a gene, a
restriction endonuclease recognition site or a nucleic acid
encoding a polypeptide (e.g., an mRNA) expressed by the native
cell. The marker may be an expressed region of a gene referred to
as a "gene expression marker", or some segment of DNA with no known
coding function. The biomarkers may be cell-derived, e.g.,
secreted, products.
[0057] The terms "biomarker signature," "signature," "biomarker
expression signature," or "expression signature" are used
interchangeably herein and refer to one or a combination of
biomarkers whose expression is an indicator of the cell type(s),
e.g., epithelial, tubular, etc. comprising a cell population, e.g.,
bioactive renal cells. The biomarker signature may serve as an
indictor of suitability of the cell population for use in the
methods and manufactures provided for herein. In some embodiments,
the biomarker signature is a "gene signature." The term "gene
signature" is used interchangeably with "gene expression signature"
and refers to one or a combination of polynucleotides whose
expression is an indicator of cell type, e.g., epithelial, tubular,
etc. In some embodiments, the biomarker signature is a "protein
signature." The term "protein signature" is used interchangeably
with "protein expression signature" and refers to one or a
combination of polypeptides whose expression is an indicator of
cell type, e.g., epithelial, tubular, etc.
[0058] The terms "level of expression" or "expression level" are
used interchangeably and generally refer to either the amount of a
polynucleotide or an amino acid product or protein in a biological
sample, or the percentage of cells expressing the polynucleotide or
an amino acid product or protein. "Expressing" or "Expression" and
grammatical variants thereof refer to the presence of a
polynucleotide or an amino acid product or protein in a detectable
amount in a biological sample. For example, a protein that is
detectable (above background or control values) may be said to
express the protein. Similarly, if a portion of cells in a sample
express the protein the sample may be said to express the protein.
In the alternative, the sample may be said to have a level of
expression relating to the percentage of cells expressing the
protein, e.g., if 60% of the cells in a sample express the protein
then the level of expression is 60%.
[0059] The terms "differentially expressed gene," "differential
gene expression" and their synonyms, which are used
interchangeably, refer to a gene whose expression is activated to a
higher or lower level in a first cell or cell population, relative
to its expression in a second cell or cell population. The terms
also include genes whose expression is activated to a higher or
lower level at different stages over time during passage of the
first or second cell in culture. It is also understood that a
differentially expressed gene may be either activated or inhibited
at the nucleic acid level or protein level, or may be subject to
alternative splicing to result in a different polypeptide product.
Such differences may be evidenced by a change in mRNA levels,
surface expression, secretion or other partitioning of a
polypeptide, for example. Differential gene expression may include
a comparison of expression between two or more genes or their gene
products, or a comparison of the ratios of the expression between
two or more genes or their gene products, or even a comparison of
two differently processed products of the same gene, which differ
between the first cell and the second cell. Differential expression
includes both quantitative, as well as qualitative, differences in
the temporal or cellular expression pattern in a gene or its
expression products among, for example, the first cell and the
second cell. For the purpose of this disclosure, "differential gene
expression" is considered to be present when there is a difference
between the expression of a given gene in the first cell and the
second cell. The differential expression of a marker may be in
cells from a patient before administration of a cell population,
admixture, or construct (the first cell) relative to expression in
cells from the patient after administration (the second cell).
[0060] The terms "inhibit", "down-regulate", "under-express" and
"reduce" are used interchangeably and mean that the expression of a
gene, or level of RNA molecules or equivalent RNA molecules
encoding one or more proteins or protein subunits, or activity of
one or more proteins or protein subunits, is reduced relative to
one or more controls, such as, for example, one or more positive
and/or negative controls. The under-expression may be in cells from
a patient before administration of a cell population, admixture, or
construct relative to cells from the patient after
administration.
[0061] The term "up-regulate" or "over-express" is used to mean
that the expression of a gene, or level of RNA molecules or
equivalent RNA molecules encoding one or more proteins or protein
subunits, or activity of one or more proteins or protein subunits,
is elevated relative to one or more controls, such as, for example,
one or more positive and/or negative controls. The over-expression
may be in cells from a patient after administration of a cell
population, admixture, or construct relative to cells from the
patient before administration.
[0062] The term "subject" shall mean any single human subject,
including a patient, eligible for treatment, who is experiencing or
has experienced one or more signs, symptoms, or other indicators of
an organ-related disease, such as kidney disease, anemia, or EPO
deficiency. Such subjects include without limitation subjects who
are newly diagnosed or previously diagnosed and are now
experiencing a recurrence or relapse, or are at risk for a kidney
disease, anemia, or EPO deficiency, no matter the cause. The
subject may have been previously treated for a kidney disease,
anemia, or EPO deficiency, or not so treated.
[0063] The term "patient" refers to any single animal, more
preferably a mammal (including such non-human animals as, for
example, dogs, cats, horses, rabbits, zoo animals, cows, pigs,
sheep, and non-human primates) for which treatment is desired. Most
preferably, the patient herein is a human.
[0064] The term "sample" or "patient sample" or "biological sample"
shall generally mean any biological sample obtained from a subject
or patient, body fluid, body tissue, cell line, tissue culture, or
other source. The term includes tissue biopsies such as, for
example, kidney biopsies. The term includes cultured cells such as,
for example, cultured mammalian kidney cells. Methods for obtaining
tissue biopsies and cultured cells from mammals are well known in
the art. If the term "sample" is used alone, it shall still mean
that the "sample" is a "biological sample" or "patient sample",
i.e., the terms are used interchangeably.
[0065] The term "test sample" refers to a sample from a subject
that has been treated by a method disclosed herein. The test sample
may originate from various sources in the mammalian subject
including, without limitation, blood, semen, serum, urine, bone
marrow, mucosa, tissue, etc.
[0066] The term "control" or "control sample" refers a negative or
positive control in which a negative or positive result is expected
to help correlate a result in the test sample. Controls that are
suitable include, without limitation, a sample known to exhibit
indicators characteristic of normal erythroid homeostasis, a sample
known to exhibit indicators characteristic of anemia, a sample
obtained from a subject known not to be anemic, and a sample
obtained from a subject known to be anemic. Additional controls
suitable for use in the methods provided herein include, without
limitation, samples derived from subjects that have been treated
with pharmacological agents known to modulate erythropoiesis (e.g.,
recombinant EPO or EPO analogs). In addition, the control may be a
sample obtained from a subject prior to being treated by a method
disclosed herein. An additional suitable control may be a test
sample obtained from a subject known to have any type or stage of
kidney disease, and a sample from a subject known not to have any
type or stage of kidney disease. A control may be a normal healthy
matched control. Those of skill in the art will appreciate other
controls suitable for use herein.
[0067] "Regeneration prognosis", "regenerative prognosis", or
"prognostic for regeneration" generally refers to a forecast or
prediction of the probable regenerative course or outcome of the
administration or implantation of a cell population, admixture or
construct described herein. For a regeneration prognosis, the
forecast or prediction may be informed by one or more of the
following: improvement of a functional organ (e.g., the kidney)
after implantation or administration, development of a functional
kidney after implantation or administration, development of
improved kidney function or capacity after implantation or
administration, and expression of certain markers by the native
kidney following implantation or administration.
[0068] "Regenerated organ" refers to a native organ after
implantation or administration of a cell population, admixture, or
construct as described herein. The regenerated organ is
characterized by various indicators including, without limitation,
development of function or capacity in the native organ,
improvement of function or capacity in the native organ, and the
expression of certain markers in the native organ. Those of
ordinary skill in the art will appreciate that other indicators may
be suitable for characterizing a regenerated organ.
[0069] "Regenerated kidney" refers to a native kidney after
implantation or administration of a cell population, admixture, or
construct as described herein. The regenerated kidney is
characterized by various indicators including, without limitation,
development of function or capacity in the native kidney,
improvement of function or capacity in the native kidney, and the
expression of certain markers in the native kidney. Those of
ordinary skill in the art will appreciate that other indicators may
be suitable for characterizing a regenerated kidney.
[0070] The term "cellular aggregate" or "spheroid" refers to an
aggregate or assembly of cells cultured to allow 3D growth as
opposed to growth as a monolayer. It is noted that the term
"spheroid" does not imply that the aggregate is a geometric sphere.
The aggregate may be highly organized with a well defined
morphology or it may be an unorganized mass; it may include a
single cell type or more than one cell type. The cells may be
primary isolates, or a permanent cell line, or a combination of the
two. Included in this definition are organoids and organotypic
cultures.
[0071] The term "ambient temperature" refers to the temperature at
which the formulations of the present disclosure will be
administered to a subject. Generally, the ambient temperature is
the temperature of a temperature-controlled environment. Ambient
temperature ranges from about 18.degree. C. to about 30.degree. C.
In one embodiment, ambient temperature is about 18.degree. C.,
about 19.degree. C., about 20.degree. C., about 21.degree. C.,
about 22.degree. C., about 23.degree. C., about 24.degree. C.,
about 25.degree. C., about 26.degree. C., about 27.degree. C.,
about 28.degree. C., about 29.degree. C., or about 30.degree.
C.
[0072] The word "label" when used herein refers to a compound or
composition that is conjugated or fused directly or indirectly to a
reagent such as a nucleic acid probe or an antibody and facilitates
detection of the reagent to which it is conjugated or fused. The
label may itself be detectable (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable. The term is intended to encompass direct
labeling of a probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin.
[0073] The term "detection" includes any means of detecting,
including direct and indirect detection.
[0074] A "kit" is any manufacture (e.g., a package or container)
comprising at least one reagent, e.g., a medicament for treatment
of an kidney disease, or a probe for specifically detecting a
biomarker gene or protein as disclosed herein. The manufacture is
preferably promoted, distributed, or sold as a unit for performing
the methods disclosed herein.
2. CELL POPULATIONS
[0075] The formulations of the present disclosure may contain
isolated, heterogeneous populations of kidney cells, and admixtures
thereof, enriched for specific bioactive components or cell types
and/or depleted of specific inactive or undesired components or
cell types for use in the treatment of kidney disease, i.e.,
providing stabilization and/or improvement and/or regeneration of
kidney function, were previously described in Presnell et al. U.S.
2011-0117162 and Ilagan et al. PCT/US2011/036347, the entire
contents of which are incorporated herein by reference. The
formulations may contain isolated renal cell fractions that lack
cellular components as compared to a healthy individual yet retain
therapeutic properties, i.e., provide stabilization and/or
improvement and/or regeneration of kidney function. The cell
populations, cell fractions, and/or admixtures of cells described
herein may be derived from healthy individuals, individuals with a
kidney disease, or subjects as described herein.
[0076] The present disclosure provides formulations which are
suitable for use with various bioactive cell populations including,
without limitation, isolated cell population(s), cell fraction(s),
admixture(s), enriched cell population(s), cellular aggregate(s),
and any combination thereof. In an embodiment, the bioactive cell
populations are bioactive renal cells.
[0077] Bioactive Cell Populations
[0078] The present disclosure contemplates therapeutic formulations
suitable for bioactive cell populations that are to be administered
to target organs or tissue in a subject in need. A bioactive cell
population generally refers to a cell population potentially having
therapeutic properties upon administration to a subject. For
example, upon administration to a subject in need, a bioactive
renal cell population can provide stabilization and/or improvement
and/or regeneration of kidney function in the subject. The
therapeutic properties may include a regenerative effect.
[0079] Bioactive cell populations include, without limitation, stem
cells (e.g., pluripotent, multipotent, oligopotent, or unipotent)
such as embryonic stem cells, amniotic stem cells, adult stem cells
(e.g., hematopoietic, mammary, intestinal, mesenchymal, placental,
lung, bone marrow, blood, umbilical cord, endothelial, dental pulp,
adipose, neural, olfactory, neural crest, testicular), induced
pluripotent stem cells; genetically modified cells; as well as cell
populations or tissue explants derived from any source of the body.
The formulations of the present disclosure may also be used with
renal adipose-derived cell populations as described in Basu et al.
PCT/US11/39859 filed on Jun. 9, 2011; and with the adipose-derived
or peripheral blood-derived smooth muscle cells described in Ludlow
et al. U.S. 2010-0131075 and Ludlow et al. PCT/US11/35058 filed on
May 3, 2011; or bladder-derived urothelial or smooth muscle cells
as described in Atala U.S. Pat. No. 6,576,019, each of which is
incorporate herein by reference in its entirety. The bioactive cell
populations may be isolated, enriched, purified, homogeneous, or
heterogeneous in nature. Those of ordinary skill in the art will
appreciate other bioactive cell populations that are suitable for
use in the formulations of the present disclosure.
[0080] In one embodiment, the source of cells is the same as the
intended target organ or tissue. For example, renal cells may be
sourced from the kidney to be used in a formulation to be
administered to the kidney. In another embodiment, the source of
cells is not the same as the intended target organ or tissue. For
example, erythropoietin-expressing cells may be sourced from renal
adipose to be used in a formulation to be administered to the
kidney.
[0081] In one aspect, the present disclosure provides formulations
containing certain subfractions of a heterogeneous population of
renal cells, enriched for bioactive components and depleted of
inactive or undesired components provide superior therapeutic and
regenerative outcomes than the starting population. For example,
bioactive renal cells described herein, e.g., 82, B4, and B3, which
are depleted of inactive or undesired components, e.g., B1 and B5,
alone or admixed, can be part of a formulation to be used for the
stabilization and/or improvement and/or regeneration of kidney
function.
[0082] In another aspect, the formulations contain a specific
subfraction, B4, depleted of or deficient in one or more cell
types, e.g., vascular, endocrine, or endothelial, i.e., B4', that
retain therapeutic properties, e.g., stabilization and/or
improvement and/or regeneration of kidney function, alone or when
admixed with other bioactive subfractions, e.g., B2 and/or B3. In a
preferred embodiment, the bioactive cell population is 82. In
certain embodiments, the B2 cell population is admixed with B4 or
B4'. In other embodiments, the B2 cell population is admixed with
B3. In other embodiments, the B2 cell population is admixed with
both B3 and B4, or specific cellular components of B3 and/or
B4.
[0083] The B2 cell population is characterized by expression of a
tubular cell marker selected from the group consisting of one or
more of the following: megalin, cubilin, hyaluronic acid synthase 2
(HAS2), Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad),
E-cadherin (Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17,
member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3),
FXYD domain-containing ion transport regulator 4 (Fxyd4), solute
carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4),
aldehyde dehydrogenase 3 family, member 81 (Aldh3b1), aldehyde
dehydrogenase 1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8),
and collecting duct marker Aquaporin-4 (Aqp4). B2 is larger and
more granulated than B3 and/or B4 and thus having a buoyant density
between about 1.045 g/ml and about 1.063 g/ml (rodent), between
about 1.045 g/ml and 1.052 g/ml (human), and between about 1.045
g/ml and about 1.058 g/ml (canine).
[0084] The B3 cell population is characterized by the expression of
vascular, glomerular and proximal tubular markers with some
EPO-producing cells, being of an intermediate size and granularity
in comparison to B2 and B4, and thus having a buoyant density
between about 1.063 g/ml and about 1.073 g/ml (rodent), between
about 1.052 g/ml and about 1.063 g/ml (human), and between about
1.058 g/ml and about 1.063 g/ml (canine). B3 is characterized by
expression of markers selected from the group consisting of one or
more of the following: aquaporin 7 (Aqp7), FXYD domain-containing
ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium
phosphate), member 3 (Slc17a3), solute carrier family 3, member 1
(Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa),
solute carrier family 2 (facilitated glucose transporter), member 2
(Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane
protein 27 (Tmem27), acyl-CoA synthetase medium-chain family member
2 (Acsm2), glutathione peroxidase 3 (Gpx3),
fructose-1,6-biphosphatase 1 (Fbp1), and alanine-glyoxylate
aminotransferase 2 (Agxt2). 83 is also characterized by the
vascular expression marker Platelet endothelial cell adhesion
molecule (Pecam) and the glomerular expression marker podocin
(Podn).
[0085] The B4 cell population is characterized by the expression of
a vascular marker set containing one or more of the following:
PECAM, VEGF, KDR, HIF1a, CD31, CD146; a glomerular marker set
containing one or more of the following: Podocin (Podn), and
Nephrin (Neph); and an oxygen-tunable EPO enriched population
compared to unfractionated (UNFX), B2 and B3. B4 is also
characterized by the expression of one or more of the following
markers: chemokine (C--X--C motif) receptor 4 (Cxcr4), endothelin
receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2),
Cadherin 5 (Cdh5), plasminogen activator, tissue (Plat),
angiopoietin 2 (Angpt2), kinase insert domain protein receptor
(Kdr), secreted protein, acidic, cysteine-rich (osteonectin)
(Sparc), serglycin (Srgn), TIMP metallopeptidase inhibitor 3
(Timp3), Wilms tumor 1 (Wt1), wingless-type MMTV integration site
family, member 4 (Wnt4), regulator of G-protein signaling 4 (Rgs4),
Platelet endothelial cell adhesion molecule (Pecam), and
Erythropoietin (Epo). B4 is also characterized by smaller, less
granulated cells compared to either B2 or B3, with a buoyant
density between about 1.073 g/ml and about 1.091 g/ml (rodent),
between about 1.063 g/ml and about 1.091 g/mL (human and
canine).
[0086] The B4' cell population is defined as having a buoyant
density of between 1.063 g/mL and 1.091 g/mL and expressing one or
more of the following markers: PECAM, vEGF, KDR, HIF1a, podocin,
nephrin, EPO, CK7, CK8/18/19. In one embodiment, the B4' cell
population is characterized by the expression of a vascular marker
set containing one or more of the following: PECAM, vEGF, KDR,
HIF1a, CD31, CD146. In another embodiment, the B4' cell population
is characterized by the expression of an endocrine marker EPO. In
one embodiment, the B4' cell population is characterized by the
expression of a glomerular marker set containing one or more of the
following: Podocin (Podn), and Nephrin (Neph). In certain
embodiments, the B4' cell population is characterized by the
expression of a vascular marker set containing one or more of the
following: PECAM, vEGF, KDR, HIF1a and by the expression of an
endocrine marker EPO. In another embodiment, B4' is also
characterized by smaller, less granulated cells compared to either
B2 or 83, with a buoyant density between about 1.073 g/ml and about
1.091 g/ml (rodent), between about 1.063 g/ml and about 1.091 g/mL
(human and canine).
[0087] In one aspect, the present disclosure provides formulations
containing an isolated, enriched B4' population of human renal
cells comprising at least one of erythropoietin (EPO)-producing
cells, vascular cells, and glomerular cells having a density
between 1.063 g/mL and 1.091 g/mL. In one embodiment, the B4' cell
population is characterized by expression of a vascular marker. In
certain embodiments, the B4' cell population is not characterized
by expression of a glomerular marker. In some embodiments, the B4'
cell population is capable of oxygen-tunable erythropoietin (EPO)
expression.
[0088] In one embodiment, formulation contains the B4' cell
population but does not include a B2 cell population comprising
tubular cells having a density between 1.045 g/mL and 1.052 g/mL.
In another embodiment, the B4' cell population formulation does not
include a B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of <1.045
g/ml. In yet another embodiment, the B4' cell population
formulation does not include a B5 cell population comprising debris
and small cells of low granularity and viability with a density
>1.091 g/ml.
[0089] In one embodiment, the B4' cell population-containing
formulation does not include a B2 cell population comprising
tubular cells having a density between 1.045 g/mL and 1.052 g/mL; a
B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of <1.045
g/ml; and a B5 cell population comprising debris and small cells of
low granularity and viability with a density >1.091 g/ml. In
some embodiments, the B4' cell population may be derived from a
subject having kidney disease.
[0090] In one aspect, the present disclosure provides formulations
containing admixtures of human renal cells comprising a first cell
population, B2, comprising an isolated, enriched population of
tubular cells having a density between 1.045 g/mL and 1.052 g/mL,
and a second cell population, B4', comprising erythropoietin
(EPO)-producing cells and vascular cells but depleted of glomerular
cells having a density between about 1.063 g/mL and 1.091 g/mL,
wherein the admixture does not include a B1 cell population
comprising large granular cells of the collecting duct and tubular
system having a density of <1.045 g/ml, or a B5 cell population
comprising debris and small cells of low granularity and viability
with a density >1.091 g/ml. In certain embodiment, the B4' cell
population is characterized by expression of a vascular marker. In
one embodiment, the B4' cell population is not characterized by
expression of a glomerular marker. In certain embodiments, B2
further comprises collecting duct epithelial cells. In one
embodiment, the formulation contains an admixture of cells that is
capable of receptor-mediated albumin uptake. In another embodiment,
the admixture of cells is capable of oxygen-tunable erythropoietin
(EPO) expression. In one embodiment, the admixture contains
HAS-2-expressing cells capable of producing and/or stimulating the
production of high-molecular weight species of hyaluronic acid (HA)
both in vitro and in vivo. In all embodiments, the first and second
cell populations may be derived from kidney tissue or cultured
kidney cells (Basu et al. Lipids in Health and Disease, 2011,
10:171).
[0091] In one embodiment, the formulation contains an admixture
that is capable of providing a regenerative stimulus upon in vivo
delivery. In other embodiments, the admixture is capable of
reducing the decline of, stabilizing, or improving glomerular
filtration, tubular resorption, urine production, and/or endocrine
function upon in vivo delivery. In one embodiment, the B4' cell
population is derived from a subject having kidney disease.
[0092] In one aspect, the present disclosure provides formulations
containing an isolated, enriched B4' population of human renal
cells comprising at least one of erythropoietin (EPO)-producing
cells, vascular cells, and glomerular cells having a density
between 1.063 g/mL and 1.091 g/mL. In one embodiment, the B4' cell
population is characterized by expression of a vascular marker. In
certain embodiments, the B4' cell population is not characterized
by expression of a glomerular marker. The glomerular marker that is
not expressed may be podocin. In some embodiments, the B4' cell
population is capable of oxygen-tunable erythropoietin (EPO)
expression.
[0093] In one embodiment, the B4' cell population-containing
formulation does not include a B2 cell population comprising
tubular cells having a density between 1.045 g/mL and 1.052 g/mL.
In another embodiment, the B4' cell population formulation does not
include a B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of <1.045
g/ml. In yet another embodiment, the B4' cell population
formulation does not include a B5 cell population comprising debris
and small cells of low granularity and viability with a density
>1.091 g/ml.
[0094] In one embodiment, the B4' cell population-containing
formulation does not include a B2 cell population comprising
tubular cells having a density between 1.045 g/mL and 1.052 g/mL; a
B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of <1.045
g/ml; and a B5 cell population comprising debris and small cells of
low granularity and viability with a density >1.091 g/ml. In
some embodiments, the B4' cell population may be derived from a
subject having kidney disease.
[0095] In one aspect, the present disclosure provides formulations
containing an admixture of human renal cells comprising a first
cell population, B2, comprising an isolated, enriched population of
tubular cells having a density between 1.045 g/mL and 1.052 g/mL,
and a second cell population, B4', comprising erythropoietin
(EPO)-producing cells and vascular cells but depleted of glomerular
cells having a density between about 1.063 g/mL and 1.091 g/mL,
wherein the admixture does not include a B1 cell population
comprising large granular cells of the collecting duct and tubular
system having a density of <1.045 g/ml, or a B5 cell population
comprising debris and small cells of low granularity and viability
with a density >1.091 g/ml. In certain embodiment, the B4' cell
population is characterized by expression of a vascular marker. In
one embodiment, the B4' cell population is not characterized by
expression of a glomerular marker. In certain embodiments, B2
further comprises collecting duct epithelial cells. In one
embodiment, the admixture of cells is capable of receptor-mediated
albumin uptake. In another embodiment, the admixture of cells is
capable of oxygen-tunable erythropoietin (EPO) expression. In one
embodiment, the admixture contains HAS-2-expressing cells capable
of producing and/or stimulating the production of high-molecular
weight species of hyaluronic acid (HA) both in vitro and in vivo.
In all embodiments, the first and second cell populations may be
derived from kidney tissue or cultured kidney cells.
[0096] In another aspect, the present disclosure provides
formulations containing a heterogeneous renal cell population
comprising a combination of cell fractions or enriched cell
populations (e.g., 81, B2, B3, B4 (or B4'), and B5). In one
embodiment, the combination has a buoyant density between about
1.045 g/ml and about 1.091 g/ml. In one other embodiment, the
combination has a buoyant density between less than about 1.045
g/ml and about 1.099 g/ml or about 1.100 g/ml. In another
embodiment, the combination has a buoyant density as determined by
separation on a density gradient, e.g., by centrifugation. In yet
another embodiment, the combination of cell fractions contains B2,
B3, and B4 (or B4') depleted of B1 and/or B5. In some embodiments,
the combination of cell fractions contains B2, B3, B4 (or B4'), and
85 but is depleted of B1. Once depleted of B1 and/or B5, the
combination may be subsequently cultured in vitro prior to the
preparation of a formulation comprising the combination of B2, 83,
and B4 (or B4') cell fractions.
[0097] The inventors of the present disclosure have surprisingly
discovered that in vitro culturing of a B1-depleted combination of
B2, B3, B4, and B5 results in depletion of 85. In one embodiment,
B5 is depleted after at least one, two, three, four, or five
passages. In one other embodiment, the B2, B3, B4, and B5 cell
fraction combination that is passaged under the conditions
described herein provides a passaged cell population having B5 at a
percentage that is less than about 5%, less than about 4%, less
than about 3%, less than about 2%, less than about 1%, or less than
about 0.5% of the passaged cell population.
[0098] In another embodiment, B4' is part of the combination of
cell fractions. In one other embodiment, the in vitro culturing
depletion of B5 is under hypoxic conditions.
[0099] In one embodiment, the formulation contains an admixture
that is capable of providing a regenerative stimulus upon in vivo
delivery. In other embodiments, the admixture is capable of
reducing the decline of, stabilizing, or improving glomerular
filtration, tubular resorption, urine production, and/or endocrine
function upon in vivo delivery. In one embodiment, the B4' cell
population is derived from a subject having kidney disease.
[0100] In a preferred embodiment, the formulation contains an
admixture that comprises B2 in combination with B3 and/or B4. In
another preferred embodiment, the admixture comprises B2 in
combination with B3 and/or B4'. In other preferred embodiments, the
admixture consists of or consists essentially of (i) B2 in
combination with B3 and/or B4; or (ii) 82 in combination with B3
and/or B4'.
[0101] The admixtures that contain a B4' cell population may
contain B2 and/or B3 cell populations that are also obtained from a
non-healthy subject. The non-healthy subject may be the same
subject from which the B4' fraction was obtained. In contrast to
the B4' cell population, the B2 and B3 cell populations obtained
from non-healthy subjects are typically not deficient in one or
more specific cell types as compared to a starting kidney cell
population derived from a healthy individual.
[0102] As described in Presnell et al. WO/2010/056328, it has been
found that the B2 and B4 cell preparations are capable of
expressing higher molecular weight species of hyaluronic acid (HA)
both in vitro and in vivo, through the actions of hyaluronic acid
synthase-2 (HAS-2)--a marker that is enriched more specifically in
the B2 cell population. Treatment with B2 in a 5/6 Nx model was
shown to reduce fibrosis, concomitant with strong expression HAS-2
expression in vivo and the expected production of
high-molecular-weight HA within the treated tissue. Notably, the
5/6 Nx model left untreated resulted in fibrosis with limited
detection of HAS-2 and little production of high-molecular-weight
HA. Without wishing to be bound by theory, it is hypothesized that
this anti-inflammatory high-molecular weight species of HA produced
predominantly by B2 (and to some degree by B4) acts synergistically
with the cell preparations in the reduction of renal fibrosis and
in the aid of renal regeneration. Accordingly, the instant
disclosure includes formulations containing the bioactive renal
cells described herein along with a biomaterial comprising
hyaluronic acid. Also contemplated by the instant disclosure is the
provision of a biomaterial component of the regenerative stimulus
via direct production or stimulation of production by the implanted
cells.
[0103] In one aspect, the present disclosure provides formulations
that contain isolated, heterogeneous populations of EPO-producing
kidney cells for use in the treatment of kidney disease, anemia
and/or EPO deficiency in a subject in need. In one embodiment, the
cell populations are derived from a kidney biopsy. In another
embodiment, the cell populations are derived from whole kidney
tissue. In one other embodiment, the cell populations are derived
from in vitro cultures of mammalian kidney cells, established from
kidney biopsies or whole kidney tissue. In all embodiments, these
populations are unfractionated cell populations, also referred to
herein as non-enriched cell populations.
[0104] In another aspect, the present disclosure provides
formulations that contain isolated populations of erythropoietin
(EPO)-producing kidney cells that are further enriched such that
the proportion of EPO-producing cells in the enriched subpopulation
is greater relative to the proportion of EPO-producing cells in the
starting or initial cell population. In one embodiment, the
enriched EPO-producing cell fraction contains a greater proportion
of interstitial fibroblasts and a lesser proportion of tubular
cells relative to the interstitial fibroblasts and tubular cells
contained in the unenriched initial population. In certain
embodiments, the enriched EPO-producing cell fraction contains a
greater proportion of glomerular cells and vascular cells and a
lesser proportion of collecting duct cells relative to the
glomerular cells, vascular cells and collecting duct cells
contained in the unenriched initial population. In such
embodiments, these populations are referred to herein as the "B4"
cell population.
[0105] In another aspect, the present disclosure provides
formulations containing an EPO-producing kidney cell population
that is admixed with one or more additional kidney cell
populations. In one embodiment, the EPO-producing cell population
is a first cell population enriched for EPO-producing cells, e.g.,
B4. In another embodiment, the EPO-producing cell population is a
first cell population that is not enriched for EPO-producing cells,
e.g., B2. In another embodiment, the first cell population is
admixed with a second kidney cell population. In some embodiments,
the second cell population is enriched for tubular cells, which may
be demonstrated by the presence of a tubular cell phenotype. In
another embodiment, the tubular cell phenotype may be indicated by
the presence of one tubular cell marker. In another embodiment, the
tubular cell phenotype may be indicated by the presence of one or
more tubular cell markers. The tubular cell markers include,
without limitation, megalin, cubilin, hyaluronic acid synthase 2
(HAS2), Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad),
E-cadherin (Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17,
member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3),
FXYD domain-containing ion transport regulator 4 (Fxyd4), solute
carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4),
aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde
dehydrogenase 1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8).
In another embodiment, the first cell population is admixed with at
least one of several types of kidney cells including, without
limitation, interstitium-derived cells, tubular cells, collecting
duct-derived cells, glomerulus-derived cells, and/or cells derived
from the blood or vasculature.
[0106] The formulations of the present disclosure may include
EPO-producing kidney cell populations containing B4 or B4' in the
form of an admixture with B2 and/or B3, or in the form of an
enriched cell population, e.g., B2+B3+B4/B4'.
[0107] In one aspect, the formulation contains EPO-producing kidney
cell populations that are characterized by EPO expression and
bioresponsiveness to oxygen, such that a reduction in the oxygen
tension of the culture system results in an induction in the
expression of EPO. In one embodiment, the EPO-producing cell
populations are enriched for EPO-producing cells. In one
embodiment, the EPO expression is induced when the cell population
is cultured under conditions where the cells are subjected to a
reduction in available oxygen levels in the culture system as
compared to a cell population cultured at normal atmospheric
(.about.21%) levels of available oxygen. In one embodiment,
EPO-producing cells cultured in lower oxygen conditions express
greater levels of EPO relative to EPO-producing cells cultured at
normal oxygen conditions. In general, the culturing of cells at
reduced levels of available oxygen (also referred to as hypoxic
culture conditions) means that the level of reduced oxygen is
reduced relative to the culturing of cells at normal atmospheric
levels of available oxygen (also referred to as normal or normoxic
culture conditions). In one embodiment, hypoxic cell culture
conditions include culturing cells at about less than 1% oxygen,
about less than 2% oxygen, about less than 3% oxygen, about less
than 4% oxygen, or about less than 5% oxygen. In another
embodiment, normal or normoxic culture conditions include culturing
cells at about 10% oxygen, about 12% oxygen, about 13% oxygen,
about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17%
oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or
about 21% oxygen.
[0108] In one other embodiment, induction or increased expression
of EPO is obtained and can be observed by culturing cells at about
less than 5% available oxygen and comparing EPO expression levels
to cells cultured at atmospheric (about 21%) oxygen. In another
embodiment, the induction of EPO is obtained in a culture of cells
capable of expressing EPO by a method that includes a first culture
phase in which the culture of cells is cultivated at atmospheric
oxygen (about 21%) for some period of time and a second culture
phase in which the available oxygen levels are reduced and the same
cells are cultured at about less than 5% available oxygen. In
another embodiment, the EPO expression that is responsive to
hypoxic conditions is regulated by HIF1.alpha.. Those of ordinary
skill in the art will appreciate that other oxygen manipulation
culture conditions known in the art may be used for the cells
described herein.
[0109] In one aspect, the formulation contains enriched populations
of EPO-producing mammalian cells characterized by
bio-responsiveness (e.g., EPO expression) to perfusion conditions.
In one embodiment, the perfusion conditions include transient,
intermittent, or continuous fluid flow (perfusion). In one
embodiment, the EPO expression is mechanically-induced when the
media in which the cells are cultured is intermittently or
continuously circulated or agitated in such a manner that dynamic
forces are transferred to the cells via the flow. In one
embodiment, the cells subjected to the transient, intermittent, or
continuous fluid flow are cultured in such a manner that they are
present as three-dimensional structures in or on a material that
provides framework and/or space for such three-dimensional
structures to form. In one embodiment, the cells are cultured on
porous beads and subjected to intermittent or continuous fluid flow
by means of a rocking platform, orbiting platform, or spinner
flask. In another embodiment, the cells are cultured on
three-dimensional scaffolding and placed into a device whereby the
scaffold is stationary and fluid flows directionally through or
across the scaffolding. Those of ordinary skill in the art will
appreciate that other perfusion culture conditions known in the art
may be used for the cells described herein.
[0110] Cellular Aggregates
[0111] In one other aspect, the formulations of the present
disclosure contain cellular aggregates or spheroids. In one
embodiment, the cellular aggregate comprises a bioactive cell
population described herein. In another embodiment, the cellular
aggregate comprises bioactive renal cells such as, for example,
renal cell admixtures, enriched renal cell populations, and
combinations of renal cell fractions.
[0112] In certain embodiments, the bioactive renal cells of the
disclosure may be cultured in 3D formats as described further
herein. In some embodiments, the term "organoid" refers to an
accumulation of cells, with a phenotype and/or function, consistent
with a native kidney. In some embodiments, organoids comprise mixed
populations of cells, of a variety of lineages, which are typically
found in vivo in a given tissue. In some embodiments, the organoids
of this disclosure are formed in vitro, via any means, whereby the
cells of the disclosure form aggregates, which in turn may form
spheroids, organoids, or a combination thereof. Such aggregates,
spheroids or organoids, in some embodiments, assume a structure
consistent with a particular organ. In some embodiments, such
aggregates, spheroids or organoids, express surface markers, which
are typically expressed by cells of the particular organ. In some
embodiments, such aggregates, spheroids or organoids, produce
compounds or materials, which are typically expressed by cells of
the particular organ. In certain embodiments, the cells of the
disclosure may be cultured on natural substrates, e.g., gelatin. In
other embodiments, the cells of the disclosure may be cultured on
synthetic substrates, e.g., PGLA.
[0113] Inactive Cell Populations
[0114] As described herein, certain subfractions of a heterogeneous
population of renal cells, enriched for bioactive components and
depleted of inactive or undesired components, provide superior
therapeutic and regenerative outcomes than the starting population.
In preferred embodiments, the formulations provided by the present
disclosure contain cellular populations that are depleted of B1
and/or B5 cell populations. For instance, the following may be
depleted of B1 and/or B5: admixtures of two or more of B2, B3, and
B4 (or B4'); an enriched cell population of B2, B3, and B4 (or
B4').
[0115] The B1 cell population comprises large, granular cells of
the collecting duct and tubular system, with the cells of the
population having a buoyant density less than about 1.045 g/m. The
B5 cell population is comprised of debris and small cells of low
granularity and viability and having a buoyant density greater than
about 1.091 g/ml.
[0116] Methods of Isolating and Culturing Cell Populations
[0117] In one aspect, the formulations of the present disclosure
contain cell populations that have been isolated and/or cultured
from kidney tissue. Methods are provided herein for separating and
isolating the renal cellular components, e.g., enriched cell
populations that will be used in the formulations for therapeutic
use, including the treatment of kidney disease, anemia, EPO
deficiency, tubular transport deficiency, and glomerular filtration
deficiency. In one embodiment, the cell populations are isolated
from freshly digested, i.e., mechanically or enzymatically
digested, kidney tissue or from heterogeneous in vitro cultures of
mammalian kidney cells.
[0118] The formulations may contain heterogeneous mixtures of renal
cells that have been cultured in hypoxic culture conditions prior
to separation on a density gradient provides for enhanced
distribution and composition of cells in both B4, including B4',
and B2 and/or B3 fractions. The enrichment of oxygen-dependent
cells in B4 from B2 was observed for renal cells isolated from both
diseased and non-diseased kidneys. Without wishing to be bound by
theory, this may be due to one or more of the following phenomena:
1) selective survival, death, or proliferation of specific cellular
components during the hypoxic culture period; 2) alterations in
cell granularity and/or size in response to the hypoxic culture,
thereby effecting alterations in buoyant density and subsequent
localization during density gradient separation; and 3) alterations
in cell gene/protein expression in response to the hypoxic culture
period, thereby resulting in differential characteristics of the
cells within any given fraction of the gradient. Thus, in one
embodiment, the formulations contain cell populations enriched for
tubular cells, e.g., B2, are hypoxia-resistant.
[0119] Exemplary techniques for separating and isolating the cell
populations include separation on a density gradient based on the
differential specific gravity of different cell types contained
within the population of interest. The specific gravity of any
given cell type can be influenced by the degree of granularity
within the cells, the intracellular volume of water, and other
factors. In one aspect, the present disclosure provides optimal
gradient conditions for isolation of the cell preparations, e.g.,
B2 and B4, including B4', across multiple species including, but
not limited to, human, canine, and rodent. In a preferred
embodiment, a density gradient is used to obtain a novel enriched
population of tubular cells fraction, i.e., B2 cell population,
derived from a heterogeneous population of renal cells. In one
embodiment, a density gradient is used to obtain a novel enriched
population of EPO-producing cells fraction, i.e., B4 cell
population, derived from a heterogeneous population of renal cells.
In other embodiments, a density gradient is used to obtain enriched
subpopulations of tubular cells, glomerular cells, and endothelial
cells of the kidney. In one embodiment, both the EPO-producing and
the tubular cells are separated from the red blood cells and
cellular debris. In one embodiment, the EPO-producing, glomerular,
and vascular cells are separated from other cell types and from red
blood cells and cellular debris, while a subpopulation of tubular
cells and collecting duct cells are concomitantly separated from
other cell types and from red blood cells and cellular debris. In
one other embodiment, the endocrine, glomerular, and/or vascular
cells are separated from other cell types and from red blood cells
and cellular debris, while a subpopulation of tubular cells and
collecting duct cells are concomitantly separated from other cell
types and from red blood cells and cellular debris.
[0120] In one aspect, the formulations of the present disclosure
contain cell populations generated by using, in part, the
OPTIPREP.RTM. (Axis-Shield) density gradient medium, comprising 60%
nonionic iodinated compound iodixanol in water, based on certain
key features described below. One of skill in the art, however,
will recognize that any density gradient or other means, e.g.,
immunological separation using cell surface markers known in the
art, comprising necessary features for isolating the cell
populations of the instant disclosure may be used. It should also
be recognized by one skilled in the art that the same cellular
features that contribute to separation of cellular subpopulations
via density gradients (size and granularity) can be exploited to
separate cellular subpopulations via flow cytometry (forward
scatter=a reflection of size via flow cytometry, and side scatter=a
reflection of granularity). Importantly, the density gradient
medium should have low toxicity towards the specific cells of
interest. While the density gradient medium should have low
toxicity toward the specific cells of interest, the instant
disclosure contemplates the use of gradient mediums which play a
role in the selection process of the cells of interest. Without
wishing to be bound by theory, it appears that the cell populations
disclosed herein recovered by the gradient comprising iodixanol are
iodixanol-resistant, as there is an appreciable loss of cells
between the loading and recovery steps, suggesting that exposure to
iodixanol under the conditions of the gradient leads to elimination
of certain cells. The cells appearing in the specific bands after
the iodixanol gradient are resistant to any untoward effects of
iodixanol and/or density gradient exposure. Accordingly, the use of
additional contrast media which are mild to moderate nephrotoxins
in the isolation and/or selection of the cell populations for the
formulations described herein is also contemplated. In addition,
the density gradient medium should also not bind to proteins in
human plasma or adversely affect key functions of the cells of
interest.
[0121] In another aspect, the present disclosure provides
formulations containing cell populations that have been enriched
and/or depleted of kidney cell types using fluorescent activated
cell sorting (FACS). In one embodiment, kidney cell types may be
enriched and/or depleted using BD FACSAria.TM. or equivalent.
[0122] In another aspect, the formulations contain cell populations
that have been enriched and/or depleted of kidney cell types using
magnetic cell sorting. In one embodiment, kidney cell types may be
enriched and/or depleted using the Miltenyi autoMACS.RTM. system or
equivalent.
[0123] In another aspect, the formulations may include renal cell
populations that have been subject to three-dimensional culturing.
In one aspect, the methods of culturing the cell populations are
via continuous perfusion. In one embodiment, the cell populations
cultured via three-dimensional culturing and continuous perfusion
demonstrate greater cellularity and interconnectivity when compared
to cell populations cultured statically. In another embodiment, the
cell populations cultured via three dimensional culturing and
continuous perfusion demonstrate greater expression of EPO, as well
as enhanced expression of renal tubule-associate genes such as
e-cadherin when compared to static cultures of such cell
populations. In yet another embodiment, the cell populations
cultured via continuous perfusion demonstrate greater levels of
glucose and glutamine consumption when compared to cell populations
cultured statically.
[0124] As described herein, low or hypoxic oxygen conditions may be
used in the methods to prepare the cell populations for the
formulations provided for herein. However, the methods of preparing
cell populations may be used without the step of low oxygen
conditioning. In one embodiment, normoxic conditions may be
used.
[0125] Those of ordinary skill in the art will appreciate that
other methods of isolation and culturing known in the art may be
used for the cells described herein.
3. BIOMATERIALS
[0126] A variety of biomaterials may be combined with an active
agent to provide the therapeutic formulations of the present
disclosure. The biomaterials may be in any suitable shape (e.g.,
beads) or form (e.g., liquid, gel, etc.). As described in Bertram
et al. U.S. Published Application 20070276507 (incorporated herein
by reference in its entirety), polymeric matrices or scaffolds may
be shaped into any number of desirable configurations to satisfy
any number of overall system, geometry or space restrictions. In
one embodiment, the matrices or scaffolds of the present disclosure
may be three-dimensional and shaped to conform to the dimensions
and shapes of an organ or tissue structure. For example, in the use
of the polymeric scaffold for treating kidney disease, anemia, EPO
deficiency, tubular transport deficiency, or glomerular filtration
deficiency, a three-dimensional (3-D) matrix may be used. A variety
of differently shaped 3-D scaffolds may be used. Naturally, the
polymeric matrix may be shaped in different sizes and shapes to
conform to differently sized patients. The polymeric matrix may
also be shaped in other ways to accommodate the special needs of
the patient. In another embodiment, the polymeric matrix or
scaffold may be a biocompatible, porous polymeric scaffold. The
scaffolds may be formed from a variety of synthetic or
naturally-occurring materials including, but not limited to,
open-cell polylactic acid (OPLA.RTM.), cellulose ether, cellulose,
cellulosic ester, fluorinated polyethylene, phenolic,
poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,
polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,
polyester, polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene,
polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,
polyphenylene oxide, polyphenylene sulfide, polypropylene,
polystyrene, polysulfide, polysulfone, polytetrafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl,
polyvinylidene fluoride, regenerated cellulose, silicone,
urea-formaldehyde, collagens, gelatin, alginate, laminins,
fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or
copolymers or physical blends thereof. Scaffolding configurations
may range from liquid suspensions to soft porous scaffolds to
rigid, shape-holding porous scaffolds. In one embodiment, the
configuration is the liquid form of a solution that is capable of
becoming a hydrogel.
[0127] Hydrogels may be formed from a variety of polymeric
materials and are useful in a variety of biomedical applications.
Hydrogels can be described physically as three-dimensional networks
of hydrophilic polymers. Depending on the type of hydrogel, they
contain varying percentages of water, but altogether do not
dissolve in water. Despite their high water content, hydrogels are
capable of additionally binding great volumes of liquid due to the
presence of hydrophilic residues. Hydrogels swell extensively
without changing their gelatinous structure. The basic physical
features of hydrogel can be specifically modified, according to the
properties of the polymers used and the additional special
equipments of the products.
[0128] Preferably, the hydrogel is made of a polymer, a
biologically derived material, a synthetically derived material or
combinations thereof, that is biologically inert and
physiologically compatible with mammalian tissues. The hydrogel
material preferably does not induce an inflammatory response.
Examples of other materials which can be used to form a hydrogel
include (a) modified alginates, (b) polysaccharides (e.g. gellan
gum and carrageenans) which gel by exposure to monovalent cations,
(c) polysaccharides (e.g., hyaluronic acid) that are very viscous
liquids or are thixotropic and form a gel over time by the slow
evolution of structure, (d) gelatin or collagen, and (e) polymeric
hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol
block copolymers and proteins). U.S. Pat. No. 6,224,893 B1 provides
a detailed description of the various polymers, and the chemical
properties of such polymers, that are suitable for making
hydrogels.
[0129] Scaffolding or biomaterial characteristics may enable cells
to attach and interact with the scaffolding or biomaterial
material, and/or may provide porous spaces into which cells can be
entrapped. In one embodiment, the porous scaffolds or biomaterials
allow for the addition or deposition of one or more populations or
admixtures of cells on a biomaterial configured as a porous
scaffold (e.g., by attachment of the cells) and/or within the pores
of the scaffold (e.g., by entrapment of the cells). In another
embodiment, the scaffolds or biomaterials allow or promote for
cell:cell and/or cell:biomaterial interactions within the scaffold
to form constructs as described herein.
[0130] In one embodiment, the biomaterial is comprised of
hyaluronic acid (HA) in hydrogel form, containing HA molecules
ranging in size from 5.1 kDA to >2.times.10.sup.6 kDa. In
another embodiment, the biomaterial is comprised of hyaluronic acid
in porous foam form, also containing HA molecules ranging in size
from 5.1 kDA to >2.times.10.sup.6 kDa. In yet another
embodiment, the biomaterial is comprised of a poly-lactic acid
(PLA)-based foam, having an open-cell structure and pore size of
about 50 microns to about 300 microns. In yet another embodiment,
the specific cell populations, preferentially B2 but also B4,
provide directly and/or stimulate synthesis of high molecular
weight Hyaluronic Acid through Hyaluronic Acid Synthase-2 (HAS-2),
especially after intra-renal implantation.
[0131] The biomaterials described herein may also be designed or
adapted to respond to certain external conditions, e.g., in vitro
or in vivo. In one embodiment, the biomaterials are
temperature-sensitive (e.g., either in vitro or in vivo). In
another embodiment, the biomaterials are adapted to respond to
exposure to enzymatic degradation (e.g., either in vitro or in
vivo). The biomaterials' response to external conditions can be
fine tuned as described herein. Temperature sensitivity of the
formulation described can be varied by adjusting the percentage of
a biomaterial in the formulation. For example, the percentage of
gelatin in a solution can be adjusted to modulate the temperature
sensitivity of the gelatin in the final formulation (e.g., liquid,
gel, beads, etc.). Alternatively, biomaterials may be chemically
cross-linked to provide greater resistance to enzymatic
degradation. For instance, a carbodiimide crosslinker may be used
to chemically crosslink gelatin beads thereby providing a reduced
susceptibility to endogenous enzymes.
[0132] In one aspect, the response by the biomaterial to external
conditions concerns the loss of structural integrity of the
biomaterial. Although temperature-sensitivity and resistance to
enzymatic degradation are provided above, other mechanisms exist by
which the loss of material integrity may occur in different
biomaterials. These mechanisms may include, but are not limited to
thermodynamic (e.g., a phase transition such as melting, diffusion
(e.g., diffusion of an ionic crosslinker from a biomaterial into
the surrounding tissue)), chemical, enzymatic, pH (e.g.,
pH-sensitive liposomes), ultrasound, and photolabile (light
penetration). The exact mechanism by which the biomaterial loses
structural integrity will vary but typically the mechanism is
triggered either at the time of implantation or
post-implantation.
[0133] Those of ordinary skill in the art will appreciate that
other types of synthetic or naturally-occurring materials known in
the art may be used to form scaffolds as described herein.
[0134] In one aspect, the constructs as described herein are made
from the above-referenced scaffolds or biomaterials.
4. CONSTRUCTS
[0135] In one aspect, the disclosure provides formulations that
contain implantable constructs having one or more of the cell
populations described herein for the treatment of kidney disease,
anemia, or EPO deficiency in a subject in need. In one embodiment,
the construct is made up of a biocompatible material or
biomaterial, scaffold or matrix composed of one or more synthetic
or naturally-occurring biocompatible materials and one or more cell
populations or admixtures of cells described herein deposited on or
embedded in a surface of the scaffold by attachment and/or
entrapment. In certain embodiments, the construct is made up of a
biomaterial and one or more cell populations or admixtures of cells
described herein coated with, deposited on, deposited in, attached
to, entrapped in, embedded in, seeded, or combined with the
biomaterial component(s). Any of the cell populations described
herein, including enriched cell populations or admixtures thereof,
may be used in combination with a matrix to form a construct.
[0136] In one aspect, the formulation contains constructs that are
made up of biomaterials designed or adapted to respond to external
conditions as described herein. As a result, the nature of the
association of the cell population with the biomaterial in a
construct will change depending upon the external conditions. For
example, a cell population's association with a
temperature-sensitive biomaterial varies with temperature. In one
embodiment, the construct contains a cell population and
biomaterial having a substantially solid state at about 8.degree.
C. or lower and a substantially liquid state at about ambient
temperature or above, wherein the cell population is suspended in
the biomaterial at about 8.degree. C. or lower.
[0137] However, the cell population is substantially free to move
throughout the volume of the biomaterial at about ambient
temperature or above. Having the cell population suspended in the
substantially solid phase at a lower temperature provides stability
advantages for the cells, such as for anchorage-dependent cells, as
compared to cells in a fluid. Moreover, having cells suspended in
the substantially solid state provides one or more of the following
benefits: i) prevents settling of the cells, ii) allows the cells
to remain anchored to the biomaterial in a suspended state; iii)
allows the cells to remain more uniformly dispersed throughout the
volume of the biomaterial; iv) prevents the formation of cell
aggregates; and v) provides better protection for the cells during
storage and transportation of the formulation. A formulation that
can retain such features leading up to the administration to a
subject is advantageous at least because the overall health of the
cells in the formulation will be better and a more uniform and
consistent dosage of cells will be administered.
[0138] In another embodiment, the deposited cell population or
cellular component of the construct is a first kidney cell
population enriched for oxygen-tunable EPO-producing cells. In
another embodiment, the first kidney cell population contains
glomerular and vascular cells in addition to the oxygen-tunable
EPO-producing cells. In one embodiment, the first kidney cell
population is a B4' cell population. In one other embodiment, the
deposited cell population or cellular component(s) of the construct
includes both the first enriched renal cell population and a second
renal cell population. In some embodiments, the second cell
population is not enriched for oxygen-tunable EPO producing cells.
In another embodiment, the second cell population is enriched for
renal tubular cells. In another embodiment, the second cell
population is enriched for renal tubular cells and contains
collecting duct epithelial cells. In other embodiments, the renal
tubular cells are characterized by the expression of one or more
tubular cell markers that may include, without limitation, megalin,
cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D3
25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),
Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogene
family (Rab17), GATA binding protein 3 (Gata3), FXYD
domain-containing ion transport regulator 4 (Fxyd4), solute carrier
family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde
dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase
1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8).
[0139] In one embodiment, the cell populations deposited on or
combined with biomaterials or scaffolds to form constructs are
derived from a variety of sources, such as autologous sources.
Non-autologous sources are also suitable for use, including without
limitation, allogeneic, or syngeneic (autogeneic or isogeneic)
sources.
[0140] Those of ordinary skill in the art will appreciate there are
several suitable methods for depositing or otherwise combining cell
populations with biomaterials to form a construct.
[0141] In one aspect, the constructs are suitable for use in the
methods of use described herein. In one embodiment, the constructs
are suitable for administration to a subject in need of treatment
for a kidney disease of any etiology, anemia, or EPO deficiency of
any etiology. In other embodiments, the constructs are suitable for
administration to a subject in need of an improvement in or
restoration of erythroid homeostasis. In another embodiment, the
constructs are suitable for administration to a subject in need of
improved kidney function.
[0142] In yet another aspect, the present disclosure provides a
construct for implantation into a subject in need of improved
kidney function comprising: a) a biomaterial comprising one or more
biocompatible synthetic polymers or naturally-occurring proteins or
peptides; and b) an admixture of mammalian renal cells derived from
a subject having kidney disease comprising a first cell population,
B2, comprising an isolated, enriched population of tubular cells
having a density between 1.045 g/mL and 1.052 g/mL and a second
cell population, B4', comprising erythropoietin (EPO)-producing
cells and vascular cells but depleted of glomerular cells having a
density between 1.063 g/mL and 1.091 g/mL, coated with, deposited
on or in, entrapped in, suspended in, embedded in and/or otherwise
combined with the biomaterial. In certain embodiments, the
admixture does not include a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml, or a B5 cell population comprising
debris and small cells of low granularity and viability with a
density >1.091 g/ml.
[0143] In one embodiment, the construct includes a B4' cell
population which is characterized by expression of a vascular
marker. In some embodiments, the B4' cell population is not
characterized by expression of a glomerular marker. In certain
embodiments, the admixture is capable of oxygen-tunable
erythropoietin (EPO) expression. In all embodiments, the admixture
may be derived from mammalian kidney tissue or cultured kidney
cells.
[0144] In one embodiment, the construct includes a biomaterial
configured as a three-dimensional (3-D) porous biomaterial suitable
for entrapment and/or attachment of the admixture. In another
embodiment, the construct includes a biomaterial configured as a
liquid or semi-liquid gel suitable for embedding, attaching,
suspending, or coating mammalian cells. In yet another embodiment,
the construct includes a biomaterial configured comprised of a
predominantly high-molecular weight species of hyaluronic acid (HA)
in hydrogel form. In another embodiment, the construct includes a
biomaterial comprised of a predominantly high-molecular weight
species of hyaluronic acid in porous foam form. In yet another
embodiment, the construct includes a biomaterial comprised of a
poly-lactic acid-based foam having pores of between about 50
microns to about 300 microns. In still another embodiment, the
construct includes one or more cell populations that may be derived
from a kidney sample that is autologous to the subject in need of
improved kidney function. In certain embodiments, the sample is a
kidney biopsy. In some embodiments, the subject has a kidney
disease. In yet other embodiments, the cell population is derived
from a non-autologous kidney sample. In one embodiment, the
construct provides erythroid homeostasis.
5. PHENOTYPIC CHARACTERIZATION OF RENAL CELLS
[0145] The cells isolated at any stage of the process may be
characterized by their phenotype.
[0146] In one embodiment, the cells are a heterogeneous renal cell
population that has been enriched. In a further embodiment, the
enriched heterogeneous renal cell population has been cultured
under hypoxic conditions for at least 24 hours. In a yet further
embodiment, the enriched heterogeneous renal cell population has
been subjected to a density gradient.
[0147] The presence (e.g., expression) and/or level/amount of
various biomarkers in a sample can be analyzed by a number of
methodologies, many of which are known in the art and understood by
the skilled artisan, including, but not limited to,
immunohistochemical ("IHC"), Western blot analysis,
immunoprecipitation, molecular binding assays, ELISA, ELIFA,
fluorescence activated cell sorting ("FACS"), MassARRAY,
proteomics, biochemical enzymatic activity assays, in situ
hybridization, Southern analysis, Northern analysis, whole genome
sequencing, polymerase chain reaction ("PCR") including
quantitative real time PCR ("qRT-PCR") and other amplification type
detection methods, such as, for example, branched DNA, SISBA, TMA
and the like), RNA-Seq, FISH, microarray analysis, gene expression
profiling, and/or serial analysis of gene expression ("SAGE"), as
well as any one of the wide variety of assays that can be performed
by protein, gene, and/or tissue array analysis. Typical protocols
for evaluating the status of genes and gene products are found, for
example in Ausubel et al., eds., 1995, Current Protocols In
Molecular Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed
immunoassays such as those available from Rules Based Medicine or
Meso Scale Discovery may also be used.
[0148] In one aspect, a method of detecting the presence of two or
more biomarkers in a heterogeneous renal cell sample is provided,
the method comprising contacting the sample with an antibody
directed to a biomarker under conditions permissive for binding of
the antibody to its cognate ligand (i.e., biomarker), and detecting
the presence of the bound antibody, e.g., by detecting whether a
complex is formed between the antibody and the biomarker. In some
embodiments, the detection of the presence of one or more
biomarkers is by immunohistochemistry.
[0149] In certain embodiments, any of the antibodies provided
herein is useful for detecting the presence of a biomarker in a
heterogeneous renal cell sample. The term "detecting" as used
herein encompasses quantitative or qualitative detection. In
certain embodiments, a biological sample comprises a SRC
sample.
[0150] In certain embodiments, the heterogeneous renal cells are
identified with one or more reagents that allow detection of a
biomarker selected from AQP1, AQP2, AQP4, Calbindin, Calponin,
CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73,
CK18, CK19, CK40 to 67, CK7, CK8, CK8/18, CK8/18/19, Connexin 43,
Cubilin, CXCR4 (Fusin), DBA, E-cadherin (CD324), EPO
(erythropoeitin), GGT1, GLEPP1 (glomerular epithelial protein 1),
Haptoglobulin, Itgb1 (Integrin .beta.1), KIM-1/TIM-1 (kidney injury
molecule-1/T-cell immunoglobulin and mucin-containing molecule),
MAP-2 (microtubule-associated protein 2), Megalin, N-cadherin,
Nephrin, NKCC (Na-K-Cl-cotransporters), OAT-1 (organic anion
transporter 1), Osteopontin, Pan-cadherin, PCLP1 (podocalyxin-like
1 molecule), Podocin, SMA (smooth muscle alpha-actin),
Synaptopodin, THP (tamm-horsfall protein), Vimentin, and
.alpha.GST-1 (alpha glutathione S-transferase). In certain
embodiments, a biomarker is detected by monoclonal or polyclonal
antibodies.
[0151] In one embodiment, an detectable label comprises a
radioactive atom to form a radioconjugate. A variety of radioactive
isotopes are available for the production of radioconjugates.
Examples include .sup.211At, .sup.131I, .sup.125I, .sup.90Y,
.sup.186Re, .sup.153Sm, .sup.212Bi, .sup.32P, .sup.212Pb and
radioactive isotopes of Lu. When the radioconjugate is used for
detection, it may comprise a radioactive atom for scintigraphic
studies, for example .sup.99Tc-m (metastable nuclear isomer) or
.sup.123I, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123, iodine-131, indium-ill, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0152] In case more than one detectable label (including a dye) is
used in one testing, it is preferred that the detectable labels are
selected such that each label can be independently detected without
substantial interference to any other detectable signals present in
the sample. For example, the detectable labels (including a dye)
may be different fluorescent molecules showing different colors
under the detection condition.
[0153] The detection can be carried out by any suitable method, for
example, those based on immunofluorescent microscopy, flow
cytometry, fiber-optic scanning cytometry, or laser scanning
cytometry.
[0154] In some embodiments, the expression of a biomarker in a cell
is determined by evaluating mRNA in a cell. Methods for the
evaluation of mRNAs in cells are well known and include, for
example, hybridization assays using complementary DNA probes (such
as in situ hybridization using labeled riboprobes specific for the
one or more genes, Northern blot and related techniques) and
various nucleic acid amplification assays (such as RT-PCR using
complementary primers specific for one or more of the genes, and
other amplification type detection methods, such as, for example,
branched DNA, SISBA, TMA and the like). In some embodiments, the
expression of a biomarker in a test sample is compared to a
reference sample. For example, the test sample may be a diseased
tissue sample and the reference sample may be from normal
tissue.
[0155] Samples from mammals can be conveniently assayed for mRNAs
using Northern, dot blot or PCR analysis. In addition, such methods
can include one or more steps that allow one to determine the
levels of target mRNA in a biological sample (e.g., by
simultaneously examining the levels a comparative control mRNA
sequence of a "housekeeping" gene such as an actin family member).
Optionally, the sequence of the amplified target cDNA can be
determined.
[0156] Optional methods include protocols which examine or detect
mRNAs, such as target mRNAs, in a tissue or cell sample by
microarray technologies. Using nucleic acid microarrays, test and
control mRNA samples from test and control samples are reverse
transcribed and labeled to generate cDNA probes. The probes are
then hybridized to an array of nucleic adds immobilized on a solid
support. The array is configured such that the sequence and
position of each member of the array is known. For example, a
selection of genes whose expression correlates with a cell
population capable of eliciting a regenerative response may be
arrayed on a solid support. Hybridization of a labeled probe with a
particular array member indicates that the sample from which the
probe was derived expresses that gene.
[0157] According to some embodiments, presence and/or level/amount
is measured by observing protein expression levels of an
aforementioned gene. In certain embodiments, the method comprises
contacting the biological sample with antibodies to a biomarker
described herein under conditions permissive for binding of the
biomarker, and detecting whether a complex is formed between the
antibodies and biomarker.
[0158] In certain embodiments, the presence and/or level/amount of
biomarker proteins in a sample are examined using IHC and staining
protocols. IHC staining of cells has been shown to be a reliable
method of determining or detecting the presence of proteins in a
sample. In one aspect, level of biomarker is determined using a
method comprising: (a) performing IHC analysis of a sample (such as
a renal cell sample) with an antibody; and b) determining level of
a biomarker in the sample. In some embodiments, IHC staining
intensity is determined relative to a reference value.
[0159] IHC may be performed in combination with additional
techniques such as morphological staining and/or fluorescence
in-situ hybridization. Two general methods of IHC are available;
direct and indirect assays. According to the first assay, binding
of antibody to the target antigen is determined directly. This
direct assay uses a labeled reagent, such as a fluorescent tag or
an enzyme-labeled primary antibody, which can be visualized without
further antibody interaction. In a typical indirect assay,
unconjugated primary antibody binds to the antigen and then a
labeled secondary antibody binds to the primary antibody. Where the
secondary antibody is conjugated to an enzymatic label, a
chromogenic or fluorogenic substrate is added to provide
visualization of the antigen. Signal amplification occurs because
several secondary antibodies may react with different epitopes on
the primary antibody.
[0160] The primary and/or secondary antibody used for IHC typically
will be labeled with a detectable moiety. Numerous labels are
available which can be generally grouped into the following
categories: (a) Radioisotopes, such as .sup.35S, .sup.14C,
.sup.125I, .sup.3H, and .sup.131I; (b) colloidal gold particles;
(c) fluorescent labels including, but are not limited to, rare
earth chelates (europium chelates), Texas Red, rhodamine,
fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,
phycocyanin, or commercially available fluorophores such SPECTRUM
ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more
of the above; (d) various enzyme-substrate labels are available and
U.S. Pat. No. 4,275,149 provides a review of some of these.
Examples of enzymatic labels include luciferases (e.g., firefly
luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),
luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase,
urease, peroxidase such as horseradish peroxidase (HRP), alkaline
phosphatase, O-galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like.
[0161] Examples of enzyme-substrate combinations include, for
example, horseradish peroxidase (HRP) with hydrogen peroxidase as a
substrate; alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and .beta.-D-galactosidase
(.beta.-D-Gal) with a chromogenic substrate (e.g.,
p-nitrophenyl-p-D-galactosidase) or fluorogenic substrate (e.g.,
4-methylumbelliferyl-.beta.-D-galactosidase). For a general review
of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0162] In an exemplary method, the sample may be contacted with an
antibody specific for the biomarker under conditions sufficient for
an antibody-biomarker complex to form, and then detecting the
complex. The presence of the biomarker may be detected in a number
of ways, such as by Western blotting and ELISA procedures for
assaying a wide variety of tissues and samples, including plasma or
serum. A wide range of immunoassay techniques using such an assay
format are available, see, e.g., U.S. Pat. Nos. 4,016,043,
4,424,279 and 4,018,653. These include both single-site and
two-site or "sandwich" assays of the non-competitive types, as well
as in the traditional competitive binding assays. These assays also
include direct binding of a labeled antibody to a target
biomarker.
[0163] The presence and/or level/amount of a selected biomarker in
a tissue or cell sample may also be examined by way of functional
or activity-based assays. For instance, if the biomarker is an
enzyme, one may conduct assays known in the art to determine or
detect the presence of the given enzymatic activity in the tissue
or cell sample.
[0164] In certain embodiments, the samples are normalized for both
differences in the amount of the biomarker assayed and variability
in the quality of the samples used, and variability between assay
runs. Such normalization may be accomplished by detecting and
incorporating the level of certain normalizing biomarkers,
including well known housekeeping genes, such as ACTB.
Alternatively, normalization can be based on the mean or median
signal of all of the assayed genes or a large subset thereof
(global normalization approach). On a gene-by-gene basis, measured
normalized amount of a subject tumor mRNA or protein is compared to
the amount found in a reference set. Normalized expression levels
for each mRNA or protein per tested tumor per subject can be
expressed as a percentage of the expression level measured in the
reference set. The presence and/or expression level/amount measured
in a particular subject sample to be analyzed will fall at some
percentile within this range, which can be determined by methods
well known in the art.
[0165] In embodiments, the cytokeratin is selected from CK8, CK18,
CK19 and combinations thereof. In certain embodiments, the
cytokeratin is CK8, CK18, CK19, CK8/CK18, CK8/CK19, CK18/CK19 or
CK8/CK18/CK19, wherein the "/" refers to a combination of the
cytokeratins adjacent thereto. In all embodiments, the cytokeratin
has a level of expression greater than about 80%, about 85%, about
90%, or about 95%.
[0166] In embodiments, the GGT is GGT-1. In all embodiments the GGT
has a level of expression greater than about 10%, about 15%, about
18%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, or about 60%.
6. METHODS OF USE
[0167] In another aspect, the formulations of the present
disclosure may be administered for the treatment of disease. For
example, bioactive cells may be administered to a native organ as
part of a formulation described herein. In one embodiment, the
bioactive cells may be sourced from the native organ that is the
subject of the administration or from a source that is not the
target native organ.
[0168] In one aspect, the present disclosure provides methods for
the treatment of a kidney disease, anemia, or EPO deficiency in a
subject in need with the formulations containing kidney cell
populations and admixtures of kidney cells as described herein. In
one embodiment, the method comprises administering to the subject a
formulation containing a composition that includes a first kidney
cell population enriched for EPO-producing cells. In another
embodiment, the first cell population is enriched for EPO-producing
cells, glomerular cells, and vascular cells. In one embodiment, the
first kidney cell population is a B4' cell population. In another
embodiment, the composition may further include one or more
additional kidney cell populations. In one embodiment, the
additional cell population is a second cell population not enriched
for EPO-producing cells. In another embodiment, the additional cell
population is a second cell population not enriched for
EPO-producing cells, glomerular cells, or vascular cells. In
another embodiment, the composition also includes a kidney cell
population or admixture of kidney cells deposited in, deposited on,
embedded in, coated with, suspended in, or entrapped in a
biomaterial to form an implantable construct, as described herein,
for the treatment of a disease or disorder described herein. In one
embodiment, the cell populations are used alone or in combination
with other cells or biomaterials, e.g., hydrogels, porous
scaffolds, or native or synthetic peptides or proteins, to
stimulate regeneration in acute or chronic disease states.
[0169] In another aspect, the effective treatment of a kidney
disease in a subject by the methods disclosed herein can be
observed through various indicators of erythropoiesis and/or kidney
function. In one embodiment, the indicators of erythroid
homeostasis include, without limitation, hematocrit (HCT),
hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell
count (RBC), reticulocyte number, reticulocyte %, mean corpuscular
volume (MCV), and red blood cell distribution width (RDW). In one
other embodiment, the indicators of kidney function include,
without limitation, serum albumin, albumin to globulin ratio (A/G
ratio), serum phosphorous, serum sodium, kidney size (measurable by
ultrasound), serum calcium, phosphorous:calcium ratio, serum
potassium, proteinuria, urine creatinine, serum creatinine, blood
nitrogen urea (BUN), cholesterol levels, triglyceride levels and
glomerular filtration rate (GFR). Furthermore, several indicators
of general health and well-being include, without limitation,
weight gain or loss, survival, blood pressure (mean systemic blood
pressure, diastolic blood pressure, or systolic blood pressure),
and physical endurance performance.
[0170] In another embodiment, an effective treatment with a
bioactive renal cell formulation is evidenced by stabilization of
one or more indicators of kidney function. The stabilization of
kidney function is demonstrated by the observation of a change in
an indicator in a subject treated by a method provided for herein
as compared to the same indicator in a subject that has not been
treated by the method herein. Alternatively, the stabilization of
kidney function may be demonstrated by the observation of a change
in an indicator in a subject treated by a method herein as compared
to the same indicator in the same subject prior to treatment. The
change in the first indicator may be an increase or a decrease in
value. In one embodiment, the treatment provided by the present
disclosure may include stabilization of blood urea nitrogen (BUN)
levels in a subject where the BUN levels observed in the subject
are lower as compared to a subject with a similar disease state who
has not been treated by the methods of the present disclosure. In
one other embodiment, the treatment may include stabilization of
serum creatinine levels in a subject where the serum creatinine
levels observed in the subject are lower as compared to a subject
with a similar disease state who has not been treated by the
methods of the present disclosure. In another embodiment, the
treatment may include stabilization of hematocrit (HCT) levels in a
subject where the HCT levels observed in the subject are higher as
compared to a subject with a similar disease state who has not been
treated by the methods of the present disclosure. In another
embodiment, the treatment may include stabilization of red blood
cell (RBC) levels in a subject where the RBC levels observed in the
subject are higher as compared to a subject with a similar disease
state who has not been treated by the methods of the present
disclosure. Those of ordinary skill in the art will appreciate that
one or more additional indicators described herein or known in the
art may be measured to determine the effective treatment of a
kidney disease in the subject.
[0171] In another aspect, the present disclosure concerns
formulations for use in methods of providing erythroid homeostasis
in a subject. In one embodiment, the method includes the step of
(a) administering to the subject a formulation containing a renal
cell population, e.g., B2 or B4', or admixture of renal cells,
e.g., B2/B4' and/or B2/B3, or an enriched renal cell population, as
described herein; and (b) determining, in a biological sample from
the subject, that the level of an erythropoiesis indicator is
different relative to the indicator level in a control, wherein the
difference in indicator level (i) indicates the subject is
responsive to the administering step (a), or (ii) is indicative of
erythroid homeostasis in the subject. In another embodiment, the
method includes the step of (a) administering to the subject a
formulation comprising a renal cell population or admixture of
renal cells as described herein; and (b) determining, in a
biological sample from the subject, that the level of an
erythropoiesis indicator is different relative to the indicator
level in a control, wherein the difference in indicator level (i)
indicates the subject is responsive to the administering step (s),
or (ii) is indicative of erythroid homeostasis in the subject. In
another embodiment, the method includes the step of (a) providing a
biomaterial or biocompatible polymeric scaffold; (b) depositing a
renal cell population or admixture of renal cells of the present
disclosure on or within the biomaterial or scaffold in a manner
described herein to form an implantable construct; (c) preparing a
formulation containing the construct; (d) implanting the construct
into the subject; and (e) determining, in a biological sample from
the subject, that the level of an erythropoiesis indicator is
different relative to the indicator level in a control, wherein the
difference in indicator level (i) indicates the subject is
responsive to the administering step (a), or (ii) is indicative of
erythroid homeostasis in the subject.
[0172] In another aspect, the present disclosure concerns
formulations for use in methods of providing both stabilization of
kidney function and restoration of erythroid homeostasis to a
subject in need, said subject having both a deficit in kidney
function and an anemia and/or EPO-deficiency. In one embodiment,
the method includes the step of administering a formulation
containing a renal cell population or admixture of renal cells as
described herein that contain at least one of the following cell
types: tubular-derived cells, glomerulus-derived cells,
insterstitium-derived cells, collecting duct-derived cells, stromal
tissue-derived cells, or cells derived from the vasculature. In
another embodiment, the population or admixture contains both
EPO-producing cells and tubular epithelial cells, the tubular cells
having been identified by at least one of the following markers:
megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D3
25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),
Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogene
family (Rab17), GATA binding protein 3 (Gata3), FXYD
domain-containing ion transport regulator 4 (Fxyd4), solute carrier
family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde
dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase
1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8). In this
embodiment, treatment of the subject would be demonstrated by an
improvement in at least one indicator of kidney function
concomitant with improvement in at least one indicator of
erythropoiesis, compared to either an untreated subject or to the
subject's pre-treatment indicators.
[0173] In one aspect, the present disclosure provides formulations
for use in methods of (i) treating a kidney disease, anemia, or an
EPO-deficiency; (ii) stabilizing kidney function, (iii) restoring
erythroid homeostasis, or (iv) any combination of thereof by
administering a renal cell population enriched for EPO-producing
cells or admixture of renal cells containing a cell population
enriched for EPO-producing cells as described herein, wherein the
beneficial effects of the administration are greater than the
effects of administering a cell population not enriched for
EPO-producing cells. In another embodiment, the enriched cell
population provides an improved level of serum blood urea nitrogen
(BUN). In another embodiment, the enriched cell population provides
an improved retention of protein in the serum. In another
embodiment, the enriched cell population provides improved levels
of serum cholesterol and/or triglycerides. In another embodiment,
the enriched cell population provides an improved level of Vitamin
D. In one embodiment, the enriched cell population provides an
improved phosphorus:calcium ratio as compared to a non-enriched
cell population. In another embodiment, the enriched cell
population provides an improved level of hemoglobin as compared to
a non-enriched cell population. In a further embodiment, the
enriched cell population provides an improved level of serum
creatinine as compared to a non-enriched cell population. In yet
another embodiment, the enriched cell population provides an
improved level of hematocrit as compared to a non-enriched cell
population. In a further embodiment, the enriched cell population
provides an improved level of red blood cell number (RBC#) as
compared to a non-enriched cell population. In one embodiment, the
improved level of hematocrit is restored to 95% normal healthy
level. In a further embodiment, the enriched cell population
provides an improved reticulocyte number as compared to a
non-enriched cell population. In other embodiments, the enriched
cell population provides an improved reticulocyte percentage as
compared to a non-enriched cell population. In yet other
embodiments, the enriched cell population provides an improved
level of red blood cell volume distribution width (RDW) as compared
to a non-enriched cell population. In yet another embodiment, the
enriched cell population provides an improved level of hemoglobin
as compared to a non-enriched cell population. In yet another
embodiment, the enriched cell population provides an erythroietic
response in the bone marrow, such that the marrow cellularity is
near-normal and the myeloid:erythroid ratio is near normal.
[0174] In another aspect, the present disclosure provides
formulations for use in methods of (i) treating a kidney disease,
anemia, or an EPO-deficiency; (ii) stabilizing kidney function,
(iii) restoring erythroid homeostasis, or (iv) any combination of
thereof by administering an enriched cell population, wherein the
beneficial effects of administering a renal cell population or
admixture of renal cell populations described herein are
characterized by improved erythroid homeostasis when compared to
the beneficial effects provided by the administering of recombinant
EPO (rEPO). In one embodiment, the population or admixture, when
administered to a subject in need provides improved erythroid
homeostasis (as determined by hematocrit, hemoglobin, or RBC#) when
compared to the administration of recombinant EPO protein. In one
embodiment, the population or admixture, when administered provides
an improved level of hematocrit, RBC, or hemoglobin as compared to
recombinant EPO, being no greater than about 10% lower or higher
than hematocrit in a control. In a further embodiment, a single
dose or delivery of the population or admixture, when administered
provides improvement in erythroid homeostasis (as determined by
increase in hematocrit, hemoglobin, or RBC#) in the treated subject
for a period of time that significantly exceeds the period of time
that a single dose or delivery of the recombinant EPO protein
provides improvement in erythroid homeostasis. In another
embodiment, the population or admixture, when administered at a
dose described herein does not result in hematocrit, hemoglobin, or
RBC# greater than about 110% of normal levels in matched healthy
controls. In a further embodiment, the population or admixture,
when administered at a dose described herein provides superior
erythroid homeostasis (as determined by hematocrit, hemoglobin, or
RBC#) compared to recombinant EPO protein delivered at a dose
described herein. In another embodiment, the recombinant EPO is
delivered at a dose of about 100 IU/kg, about 200 IU/kg, about 300
IU/kg, about 400 IU/kg, or about 500 IU/kg. Those of ordinary skill
in the art will appreciate that other dosages of recombinant EPO
known in the art may be suitable.
[0175] Another embodiment of the present disclosure is directed to
the use of formulations containing at least one cell population,
including enriched cell populations and admixtures thereof,
described herein, or an implantable construct described herein, or
secreted products as described herein, for the preparation of a
medicament for the treatment of a kidney disease, anemia, or EPO
deficiency in a subject in need, the providing of erythroid
homeostasis in a subject in need, the improvement of kidney
function in a subject in need, or providing a regenerative effect
to a native kidney.
[0176] Another embodiment of the present disclosure is directed to
formulations containing specific enriched cell population(s)
(described herein) for the treatment of a kidney disease of a
specific etiology, based on selection of specific cell
subpopulation(s) based on specific verified therapeutic
attributes.
[0177] In yet another aspect, the present disclosure provides
formulations for use in methods of treating a kidney disease in a
subject in need, comprising: administering to the subject a
formulation comprising an admixture of mammalian renal cells
comprising a first cell population, B2, comprising an isolated,
enriched population of tubular cells having a density between 1.045
g/mL and 1.052 g/mL, and a second cell population, B4', comprising
erythropoietin (EPO)-producing cells and vascular cells but
depleted of glomerular cells having a density between 1.063 g/mL
and 1.091 g/mL, wherein the admixture does not include a B1 cell
population comprising large granular cells of the collecting duct
and tubular system having a density of <1.045 g/ml, or a B5 cell
population comprising debris and small cells of low granularity and
viability with a density >1.091 g/ml. In certain embodiments,
the method includes determining in a test sample from the subject
that the level of a kidney function indicator is different relative
to the indicator level in a control, wherein the difference in
indicator level is indicative of a reduction in decline,
stabilization, or an improvement of one or more kidney functions in
the subject. In one embodiment, the B4' cell population used in the
method is characterized by expression of a vascular marker. In
certain embodiments, the B4' cell population used in the method is
not characterized by expression of a glomerular marker. In one
embodiment, the admixture of cells used in the method is capable of
oxygen-tunable erythropoietin (EPO) expression. In certain
embodiments, the kidney disease to be treated by the methods of the
disclosure is accompanied by an erythropoietin (EPO) deficiency. In
certain embodiments, the EPO deficiency is anemia. In some
embodiments, the EPO deficiency or anemia occurs secondary to renal
failure in the subject. In some other embodiments, the EPO
deficiency or anemia occurs secondary to a disorder selected from
the group consisting of chronic renal failure, primary EPO
deficiency, chemotherapy or anti-viral therapy, non-myeloid cancer,
HIV infection, liver disease, cardiac failure, rheumatoid
arthritis, or multi-organ system failure. In certain embodiments,
the composition used in the method further comprises a biomaterial
comprising one or more biocompatible synthetic polymers and/or
naturally-occurring proteins or peptides, wherein the admixture is
coated with, deposited on or in, entrapped in, suspended in,
embedded in and/or otherwise combined with the biomaterial. In
certain embodiments, the admixture used in the formulations of the
disclosure is derived from mammalian kidney tissue or cultured
mammalian kidney cells. In other embodiments, the admixture is
derived from a kidney sample that is autologous to the subject in
need. In one embodiment, the sample is a kidney biopsy. In other
embodiments, the formulation contains an admixture derived from a
non-autologous kidney sample.
[0178] In yet another aspect, the disclosure provides a use of a
formulation containing the cell preparations and admixtures
described herein or an implantable construct of the instant
disclosure for the preparation of a medicament useful in the
treatment of a kidney disease, anemia or EPO deficiency in a
subject in need thereof.
[0179] In another aspect, the present disclosure provides
formulations for use in methods for the regeneration of a native
kidney in a subject in need thereof. In one embodiment, the method
includes the step of administering or implanting a cell population,
admixture, or construct described herein to the subject. A
regenerated native kidney may be characterized by a number of
indicators including, without limitation, development of function
or capacity in the native kidney, improvement of function or
capacity in the native kidney, and the expression of certain
markers in the native kidney. In one embodiment, the developed or
improved function or capacity may be observed based on the various
indicators of erythroid homeostasis and kidney function described
above. In another embodiment, the regenerated kidney is
characterized by differential expression of one or more stem cell
markers. The stem cell marker may be one or more of the following:
SRY (sex determining region Y)-box 2 (Sox2); Undifferentiated
Embryonic Cell Transcription Factor (UTF1); Nodal Homolog from
Mouse (NODAL); Prominin 1 (PROM1) or CD133 (CD133); CD24; and any
combination thereof (see Ilagan et al. PCT/US2011/036347
incorporated herein by reference in its entirety). In another
embodiment, the expression of the stem cell marker(s) is
up-regulated compared to a control.
[0180] The cell populations described herein, including enriched
cell populations and admixtures thereof, as well as constructs
containing the same may be used to provide a regenerative effect to
a native kidney. The effect may be provided by the cells themselves
and/or by products secreted from the cells. The regenerative effect
may be characterized by one or more of the following: a reduction
in epithelial-mesenchymal transition (which may be via attenuation
of TGF-.beta. signaling); a reduction in renal fibrosis; a
reduction in renal inflammation; differential expression of a stem
cell marker in the native kidney; migration of implanted cells
and/or native cells to a site of renal injury, e.g., tubular
injury, engraftment of implanted cells at a site of renal injury,
e.g., tubular injury; stabilization of one or more indicators of
kidney function (as described herein); restoration of erythroid
homeostasis (as described herein); and any combination thereof.
7. METHODS OF MONITORING REGENERATION
[0181] In another aspect, the present disclosure provides a
prognostic method for monitoring regeneration of a native kidney
following administration or implantation of a formulation
containing a cell population, admixture, or construct described
herein to the subject. In one embodiment, the method includes the
step of detecting the level of marker expression in a test sample
obtained from the subject and in a control sample, wherein a higher
level of expression of the marker in the test sample, as compared
to the control sample, is prognostic for regeneration of the native
kidney in the subject. In another embodiment, the method includes
the detection of expression of one or more stem cell markers in the
sample. The stem cell marker may be selected from Sox2; UTF1;
NODAL; CD133; CD24; and any combination thereof (see Example 11 of
Ilagan et al. PCT/US2011/036347). The detecting step may include
determining that expression of the stem cell marker(s) is
up-regulated or higher in the test sample relative to a control
sample, wherein the higher level of expression is prognostic for
regeneration of the subject's native kidney. In one other
embodiment, mRNA expression of the stem cell marker(s) is detected.
In other embodiments, the detection of mRNA expression may be via a
PCR-based method, e.g., qRT-PCR. In situ hybridization may also be
used for the detection of mRNA expression. In another embodiment,
polypeptide expression of the stem cell marker may also be detected
using an anti-stem cell marker agent. In one other embodiment, the
agent is an antibody against the marker. In another embodiment,
stem cell marker polypeptide expression is detected using
immunohistochemistry or a Western Blot. Those of ordinary skill in
the art will appreciate other methods for detecting mRNA and/or
polypeptide expression of markers.
[0182] In another aspect, the disclosure provides methods for
prognostic evaluation of a patient following implantation or
administration of a formulation containing a cell population,
admixture, or construct described herein. In one embodiment, the
method includes the step of detecting the level of marker
expression in a test sample obtained from said subject; (b)
determining the expression level in the test sample relative to the
level of marker expression relative to a control sample (or a
control reference value); and (c) predicting regenerative prognosis
of the patient based on the determination of marker expression
levels, wherein a higher level of expression of marker in the test
sample, as compared to the control sample (or a control reference
value), is prognostic for regeneration in the subject.
[0183] In one other aspect, the present disclosure provides
prognostic methods for monitoring regeneration of a native kidney
following administration or implantation of a formulation
containing a cell population, admixture, or construct described
herein to the subject, in which a non-invasive method is used. As
an alternative to a tissue biopsy, a regenerative outcome in the
subject receiving treatment can be assessed from examination of a
bodily fluid, e.g., urine. It has been discovered that
microvesicles obtained from subject-derived urine sources contain
certain components including, without limitation, specific proteins
and miRNAs that are ultimately derived from the renal cell
populations impacted by treatment with the cell populations of the
present disclosure. These components may include factors involved
in stem cell replication and differentiation, apoptosis,
inflammation and immuno-modulation. A temporal analysis of
microvesicle-associated miRNA/protein expression patterns allows
for continuous monitoring of regenerative outcomes within the
kidney of subjects receiving the cell populations, admixtures, or
constructs of the present disclosure.
[0184] These kidney-derived vesicles and/or the luminal contents of
kidney derived vesicles shed into the urine of a subject may be
analyzed for biomarkers indicative of regenerative outcome.
[0185] In one embodiment, the present disclosure provides methods
of assessing whether a kidney disease (KD) patient is responsive to
treatment with a therapeutic formulation. The method may include
the step of determining or detecting the amount of vesicles or
their luminal contents in a test sample obtained from a KD patient
treated with the therapeutic, as compared to or relative to the
amount of vesicles in a control sample, wherein a higher or lower
amount of vesicles or their luminal contents in the test sample as
compared to the amount of vesicles or their luminal contents in the
control sample is indicative of the treated patient's
responsiveness to treatment with the therapeutic.
[0186] The present disclosure also provides a method of monitoring
the efficacy of treatment with a therapeutic in a KD patient. In
one embodiment, the method includes the step of determining or
detecting the amount of vesicles in a test sample obtained from a
KD patient treated with the therapeutic, as compared to or relative
to the amount of vesicles or their luminal contents in a control
sample, wherein a higher or lower amount of vesicles or their
luminal contents in the test sample as compared to the amount of
vesicles or their luminal contents in the control sample is
indicative of the efficacy of treatment with the therapeutic in the
KD patient.
[0187] The present disclosure provides a method of identifying a
patient subpopulation for which an agent is effective to treat
kidney disease (KD). In one embodiment, the method includes the
step of determining a correlation between efficacy of the agent and
the presence of an amount of vesicles or their luminal contents in
samples from the patient subpopulation as compared to the amount of
vesicles or their luminal contents in a sample obtained from a
control sample, wherein a higher or lower amount of vesicles in the
samples from the patient subpopulation as compared to the amount of
vesicles or their luminal contents in the control sample is
indicative that the agent is effective to treat KD in the patient
subpopulation.
[0188] The determining or detecting step may include analyzing the
amount of miRNA or other secreted products that may exist in the
test sample, e.g., urine.
[0189] The non-invasive prognostic methods may include the step of
obtaining a urine sample from the subject before and/or after
administration or implantation of a cell population, admixture, or
construct described herein. Vesicles and other secreted products
may be isolated from the urine samples using standard techniques
including without limitation, centrifugation to remove unwanted
debris (Zhou et al. 2008. Kidney Int. 74(5):613-621; Skog et al.
U.S. Published Patent Application No. 20110053157, each of which is
incorporated herein by reference in its entirety).
[0190] The present disclosure relates to non-invasive methods to
detect regenerative outcome in a subject following treatment. The
methods involve detection of vesicles or their luminal contents in
urine from a treated subject. The luminal contents may be one or
more miRNAs. The detection of combinations or panels of the
individual miRNAs may be suitable for such prognostic methods.
Exemplary combinations include two or more of the following:
miR-24; miR-195; miR-871; miR-30b-5p; miR-19b; miR-99a; miR-429;
let-7f; miR-200a; miR-324-5p; miR-10a-5p; and any combination
thereof. In one embodiment, the combination of miRNAs may include
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more individual miRNAs. Those of
ordinary skill in the art will appreciate that other miRNAs and
combinations of miRNAs may be suitable for use in such prognostic
methods. Sources of additional miRNAs include miRBase at
http://mirbase.org, which is hosted and maintained in the Faculty
of Life Sciences at the University of Manchester.
[0191] Those of skill in the art will appreciate that the
prognostic methods for detecting regeneration may be suitable for
subjects treated with other therapeutics known in the art, apart
from the cell populations and constructs described herein.
[0192] In some embodiments, the determining step comprises the use
of a software program executed by a suitable processor for the
purpose of (i) measuring the differential level of marker
expression (or vesicles/vesicle contents) in a test sample and a
control; and/or (ii) analyzing the data obtained from measuring
differential level of marker expression in a test sample and a
control. Suitable software and processors are well known in the art
and are commercially available. The program may be embodied in
software stored on a tangible medium such as CD-ROM, a floppy disk,
a hard drive, a DVD, or a memory associated with the processor, but
persons of ordinary skill in the art will readily appreciate that
the entire program or parts thereof could alternatively be executed
by a device other than a processor, and/or embodied in firmware
and/or dedicated hardware in a well known manner.
[0193] Following the determining step, the measurement results,
findings, diagnoses, predictions and/or treatment recommendations
are typically recorded and communicated to technicians, physicians
and/or patients, for example. In certain embodiments, computers
will be used to communicate such information to interested parties,
such as, patients and/or the attending physicians. In some
embodiments, the assays will be performed or the assay results
analyzed in a country or jurisdiction which differs from the
country or jurisdiction to which the results or diagnoses are
communicated.
[0194] In a preferred embodiment, a prognosis, prediction and/or
treatment recommendation based on the level of marker expression
measured in a test subject having a differential level of marker
expression is communicated to the subject as soon as possible after
the assay is completed and the prognosis and/or prediction is
generated. The results and/or related information may be
communicated to the subject by the subject's treating physician.
Alternatively, the results may be communicated directly to a test
subject by any means of communication, including writing,
electronic forms of communication, such as email, or telephone.
Communication may be facilitated by use of a computer, such as in
case of email communications. In certain embodiments, the
communication containing results of a prognostic test and/or
conclusions drawn from and/or treatment recommendations based on
the test, may be generated and delivered automatically to the
subject using a combination of computer hardware and software which
will be familiar to artisans skilled in telecommunications. One
example of a healthcare-oriented communications system is described
in U.S. Pat. No. 6,283,761; however, the present disclosure is not
limited to methods which utilize this particular communications
system. In certain embodiments of the methods of the disclosure,
all or some of the method steps, including the assaying of samples,
prognosis and/or prediction of regeneration, and communicating of
assay results or prognoses, may be carried out in diverse (e.g.,
foreign) jurisdictions.
[0195] In another aspect, the prognostic methods described herein
provide information to an interested party concerning the
regenerative success of the implantation or administration.
[0196] In all embodiments, the methods of providing a regenerated
kidney to a subject in need of such treatment as described herein
may include the post-implantation step of prognostic evaluation of
regeneration as described above.
8. BIOACTIVE CELL FORMULATIONS
[0197] The formulations described herein incorporate biomaterials
having properties which create a favorable environment for the
active agent, such as bioactive renal cells, to be administered to
a subject. In one embodiment, the formulation contains a first
biomaterial that provides a favorable environment from the time the
active agent is formulated with the biomaterial up until the point
of administration to the subject. In one other embodiment, the
favorable environment concerns the advantages of having bioactive
cells suspended in a substantially solid state versus cells in a
fluid (as described herein) prior to administration to a subject.
In another embodiment, the first biomaterial is a
temperature-sensitive biomaterial. The temperature-sensitive
biomaterial may have (i) a substantially solid state at about
8.degree. C. or below, and (ii) a substantially liquid state at
ambient temperature or above. In one embodiment, the ambient
temperature is about room temperature.
[0198] In another aspect, the formulation contains bioactive cells
combined with a second biomaterial that provides a favorable
environment for the combined cells from the time of formulation up
until a point after administration to the subject. In one
embodiment, the favorable environment provided by the second
biomaterial concerns the advantages of administering cells in a
biomaterial that retains structural integrity up until the point of
administration to a subject and for a period of time after
administration. In one embodiment, the structural integrity of the
second biomaterial following implantation is minutes, hours, days,
or weeks. In one embodiment, the structural integrity is less than
one month, less than one week, less than one day, or less than one
hour. The relatively short term structural integrity provides a
formulation that can deliver the active agent and biomaterial to a
target location in a tissue or organ with controlled handling,
placement or dispersion without being a hindrance or barrier to the
interaction of the incorporated elements with the tissue or organ
into which it was placed.
[0199] In another embodiment, the second biomaterial is a
temperature-sensitive biomaterial that has a different sensitivity
than the first biomaterial. The second biomaterial may have (i) a
substantially solid state at about ambient temperature or below,
and (ii) a substantially liquid state at about 37.degree. C. or
above. In one embodiment, the ambient temperature is about room
temperature.
[0200] In one embodiment, the second biomaterial is crosslinked
beads. The crosslinked beads may have finely tunable in vivo
residence times depending on the degree of crosslinking, as
described herein. In another embodiment, the crosslinked beads
comprise bioactive cells and are resistant to enzymatic degradation
as described herein.
[0201] The formulations of the present disclosure may include the
first biomaterial combined with an active agent, e.g., bioactive
cells, with or without a second biomaterial combined with an active
agent, e.g., bioactive cells. Where a formulation includes a second
biomaterial, it may be a temperature sensitive bead and/or a
crosslinked bead. Various representative formulations are provided
in the examples below (see also FIGS. 3-7).
[0202] The bioactive cell preparations, admixtures, and/or
constructs described herein can be administered as bioactive cell
formulations. In one aspect, the formulations include the cells and
one or more biomaterials that provide stability to the bioactive
cell preparations, admixtures, and/or constructs described herein.
In one embodiment, the biomaterial is a temperature-sensitive
biomaterial that can maintain at least two different phases or
states depending on temperature. The biomaterial is capable of
maintaining a first state at a first temperature, a second state at
a second temperature, and/or a third state at a third temperature.
The first, second or third state may be a substantially solid, a
substantially liquid, or a substantially semi-solid or semi-liquid
state. In one embodiment, the biomaterial has a first state at a
first temperature and a second state at a second temperature,
wherein the first temperature is lower than the second
temperature.
[0203] In one other embodiment, the state of the
temperature-sensitive biomaterial is a substantially solid state at
a temperature of about 8.degree. C. or below. In other embodiments,
the substantially solid state is maintained at about 1.degree. C.,
about 2.degree. C., about 3.degree. C., about 4.degree. C., about
5.degree. C., about 6.degree. C., about 7.degree. C., or about
8.degree. C. In one embodiment, the substantially solid state has
the form of a gel. In other embodiments, the state of the
temperature-sensitive biomaterial is a substantially liquid state
at ambient temperature or above. In one embodiment, the
substantially liquid state is maintained at about 31.degree. C.,
about 32.degree. C., about 33.degree. C., about 34.degree. C.,
about 35.degree. C., about 36.degree. C., or about 37.degree. C. In
one embodiment, the ambient temperature is about room
temperature.
[0204] In another embodiment, the state of the
temperature-sensitive biomaterial is a substantially solid state at
a temperature of about ambient temperature or below. In one
embodiment, the ambient temperature is about room temperature. In
another embodiment, the substantially solid state is maintained at
about 17.degree. C., about 16.degree. C., about 15.degree. C.,
about 14.degree. C., about 13.degree. C., about 12.degree. C.,
about 11.degree. C., about 10.degree. C., about 9.degree. C., about
8.degree. C., about 7.degree. C., about 6.degree. C., about
5.degree. C., about 4.degree. C., about 3.degree. C., about
2.degree. C., or about 1.degree. C. In one embodiment, the
substantially solid state has the form of a bead. In another
embodiment, the state of the temperature-sensitive biomaterial is a
substantially liquid state at a temperature of about 37.degree. C.
or above. In one other embodiment, the substantially solid state is
maintained at about 37.degree. C., about 38.degree. C., about
39.degree. C., or about 40.degree. C.
[0205] The temperature-sensitive biomaterials may be provided in
the form of a solution, in the form of beads, or in other suitable
forms described herein and/or known to those of ordinary skill in
the art. The cell populations and preparations described herein may
be coated with, deposited on, embedded in, attached to, seeded,
suspended in, or entrapped in a temperature-sensitive biomaterial.
Alternatively, the temperature-sensitive biomaterial may be
provided without any cells, such as, for example in the form of
spacer beads.
[0206] In other embodiments, the temperature-sensitive biomaterial
has a transitional state between a first state and a second state.
In one embodiment, the transitional state is a solid-to-liquid
transitional state between a temperature of about 8.degree. C. and
about ambient temperature. In one embodiment, the ambient
temperature is about room temperature. In one other embodiment, the
solid-to-liquid transitional state occurs at one or more
temperatures of about 8.degree. C., about 9.degree. C., about 10*C,
about 11.degree. C., about 12.degree. C., about 13.degree. C.,
about 14.degree. C., about 15.degree. C., about 16.degree. C.,
about 17.degree. C., and about 18.degree. C.
[0207] The temperature-sensitive biomaterials have a certain
viscosity at a given temperature measured in centipoise (cP). In
one embodiment, the biomaterial has a viscosity at 25.degree. C. of
about 1 cP to about 5 cP, about 1.1 cP to about 4.5 cP, about 1.2
cP to about 4 cP, about 1.3 cP to about 3.5 cP, about 1.4 cP to
about 3.5 cP, about 1.5 cP to about 3 cP, about 1.55 cP to about
2.5 cP, or about 1.6 cP to about 2 cP. In another embodiment, the
0.75% (w/v) solution has a viscosity at 37.degree. C. of about 1.0
cP to about 1.15 cP. The viscosity at 37.degree. C. may be about
1.0 cP, about 1.01 cP, about 1.02 cP, about 1.03 cP, about 1.04 cP,
about 1.05 cP, about 1.06 cP, about 1.07 cP, about 1.08 cP, about
1.09 cP, about 1.10 cP, about 1.11 cP, about 1.12 cP, about 1.13
cP, about 1.14 cP, or about 1.15 cP. In one other embodiment, the
biomaterial is a gelatin solution. The gelatin is present at about
0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about
0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95% or about
1%, (w/v) in the solution. In one example, the biomaterial is a
0.75% (w/v) gelatin solution in PBS. In one embodiment, the 0.75%
(w/v) solution has a viscosity at 25.degree. C of about 1.6 cP to
about 2 cP. In one embodiment, the 0.75% (w/v) solution has a
viscosity at 37.degree. C. of about 1.07 cP to about 1.08 cP. The
gelatin solution may be provided in PBS, DMEM, or another suitable
solvent.
[0208] In one aspect, the bioactive cell formulation also includes
a cell viability agent. In one embodiment, the cell viability agent
is selected from the group consisting of an antioxidant, an oxygen
carrier, an immunomodulatory factor, a cell recruitment factor, a
cell attachment factor, an anti-inflammatory agent, an angiogenic
factor, a wound healing factor, and products secreted from
bioactive cells.
[0209] Antioxidants are characterized by the ability to inhibit
oxidation of other molecules. Antioxidants include, without
limitation, one or more of
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox.RTM.), carotenoids, flavonoids, isoflavones, ubiquinone,
glutathione, lipoic acid, superoxide dismutase, ascorbic acid,
vitamin E, vitamin A, mixed carotenoids (e.g., beta carotene, alpha
carotene, gamma carotene, lutein, lycopene, phytopene, phytofluene,
and astaxanthin), selenium, Coenzyme Q10, indole-3-carbinol,
proanthocyanidins, resveratrol, quercetin, catechins, salicylic
acid, curcumin, bilirubin, oxalic acid, phytic acid, lipoic acid,
vanilic acid, polyphenols, ferulic acid, theaflavins, and
derivatives thereof. Those of ordinary skill in the art will
appreciate other suitable antioxidants for use in the present
disclosure.
[0210] Oxygen carriers are agents characterized by the ability to
carry and release oxygen. They include, without limitation,
perfluorocarbons and pharmaceuticals containing perfluorocarbons.
Suitable perfluorocarbon-based oxygen carriers include, without
limitation, perfluorooctyl bromide (C8F17Br); perfluorodichorotane
(C8F16C12); perfluorodecyl bromide; perfluobron; perfluorodecalin;
perfluorotripopylamine; perfluoromethylcyclopiperidine;
Fluosol.RTM. (perfluorodecalin & perfluorotripopylamine);
Perftoran.RTM. (perfluorodecalin &
perfluoromethylcyclopiperidine); Oxygent.RTM. (perfluorodecyl
bromide & perfluobron); Ocycyte.TM. (perfluoro
(tert-butylcyclohexane)). Those of ordinary skill in the art will
appreciate other suitable perfluorocarbon-based oxygen carriers for
use in the present disclosure.
[0211] Immunomodulatory factors include, without limitation,
osteopontin, FAS Ugand factors, interleukins, transforming growth
factor beta, platelet derived growth factor, clusterin,
transferrin, regulated upon action, normal T-cell expressed,
secreted protein (RANTES), plasminogen activator inhibitor-1
(Pai-1), tumor necrosis factor alpha (TNF-alpha), interleukin 6
(IL-6), alpha-1 microglobulin, and beta-2-microglobulin. Those of
ordinary skill in the art will appreciate other suitable
immunomodulatory factors for use in the present disclosure.
[0212] Anti-inflammatory agents or immunosuppressant agents
(described below) may also be part of the formulation. Those of
ordinary skill in the art will appreciate other suitable
antioxidants for use in the present formulations and/or
treatments.
[0213] Cell recruitment factors include, without limitation,
monocyte chemotatic protein 1 (MCP-1), and CXCL-1. Those of
ordinary skill in the art will appreciate other suitable cell
recruitment factors for use in the present formulations and/or
treatments.
[0214] Cell attachment factors include, without limitation,
fibronectin, procollagen, collagen, ICAM-1, connective tissue
growth factor, laminins, proteoglycans, specific cell adhesion
peptides such as RGD and YSIGR. Those of ordinary skill in the art
will appreciate other suitable cell attachment factors for use in
the present formulations and/or treatments.
[0215] Angiogenic factors include, without limitation, matrix
metalloprotease 1 (MMP1), matrix metalloprotease 2 (MMP2), vascular
endothelial growth factor F (VEGF), matrix metalloprotease 9
(MMP-9), tissue inhibitor or matalloproteases-1 (TIMP-1) vascular
endothelial growth factor F (VEGF), angiopoietin-2 (ANG-2). Those
of ordinary skill in the art will appreciate other suitable
angiogenic factors for use in the present formulations and/or
treatments.
[0216] Wound healing factors include, without limitation,
keratinocyte growth factor 1 (KGF-1), tissue plasminogen activator
(tPA), calbindin, clusterin, cystatin C, trefoil factor 3. Those of
ordinary skill in the art will appreciate other suitable wound
healing factors for use in the present formulations and/or
treatments.
[0217] Secreted products from bioactive cells described herein may
also be added to the bioactive cell formulation as a cell viability
agent.
[0218] In one other aspect, the formulation includes a
temperature-sensitive biomaterial described herein and a population
of biocompatible beads containing a biomaterial. In one embodiment,
the beads are crosslinked. Crosslinking may be achieved using any
suitable crosslinking agent known to those of ordinary skill in the
art, such as, for example, carbodiimides; aldehydes (e.g. furfural,
acrolein, formaldehyde, glutaraldehyde, glyceryl aldehyde),
succinimide-based crosslinkers {Bis(sulfosuccinimidyl) suberate
(BS3), Disuccinimidyl glutarate (DSG), Disuccinimidyl suberate
(DSS), Dithiobis(succinimidyl propionate), Ethylene
glycolbis(sulfosuccinimidylsuccinate), Ethylene
glycolbis(succinimidylsuccinate) (EGS), Bis(Sulfosuccinimidyl)
glutarate (BS2G), Disuccinimidyl tartrate (DST)}; epoxides
(Ethylene glycol diglycidyl ether, 1,4 Butanediol diglycidyl
ether); saccharides (glucose and aldose sugars); sulfonic acids and
p-toluene sulfonic acid; carbonyldiimidazole; genipin; imines;
ketones; diphenylphosphorylazide (DDPA); terephthaloyl chloride;
cerium (III) nitrate hexahydrate; microbial transglutaminase; and
hydrogen peroxide. Those of ordinary skill in the art will
appreciate other suitable crosslinking agents and crosslinking
methods for use in the present methods, formulations and/or
treatments.
[0219] In one embodiment, the beads are carbodiimide-crosslinked
beads. The carbodiimide-crosslinked beads may be crosslinked with a
carbodiimide selected from the group consisting of
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
DCC-N,N'-dicyclohexylcarbodiimide (DCC), and
N,N'-Diisopropylcarbodiimide (DIPC). Beads treated with lower
concentration of EDC were expected to have a higher number of free
primary amines, while samples treated with high concentrations of
crosslinker would have most of the primary amines engaged in amide
bonds. The intensity of the orange color developed by the covalent
bonding between the primary amine and picrylsulfonic acid,
detectable spectrophotometrically at 335 nm, is proportional to the
number of primary amines present in the sample. When normalized per
milligram of protein present in the sample, an inverse correlation
between the number of free amines present and the initial
concentration of EDC used for crosslinking can be observed. This
result is indicative of differential bead crosslinking, dictated by
the amount of carbodiimide used in the reaction. In general,
crosslinked beads exhibit a reduced number of free primary amines
as compared to non-crosslinked beads. The number of free primary
amines may be detected spectrophotometrically at about 335 nm.
[0220] The crosslinked beads have a reduced susceptibility to
enzymatic degradation as compared to non-crosslinked biocompatible
beads, thereby providing beads with finely tunable in vivo
residence times. For example, the cross-linked beads are resistant
to endogenous enzymes, such as collagenases. The provision of
crosslinked beads is part of a delivery system focused on the
development and production of biomaterials that facilitate one or
more of: (a) delivery of attached cells to the desired sites and
creation of space for regeneration and ingrowth of native tissue
and vascular supply; (b) ability to persist at the site long enough
to allow cells to establish, function, remodel their
microenvironment and secrete their own extracellular matrix (ECM);
(c) promotion of integration of the transplanted cells with the
surrounding tissue; (d) ability to implant cells in a substantially
solid form; (e) short term structural integrity that does not
provide a significant barrier to tissue ingrowth or integration of
delivered cells/materials with the host tissue; (f) localized in
vivo delivery in a substantially solid form thereby preventing
dispersion of cells within the tissue during implantation; (g)
improved stability and viability of anchorage dependent cells
compared to cells suspended in a fluid; and (h) biphasic release
profile when cells are delivered i) in a substantially solid form
(e.g., attached to beads), and ii) in a substantially liquid form
(e.g., suspended in a fluid).
[0221] In one embodiment, the present disclosure provides
crosslinked beads containing gelatin. Non-crosslinked gelatin beads
are not suitable for a bioactive cell formulation because they
rapidly lose integrity and cells dissipate from the injection site.
In contrast, highly crosslinked gelatin beads may persist too long
at the injection site and may hinder the de-novo ECM secretion,
cell integration and tissue regeneration. The present disclosure
allows for the in vivo residence time of the crosslinked beads to
be finely tuned. In order to tailor the biodegradability of
biomaterials, different crosslinker concentrations of carbodiimide
are used while the overall reaction conditions were kept constant
for all samples. For example, the enzymatic susceptibility of
carbodiimide-crosslinked beads can be finely tuned by varying the
concentration of crosslinking agent from about zero to about 1M. In
some embodiments, the concentration is about 5 mM, about 6 mM,
about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about
12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17
mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22
mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27
mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32
mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37
mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42
mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47
mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60
mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85
mM, about 90 mM, about 95 mM, or about 100 mM. The crosslinker
concentration may also be about 0.15 M, about 0.2 M, about 0.25 M,
about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, about 0.5 M,
about 0.55 M, about 0.6 M, about 0.65 M, about 0.7 M, about 0.75 M,
about 0.8 M, about 0.85 M, about 0.9 M, about 0.95 M, or about 1 M.
In another embodiment, the crosslinking agent is
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC).
In one embodiment, the EDC-crosslinked beads are gelatin beads.
[0222] Cross-linked beads may have certain characteristics that
favor the seeding, attachment, or encapsulation. For example, the
beads may have a porous surface and/or may be substantially hollow.
The presence of pores provides an increased cell attachment surface
allowing for a greater number of cells to attach as compared to a
non-porous or smooth surface. In addition, the pore structure can
support host tissue integration with the porous beads supporting
the formation of de novo tissue. The beads have a size distribution
that can be fitted to a Weibull plot corresponding to the general
particle distribution pattern. In one embodiment, the cross-linked
beads have an average diameter of less than about 120 .mu.m, about
115 .mu.m, about 110 .mu.m, about 109 .mu.m, about 108 .mu.m, about
107 .mu.m, about 106 .mu.m, about 105 .mu.m, about 104 .mu.m, about
103 .mu.m, about 102 .mu.m, about 101 .mu.m, about 100 .mu.m, about
99 .mu.m, about 98 .mu.m, about 97 .mu.m, about 96 .mu.m, about 95
.mu.m, about 94 .mu.m, about 93 .mu.m, about 92 .mu.m, about 91
.mu.m, or about 90 .mu.m. The characteristics of the cross-linked
beads vary depending upon the casting process. For instance, a
process in which a stream of air is used to aerosolize a liquid
gelatin solution and spray it into liquid nitrogen with a thin
layer chromatography reagent sprayer (ACE Glassware) is used to
provide beads having the aforementioned characteristics. Those of
skill in the art will appreciate that modulating the parameters of
the casting process provides the opportunity to tailor different
characteristics of the beads, e.g., different size
distributions.
[0223] The cytocompatibility of the cross-linked beads is assessed
in vitro prior to formulation using cell culture techniques in
which beads are cultured with cells that correspond to the final
bioactive cell formulation. For instance, the beads are cultured
with primary renal cells prior to preparation of a bioactive renal
cell formulation and live/dead cell assays are used to confirm
cytocompatibility. In certain formulations, the biocompatible
cross-linked beads are combined with a temperature-sensitive
biomaterial in solution at about 5% (w/w) to about 15% (w/w) of the
volume of the solution. The cross-linked beads may be present at
about 5% (w/w), about 5.5% (w/w), about 6% (w/w), about 6.5% (w/w),
about 7% (w/w), about 7.5% (w/w), about 8% (w/w), about 8.5% (w/w),
about 9% (w/w), about 9.5% (w/w), about 10% (w/w), about 10.5%
(w/w), about 11% (w/w), about 11.5% (w/w), about 12% (w/w), about
12.5% (w/w), about 13% (w/w), about 13.5% (w/w), about 14% (w/w),
about 14.5% (w/w), or about 15% (w/w) of the volume of the
solution.
[0224] In another aspect, the present disclosure provides
formulations that contain biomaterials which degrade over a period
time on the order of minutes, hours, or days. This is in contrast
to a large body or work focusing on the implantation of solid
materials that then slowly degrade over days, weeks, or months.
[0225] In another aspect, the present disclosure provides
formulations having biocompatible cross-linked beads seeded with
bioactive cells together with a delivery matrix. In one embodiment,
the delivery matrix has one or more of the following
characteristics: biocompatibility, biodegradeable/bioresorbable, a
substantially solid state prior to and during implantation into a
subject, loss of structural integrity (substantially solid state)
after implantation, and cytocompatible environment to support
cellular viability. The delivery matrix's ability to keep implanted
particles (e.g., crosslinked beads) spaced out during implantation
enhances native tissue ingrowth. If the delivery matrix is absent,
then compaction of cellularized beads during implantation can lead
to inadequate room for sufficient tissue ingrowth. The delivery
matrix facilitates implantation of solid formulations. In addition,
the short duration of the structural integrity means that soon
after implantation, the matrix does not provide a significant
barrier to tissue ingrowth or integration of the delivered
cells/materials with host tissue. The delivery matrix provides for
localization of the formulation described herein since inserted of
a solid unit helps prevent the delivered materials from dispersing
within the tissue during implantation. For cell-based formulations,
a solid delivery matrix improves stability and viability of
anchorage dependent cells compared to cells suspended in a
fluid.
[0226] In one embodiment, the delivery matrix is a population of
biocompatible beads that is not seeded with cells. In another
embodiment, the unseeded beads are dispersed throughout and in
between the individual cell-seeded beads. The unseeded beads act as
"spacer beads" between the cell-seeded beads prior to and
immediately after transplantation. The spacer beads contain a
temperature-sensitive biomaterial having a substantially solid
state at a first temperature and a substantially liquid state at a
second temperature, wherein the first temperature is lower than the
second temperature. For example, the spacer beads contain a
biomaterial having a substantially solid state at about ambient
temperature or below and a substantially liquid state at about
37.degree. C., such as that described herein. In one embodiment,
the ambient temperature is about room temperature. In another
embodiment, the biomaterial is a gelatin solution. The gelatin
solution is present at about 4%, about 4.5%, about 5%, about 5.5%,
about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%,
about 9%, about 9.5%, about 10%, about 10.5%, or about 11%, (w/v).
The gelatin solution may be provided in PBS, cell culture media
(e.g., DMEM), or another suitable solvent.
[0227] In one aspect, the present disclosure provides formulations
that contain biomaterials which are implanted in a substantially
solid form (e.g., spacer beads) and then liquefy/melt or otherwise
lose structural integrity following implantation into the body.
This is in contrast to the significant body of work focusing on the
use of materials that can be injected as a liquid, which then
solidify in the body.
[0228] The temperature-sensitivity of spacer beads can be assessed
in vitro prior to formulation. Spacer beads can be labeled and
mixed with unlabeled non-temperature-sensitive beads. The mixture
is then incubated at 37.degree. C. to observe changes in physical
transition. The loss of shape of the labeled temperature-sensitive
beads at the higher temperature is observed over time. For example,
temperature-sensitive gelatin beads may be made with Alcian blue
dye to serve as a marker of physical transition. The blue gelatin
beads are mixed with Cultispher S beads (white), loaded into a
catheter, then extruded and incubated in 1.times.PBS, pH 7.4, at
37.degree. C. The loss of shape of the blue gelatin beads is
followed microscopically at different time points. Changes in the
physical state of the blue gelatin beads are visible after 30 min
becoming more pronounced with prolonged incubation times. The beads
do not completely dissipate because of the viscosity of the
material.
[0229] The bioactive cell formulations described herein may be used
to prepare renal cell-based formulations for injection into the
kidney. However, those of ordinary skill in the art will appreciate
that the formulations will be suitable for many other types of
bioactive cell populations. For example, the present disclosure
contemplates formulations for bioactive cells for injection into
any solid organ or tissue.
[0230] In one aspect, the bioactive cell formulations described
herein will contain a set number of cells. In one embodiment, the
total number of cells for the formulation is about 10.sup.4, about
10.sup.5, about 10.sup.6, about 10.sup.7, about 10.sup.8, or about
10.sup.9. In one embodiment, the dosage of cells for a formulation
described herein may be calculated based on the estimated mass or
functional mass of the target organ or tissue. In certain
embodiments, the bioactive cell formulations contain a dosage
corresponding to a number of cells based upon the weight of the
host organ that will be the subject of treatment by the
formulation. For example, a bioactive renal cell formulation is
based upon an average weight of about 150 grams for a human kidney.
In one embodiment, the number of cells per gram (g) of kidney is
about 600 cells/g to about 7.0.times.10.sup.7 cells/g. In some
embodiments, the number of cells per gram of kidney is about 600
cells/g, about 1000 cells/g, about 1500 cells/g, about 2000
cells/g, about 2500 cells/g, about 3000 cells/g, about 3500
cells/g, about 4000 cells/g, about 4500 cells/g, about 5000
cells/g, about 5500 cells/g, about 6000 cells/g, about 6500
cells/g, about 7000 cells/g, about 7500 cells/g, about 8000
cells/g, about 8500 cells/g, about 9000 cells/g, about 9500
cells/g, or about 10,000 cells/g.
[0231] In other embodiments, the number of cells per gram of kidney
is about 1.5.times.10.sup.4 cells/g, about 2.0.times.10.sup.4
cells/g, about 2.5.times.10' cells/g, about 3.0.times.10.sup.4
cells/g, about 3.5.times.10.sup.4 cells/g, about 4.0.times.10.sup.4
cells/g, about 4.5.times.10.sup.4 cells/g, about 5.0.times.10.sup.4
cells/g, about 5.5.times.10.sup.4 cells/g, about 6.0.times.10.sup.4
cells/g, about 6.5.times.10.sup.4 cells/g, about 7.0.times.10'
cells/g, about 7.5.times.10.sup.4 cells/g, about 8.0.times.10.sup.4
cells/g, about 9.5.times.10.sup.4 cells/g.
[0232] In other embodiments, the number of cells per gram of kidney
is about 1.0.times.10.sup.5 cells/g, about 1.5.times.10.sup.5
cells/g, about 2.0.times.10.sup.5 cells/g, about 2.5.times.10.sup.5
cells/g, about 3.0.times.10.sup.5 cells/g, about 3.5.times.10.sup.5
cells/g, about 4.0.times.10.sup.5 cells/g, about 4.5.times.10.sup.5
cells/g, about 5.0.times.10.sup.5 cells/g, about 5.5.times.10.sup.5
cells/g, about 6.0.times.10.sup.5 cells/g, about 6.5.times.10.sup.5
cells/g, about 7.0.times.10 cells/g, about 7.5.times.10.sup.5
cells/g, about 8.0.times.10.sup.5 cells/g, about 8.5.times.10.sup.5
cells/g, about 9.0.times.10.sup.5 cells/g, or about
9.5.times.10.sup.5 cells/g.
[0233] In other embodiments, the number of cells per gram of kidney
is about 1.0.times.10.sup.6 cells/g, about 1.5.times.10.sup.6
cells/g, about 2.0.times.10.sup.6 cells/g, about 2.5.times.10.sup.6
cells/g, about 3.0.times.10.sup.6 cells/g, about 3.5.times.10.sup.6
cells/g, about 4.0.times.10.sup.6 cells/g, about 4.5.times.10.sup.6
cells/g, about 5.0.times.10.sup.6 cells/g, about 5.5.times.106
cells/g, about 6.0.times.10.sup.6 cells/g, about 6.5.times.10.sup.6
cells/g, about 7.0.times.10.sup.6 cells/g, about 7.5.times.10.sup.6
cells/g, about 8.0.times.10.sup.6 cells/g, about 8.5.times.10.sup.6
cells/g, about 9.0.times.10.sup.6 cells/g, about 9.5.times.10.sup.6
cells/g, 1.0.times.10.sup.7 cells/g, or about 1.5.times.10.sup.7
cells/g.
[0234] A total number of cells may be selected for the formulation
and the volume of the formulation may be adjusted to reach the
proper dosage.
[0235] in some embodiments, the formulation may contain a dosage of
cells to a subject that is a single dosage or a single dosage plus
additional dosages. In other embodiments, the dosages may be
provided by way of a construct as described herein. The
therapeutically effective amount of the renal cell populations or
admixtures of renal cell populations described herein can range
from the maximum number of cells that is safely received by the
subject to the minimum number of cells necessary for treatment of
kidney disease, e.g., stabilization, reduced rate-of-decline, or
improvement of one or more kidney functions.
[0236] The therapeutically effective amount of the renal cell
populations or admixtures thereof described herein can be suspended
in a pharmaceutically acceptable carrier or excipient. Such a
carrier includes, but is not limited to basal culture medium plus
1% serum albumin, saline, buffered saline, dextrose, water,
collagen, alginate, hyaluronic acid, fibrin glue,
polyethyleneglycol, polyvinylalcohol, carboxymethylcellulose and
combinations thereof. The formulation should suit the mode of
administration.
[0237] Accordingly, the disclosure provides a use of a formulation
containing renal cell populations or admixtures thereof, for
example, the B2 cell population alone or admixed with the B3 and/or
B4 or B4' cell population, for the manufacture of a medicament to
treat kidney disease in a subject. In some embodiments, the
medicament further comprises recombinant polypeptides, such as
growth factors, chemokines or cytokines. In further embodiments,
the medicaments comprise a human kidney-derived cell population.
The cells used to manufacture the medicaments can be isolated,
derived, or enriched using any of the variations provided for the
methods described herein.
[0238] The renal cell preparation(s), or admixtures thereof, or
compositions are formulated in accordance with routine procedures
as a pharmaceutical composition adapted for administration to human
beings. Typically, compositions for Intravenous administration,
intra-arterial administration or administration within the kidney
capsule, for example, are solutions in sterile isotonic aqueous
buffer. Where necessary, the composition can also include a local
anesthetic to ameliorate any pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed
together in unit dosage form, for example, as a cryopreserved
concentrate in a hermetically sealed container such as an ampoule
indicating the quantity of active agent. When the composition is to
be administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
[0239] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there are a wide variety of suitable formulations of pharmaceutical
compositions (see, e.g., Alfonso R Gennaro (ed), Remington: The
Science and Practice of Pharmacy, formerly Remington's
Pharmaceutical Sciences 20th ed., Lippincott, Williams &
Wilkins, 2003, incorporated herein by reference in its entirety).
The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
[0240] One aspect further provides a pharmaceutical formulation,
comprising a renal cell preparation, for example, the B2 cell
preparation alone or incombination with the B3 and/or B4 or B4'
cell preparation, and a pharmaceutically acceptable carrier. In
some embodiments, the formulation comprises from 10.sup.4 to
10.sup.9 mammalian kidney-derived cells.
[0241] Modified Release Formulations
[0242] In one aspect, the formulations of the present disclosure
are provided as modified release formulations. In general, the
modified release is characterized by an initial release of a first
active agent upon administration following by at least one
additional, subsequent release of a second active agent. The first
and second active agents may be the same or they may be different.
In one embodiment, the formulations provide modified release
through multiple components in the same formulation. In another
embodiment, the modified release formulation contains an active
agent as part of a first component that allows the active agent to
move freely throughout the volume of the formulation, thereby
permitting immediate release at the target site upon
administration. The first component may be a temperature-sensitive
biomaterial having a substantially liquid phase and a substantially
solid phase, wherein the first component is in a substantially
liquid phase at the time of administration. In one embodiment, the
active agent in the substantially liquid phase such that it is
substantially free to move throughout the volume of the
formulation, and therefore is immediately released to the target
site upon administration.
[0243] In another embodiment, the modified release formulation has
an active agent as part of a second component in which the active
agent is attached to, deposited on, coated with, embedded in,
seeded upon, or entrapped in the second component, which persists
before and after administration to the target site. The second
component contains structural elements with which the active agent
is able to associate with, thereby preventing immediate release of
the active agent from the second component at the time of
administration. For example, the second component is provided in a
substantially solid form, e.g., biocompatible beads, which may be
crosslinked to prevent or delay in vivo enzymatic degradation. In
one embodiment, the active agent in the substantially solid phase
retains its structural integrity within the formulation before and
after administration and therefore it does not immediately release
the active agent to the target site upon administration. Suitable
carriers for modified release formulations have been described
herein but those of ordinary skill in the art will appreciate other
carriers that are appropriate for use herein.
[0244] In one embodiment, the formulation provides an initial rapid
delivery/release of delivered elements, including cells,
nanoparticles, therapeutic molecules, etc. followed by a later
delayed release of elements. The formulations of the present
disclosure can be designed for such biphasic release profile where
the agent to be delivered is provided in both an unattached form
(e.g., cells in a solution) and an attached form (e.g., cells
together with beads or another suitable carrier). Upon initial
administration, the unencumbered agent is provided immediately to
the site of delivery while release of the encumbered agent is
delayed until structural integrity of the carrier (e.g., beads)
fails at which point the previously attached agent is released. As
discussed below, other suitable mechanisms of release will be
appreciated by those of ordinary skill in the art.
[0245] The time delay for release can be adjusted based upon the
nature of the active agent. For example, the time delay for release
in a bioactive cell formulation may be on the order of seconds,
minutes, hours, or days. In some circumstances, a delay on the
order of weeks may be appropriate. For other active agents, such as
small or large molecules, the time delay for release in a
formulation may be on the order of seconds, minutes, hours, days,
weeks, or months. It is also possible for the formulation to
contain different biomaterials that provide different time delay
release profiles. For example, a first biomaterial with a first
active agent may have a first release time and a second biomaterial
with a second active agent may have a second release time. The
first and second active agent may be the same or different.
[0246] As discussed herein, the time period of delayed release may
generally correspond to the time period for loss of structural
integrity of a biomaterial. However, those of ordinary skill in the
art will appreciate other mechanisms of delayed release. For
example, an active agent may be continually released over time
independent of the degradation time of any particular biomaterial,
e.g., diffusion of a drug from a polymeric matrix. In addition,
bioactive cells can migrate away from a formulation containing a
biomaterial and the bioactive cells to native tissue. In one
embodiment, bioactive cells migrate off of a biomaterial, e.g., a
bead, to the native tissue.
[0247] Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Prolonged
absorption of injectable formulations can be brought about by
including in the formulation an agent that delays absorption, for
example, monostearate salts and gelatin. Many methods for the
preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker,
Inc., New York, 1978. Additional methods applicable to the
controlled or extended release of polypeptide agents are described,
for example, in U.S. Pat. Nos. 6,306,406 and 6,346,274, as well as,
for example, in U.S. Patent Application Nos. US20020182254 and
US20020051808, all of which are incorporated herein by
reference.
9. METHODS AND ROUTES OF ADMINISTRATION
[0248] The bioactive cell formulations of the present disclosure
can be administered alone or in combination with other bioactive
components. The formulations are suitable for injection or
implantation of incorporated tissue engineering elements to the
interior of solid organs to regenerate tissue. In addition, the
formulations are used for the injection or implantation of tissue
engineering elements to the wall of hollow organs to regenerate
tissue.
[0249] In one aspect, the present disclosure provides methods of
providing a bioactive cell formulation described herein to a
subject in need. In one embodiment, the source of the bioactive
cell may be autologous or allogeneic, syngeneic (autogeneic or
isogeneic), and any combination thereof. In instances where the
source is not autologous, the methods may include the
administration of an immunosuppressant agent. Suitable
immunosuppressant drugs include, without limitation, azathioprine,
cyclophosphamide, mizoribine, ciclosporin, tacrolimus hydrate,
chlorambucil, lobenzarit disodium, auranofin, alprostadil,
gusperimus hydrochloride, biosynsorb, muromonab, alefacept,
pentostatin, daclizumab, sirolimus, mycophenolate mofetil,
leflonomide, basiliximab, dornase .alpha., bindarid, cladribine,
pimecrolimus, ilodecakin, cedelizumab, efalizumab, everolimus,
anisperimus, gavilimomab, faralimomab, clofarabine, rapamycin,
siplizumab, saireito, LDP-03, CD4, SR-43551, SK&F-106615,
IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-802715,
GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT-1345,
CNI-1493, CBP-2011, J-695, UP-920, L-732531, ABX-RB2, AP-1903,
IDPS, BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-38925,
ISAtx-247, RDP-58, PNU-156804, UP-1082, TMC-95A, TV-4710,
PTR-262-MG, and AGI-1096 (see U.S. Pat. No. 7,563,822). Those of
ordinary skill in the art will appreciate other suitable
immunosuppressant drugs.
[0250] The treatment methods of the subject disclosure involve the
delivery of a bioactive cell formulation described herein. In one
embodiment, direct administration of cells to the site of intended
benefit is preferred. A subject in need may also be treated by in
vivo contacting of a native kidney with a bioactive cell
formulation described herein together with products secreted from
one or more enriched renal cell populations, and/or an admixture or
construct containing the same.
[0251] The step of contacting a native kidney in vivo with secreted
products may be accomplished through the use/administration of a
formulation containing a population of secreted products from cell
culture media, e.g., conditioned media, or by implantation of an
enriched cell population, and admixture, or a construct capable of
secreting the products in vivo. The step of in vivo contacting
provides a regenerative effect to the native kidney.
[0252] A variety of means for administering cells and/or secreted
products to subjects will, in view of this specification, be
apparent to those of skill in the art. Such methods include
injection of the cells into a target site in a subject.
[0253] Cells and/or secreted products can be inserted into a
delivery device or vehicle, which facilitates introduction by
injection or implantation into the subjects. In certain
embodiments, the delivery vehicle can include natural materials. In
certain other embodiments, the delivery vehicle can include
synthetic materials. In one embodiment, the delivery vehicle
provides a structure to mimic or appropriately fit into the organ's
architecture. In other embodiments, the delivery vehicle is
fluid-like in nature. Such delivery devices can include tubes,
e.g., catheters, for injecting cells and fluids into the body of a
recipient subject. In a preferred embodiment, the tubes
additionally have a needle, e.g., a syringe, through which the
cells can be introduced into the subject at a desired location. In
some embodiments, mammalian kidney-derived cell populations are
formulated for administration into a blood vessel via a catheter
(where the term "catheter" is intended to include any of the
various tube-like systems for delivery of substances to a blood
vessel). Alternatively, the cells can be inserted into or onto a
biomaterial or scaffold, including but not limited to textiles,
such as weaves, knits, braids, meshes, and non-wovens, perforated
films, sponges and foams, and beads, such as solid or porous beads,
microparticles, nanoparticles, and the like (e.g., Cultispher-S
gelatin beads--Sigma). The cells can be prepared for delivery in a
variety of different forms. For example, the cells can be suspended
in a solution or gel. Cells can be mixed with a pharmaceutically
acceptable carrier or diluent in which the cells remain viable.
Pharmaceutically acceptable carriers and diluents include saline,
aqueous buffer solutions, solvents and/or dispersion media. The use
of such carriers and diluents is well known in the art. The
solution is preferably sterile and fluid, and will often be
isotonic. Preferably, the solution is stable under the conditions
of manufacture and storage and preserved against the contaminating
action of microorganisms such as bacteria and fungi through the use
of, for example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. One of skill in the art will appreciate
that the delivery vehicle used in the delivery of the cell
populations and admixtures thereof can include combinations of the
above-mentioned characteristics.
[0254] Modes of administration of the formulations containing
isolated renal cell population(s), for example, the B2 cell
population alone or admixed with B4' and/or B3, include, but are
not limited to, systemic, intra-renal (e.g., parenchymal),
intravenous or intra-arterial injection and injection directly into
the tissue at the intended site of activity. Additional modes of
administration to be used include single or multiple injection(s)
via direct laparotomy, via direct laparoscopy, transabdominal, or
percutaneous. Still yet additional modes of administration to be
used include, for example, retrograde and ureteropelvic infusion.
Surgical means of administration include one-step procedures such
as, but not limited to, partial nephrectomy and construct
implantation, partial nephrectomy, partial pyelectomy,
vascularization with omentum.+-.peritoneum, multifocal biopsy
needle tracks, cone or pyramidal, to cylinder, and renal pole-like
replacement, as well as two-step procedures including, for example,
organoid-internal bioreactor for replanting. In one embodiment, the
formulations containing admixtures of cells are delivered via the
same route at the same time. In another embodiment, each of the
cell compositions comprising the controlled admixture are delivered
separately to specific locations or via specific methodologies,
either simultaneously or in a temporally-controlled manner, by one
or more of the methods described herein.
[0255] The appropriate cell implantation dosage in humans can be
determined from existing information relating to either the
activity of the cells, for example EPO production, or extrapolated
from dosing studies conducted in preclinical studies. From in vitro
culture and in vivo animal experiments, the amount of cells can be
quantified and used in calculating an appropriate dosage of
implanted material. Additionally, the patient can be monitored to
determine if additional implantation can be made or implanted
material reduced accordingly.
[0256] One or more other components can be added to the cell
populations and admixtures thereof, including selected
extracellular matrix components, such as one or more types of
collagen or hyaluronic acid known in the art, and/or growth
factors, platelet-rich plasma and drugs.
[0257] Those of ordinary skill in the art will appreciate the
various formulations and methods of administration suitable for the
secreted products described herein.
10. ARTICLES OF MANUFACTURE AND KITS
[0258] The instant disclosure further includes kits comprising the
polymeric matrices and scaffolds as disclosed herein and related
materials, and/or cell culture media and instructions for use. The
instructions for use may contain, for example, instructions for
culture of the cells or administration of the cells and/or cell
products. In one embodiment, the present disclosure provides a kit
comprising a scaffold as described herein and instructions. In yet
another embodiment, the kit includes an agent for detection of
marker expression, reagents for use of the agent, and instructions
for use. This kit may be used for the purpose of determining the
regenerative prognosis of a native kidney in a subject following
the implantation or administration of a cell population, an
admixture, or a construct described herein. The kit may also be
used to determine the biotherapeutic efficacy of a cell population,
admixture, or construct described herein.
[0259] Another embodiment is an article of manufacture containing
bioactive cells useful for treatment of subjects in need. The
article of manufacture comprises a container and a label or package
insert on or associated with the container. Suitable containers
include, for example, bottles, vials, syringes, etc. The containers
may be formed from a variety of materials such as glass or plastic.
The container holds a composition which is effective for treating a
condition and may have a sterile access port (for example the
container may be a solution bag or a vial having a stopper
pierceable by an injection needle). At least one active agent in
the formulation is a bioactive cell population as provided for
herein. The label or package insert indicates that the formulation
is used for treating the particular condition. The label or package
insert will further comprise instructions for administering the
formulation to the patient. Articles of manufacture and kits
comprising combinatorial therapies described herein are also
contemplated. Package insert refers to instructions customarily
included in commercial packages of therapeutic products that
contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the
use of such therapeutic products. In one embodiment, the package
insert indicates that the formulation is used for treating a
disease or disorder, such as, for example, a kidney disease or
disorder. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes. Kits are also provided that are
useful for various purposes, e.g., for assessment of regenerative
outcome. Kits can be provided which contain detection agents for
urine-derived vesicles and/or their contents, e.g., nucleic acids
(such as miRNA), vesicles, exosomes, etc., as described herein.
Detection agents include, without limitation, nucleic acid primers
and probes, as well as antibodies for in vitro detection of the
desired target. As with the article of manufacture, the kit
comprises a container and a label or package insert on or
associated with the container. The container holds a composition
comprising at least one detection agent. Additional containers may
be included that contain, e.g., diluents and buffers or control
detection agents. The label or package insert may provide a
description of the composition as well as instructions for the
intended in vitro, prognostic, or diagnostic use.
11. REPORTS
[0260] The methods of this disclosure, when practiced for
commercial purposes generally produce a report or summary of the
regenerative prognosis. The methods of this disclosure will produce
a report comprising a prediction of the probable course or outcome
of regeneration before and after any administration or implantation
of a formulation containing a cell population, an admixture, or a
construct described herein. The report may include information on
any indicator pertinent to the prognosis. The methods and reports
of this disclosure can further include storing the report in a
database. Alternatively, the method can further create a record in
a database for the subject and populate the record with data. In
one embodiment the report is a paper report, in another embodiment
the report is an auditory report, in another embodiment the report
is an electronic record. It is contemplated that the report is
provided to a physician and/or the patient. The receiving of the
report can further include establishing a network connection to a
server computer that includes the data and report and requesting
the data and report from the server computer. The methods provided
for herein may also be automated in whole or in part.
[0261] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. Thus, for an embodiment of the
invention using one of the terms, the invention also includes
another embodiment wherein one of these terms is replaced with
another of these terms. In each embodiment, the terms have their
established meaning. Thus, for example, one embodiment may
encompass a formulation "comprising" a number of components,
another embodiment would encompass a formulation "consisting
essentially of" the same components, and a third embodiment would
encompass a formulation "consisting of" the same components. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
[0262] The foregoing written description is considered to be
sufficient to enable one skilled in the art to practice the
invention. The following Examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0263] All patents, patent applications, and literature references
cited in the present specification are hereby incorporated by
reference in their entirety.
EXAMPLES
Example 1
Preparation of Solutions
[0264] This example provides the compositions of the various media
formulations and solutions used in the following Examples for the
isolation and characterization of the heterogeneous renal cell
population, and manufacture of the regenerative therapy
product.
TABLE-US-00001 TABLE 1.1 Culture Media and Solutions Material
Composition Tissue Transport Medium Viaspan .TM. or
HypoThermosol-FRS .RTM. or DMEM Kanamycin: 100 .mu.g/mL Renal Cell
Growth Medium DMEM:KSFM (50:50) 5% FBS Growth Supplements: HGF: 10
mg/L EGF: 2.5 .mu.g/L Insulin: 10.0 mg/L, Transferrin: 5.5 mg/L
Selenium: 670 .mu.g/L Kanamycin: 100 .mu.g/mL Tissue Wash Solution
DMEM Kanamycin: 100 .mu.g/mL Digestion Solution Collagenase IV: 300
Units Dispase: 5 mg/mL Calcium Chloride: 5 mM Cell Dissociation
Solution TrypLE Density Gradient Solution 7% OptiPrep OptiMEM
Cryopreservation Solution DMEM or HypoThermosol .RTM. FRS 10% DMSO
10% FBS
Example 2
Isolation of the Heterogeneous Unfractionated Renal Cell
Population
[0265] This example illustrates the isolation of an unfractionated
(UNFX) heterogeneous renal cell population from human. Initial
tissue dissociation was performed to generate heterogeneous cell
suspensions from human kidney tissue.
[0266] Renal tissue via kidney biopsy provided the source material
for a heterogeneous renal cell population. Renal tissue comprising
one or more of cortical, corticomedullary junction or medullary
tissue may be used. It is preferred that the corticomedullary
junction tissue is used. Multiple biopsy cores (minimum 2),
avoiding scar tissue, were required from a CKD kidney. Renal tissue
was obtained by the clinical investigator from the patient at the
clinical site approximately 4 weeks in advance of planned
implantation of the final NKA. The tissue was transported in the
Tissue Transport Medium of Example 1.
[0267] The tissue was then washed with Tissue Wash Solution of
Example 1 in order to reduce incoming bioburden before processing
the tissue for cell extractions.
[0268] Renal tissue was minced, weighed, and dissociated in the
Digestion Solution of Example 1. The resulting cell suspension was
neutralized in Dulbecco's Modified Eagle Medium (DMEM)+10% fetal
bovine serum (FBS) (Invitrogen, Carlsbad Calif.), washed, and
resuspended in serum-free, supplement-free, Keratinocyte Media
(KSFM) (Invitrogen). Cell suspensions were then subjected to a 15%
(w/v) iodixanol (OptiPrep.TM., Sigma) gradient to remove red blood
cells and debris prior to initiation of culture onto tissue culture
treated polystyrene flasks or dishes at a density of 25,000 cells
per cm.sup.2 in Renal Cell Growth Medium of Example 1. For example,
cells may be plated onto T500 Nunc flask at 25.times.10.sup.6
cells/flask in 150 ml of 50:50 media.
Example 3
Cell Expansion of the Isolated Renal Cell Population
[0269] Renal cell expansion is dependent on the amount of tissue
received and on the success of isolating renal cells from the
incoming tissue. Isolated cells can be cryopreserved, if required
(see infra). Renal cell growth kinetics may vary from sample to
sample due to the inherent variability of cells isolated from
individual patients.
[0270] A defined cell expansion process was developed that
accommodates the range of cell recoveries resulting from the
variability of incoming tissue Table 3.1. Expansion of renal cells
involves serial passages in closed culture vessels (e.g., T-flasks,
Cell Factories, HyperStacks.RTM.) in Renal Cell Growth Medium Table
1.1 using defined cell culture procedures.
[0271] A BPE-free medium was developed for human clinical trials to
eliminate the inherent risks associated with the use of BPE. Cell
growth, phenotype (CK18) and cell function (GGT and LAP enzymatic
activity) were evaluated in BPE-free medium and compared to BPE
containing medium used in the animal studies. Renal cell growth,
phenotype and function were
TABLE-US-00002 TABLE 3.1 Cell Recovery from Human Kidney Biopsies
Renal cells Source (cells/10 mg tissue) Human Kidney Tissue Passage
0 Passage 1 Samples 1.51-5.36 .times. 106 2.40-7.48 .times. 107 (n
= 6)
[0272] Once cell growth was observed in the initial T-flasks
(passage 0) and there were no visual signs of contamination,
culture medium was replaced and changed thereafter every 2-4 days
FIG. 2B. Cells were assessed to verify renal cell morphology by
visual observation of cultures under the microscope. Cultures
characteristically demonstrated a tight pavement or cobblestone
appearance, due to the cells clustering together. These
morphological characteristics vary during expansion and may not be
present at every passage. Cell culture confluence was estimated
using an Image Library of cells at various levels of confluence in
the culture vessels employed throughout cell expansions.
[0273] Renal cells were passaged by trypsinization when culture
vessels are at least 50% confluent FIG. 2B. Detached cells were
collected into vessels containing Renal Cell Growth Medium, counted
and cell viability calculated. At each cell passage, cells were
seeded at 500-4000 cells/cm2 in a sufficient number of culture
vessels in order to expand the cell number to that required for
formulation of NKA FIG. 2B. Culture vessels were placed in a
37.degree. C. incubator in a 5% CO2 environment. As described
above, cell morphology and confluence was monitored and tissue
culture media was replaced every 2-4 days. Table 3.2 lists the
viability of human renal cells observed during cell isolation and
expansion of six kidney biopsies from
TABLE-US-00003 TABLE 3.2 Cell Viability Human Renal Cells in
Culture Passage (n = 6) Cell Viability (Average %) Range (%) P0 88
84-93 P1 91 80-98 P2 94 92-99 P3 98 97-99
[0274] Inherent variability of tissue from different patients
resulted in different cell yield in culture. Therefore, it is not
practical to strictly define the timing of cell passages or number
and type of culture vessels required at each passage to attain
target cell numbers. Typically renal cells undergo 2 or 3 passages;
however, duration of culture and cell yield can vary depending on
the cell growth rate. This is exemplified in FIG. 3 where the
culture duration and cell yields (calculation) from 6 patients are
shown.
[0275] Cells were detached for harvest or passage with 0.25%
Trypsin with EDTA (Invitrogen). Viability was assessed via Trypan
Blue exclusion and enumeration was performed manually using a
hemacytometer or using the automated Cellometer.RTM. counting
system (Nexcelom Bioscience, Lawrence Mass.).
Example 4
Cryopreservation of Cultured Cells
[0276] Expanded renal cells were routinely cryopreserved to
accommodate for inherent variability of cell growth from individual
patients and to deliver product on a pre-determined clinical
schedule (FIG. 2B). Cryopreserved cells also provide a backup
source of cells in the event that another NKA is needed (e.g.,
delay due to patient sickness, unforeseen process events, etc.).
Conditions were established that have been used to cryopreserve
cells and recover viable, functional cells upon thawing.
[0277] For cryopreservation, cells were suspended to a final
concentration of about 50.times.10.sup.5 cells/mL in
Cryopreservation Solution (see Example 1) and dispensed into vials.
One mL vials containing about 50.times.10.sup.6 cells/mL were
placed in the freezing chamber of a controlled rate freezer and
frozen at a pre-programmed rate. After freezing, the cells were
transferred to a liquid nitrogen freezer for in-process
storage.
Example 5
Preparation of SRC Cell Population
[0278] Selected Renal Cells (SRC) can be prepared from the final
culture vessels that are grown from cryopreserved cells or directly
from expansion cultures depending on scheduling FIG. 2B.
[0279] If using cryopreserved cells, the cells were thawed and
plated on tissue culture vessels for one final expansion step. When
the final culture vessels were approximately 50-100% confluent
cells were ready for processing for SRC separation. Media exchanges
and final washes of NKA dilute any residual Cryopreservation
Solution in the final product.
[0280] Once the final cell culture vessels have reached at least
50% confluence the culture vessels were transferred to a hypoxic
incubator set for 2% oxygen in a 5% CO2 environment at 37.degree.
C. and cultured overnight. See FIG. 2C. Cells may be held in the
oxygen-controlled incubator set to 2% oxygen for as long as 48
hours. Exposure to the more physiologically relevant low-oxygen
(2%) environment improved cell separation efficiency and enabled
greater detection of hypoxia-induced markers such as VEGF.
[0281] After the cells have been exposed to the hypoxic conditions
for a sufficient time (e.g., overnight to 48 hours), the cells were
detached with 0.25% Trypsin with EDTA (Invitrogen). Viability was
assessed via Trypan Blue exclusion and enumeration was performed
manually using a hemacytometer or using the automated
Cellometer.RTM. counting system (Nexcelom Bioscience, Lawrence
Mass.). Cells were washed once with DPBS and resuspended to about
850.times.10.sup.6 cells/mL in DPBS.
[0282] Density gradient centrifugation was used to separate
harvested renal cell populations based on cell buoyant density.
Renal cell suspensions were separated on single-step 7% iodixanol
Density Gradient Solution (OptiPrep; 60% (w/v) in OptiMEM; see
Example 1).
[0283] The 7% OptiPrep gradient solution was prepared and
refractive index indicative of desired density was measured (R.I.
1.3456+/-0.0004) prior to use. Harvested renal cells were layered
on top of the gradient solution. The density gradient was
centrifuged at 800 g for 20 min at room temperature (without brake)
in either centrifuge tubes or a cell processor (e.g., COBE 2991).
The cellular fraction exhibiting buoyant density greater than
approximately 1.045 g/mL was collected after centrifugation as a
distinct pellet FIG. 4. Cells maintaining a buoyant density of less
than 1.045 g/mL were excluded and discarded.
[0284] The SRC pellet was re-suspended in DPBS (FIG. 2C). The
carry-over of residual OptiPrep, FBS, culture medium and ancillary
materials in the final product is minimized by 4 DPBS wash and 1
Gelatin Solution steps.
Example 6
[0285] Cells with Therapeutic Potential can be Isolated and
Propagated from Normal and Chronically-Diseased Kidney Tissue
[0286] The objective of the present study was to determine the
functional characterization of human NKA cells through high content
analysis (HCA). High-content imaging (HCl) provides simultaneous
imaging of multiple sub-cellular events using two or more
fluorescent probes (multiplexing) across a number of samples.
High-content Analysis (HCA) provides simultaneous quantitative
measurement of multiple cellular parameters captured in
High-Content Images. In brief, unfractionated (UNFX) cultures were
generated (Aboushwareb et al., World J Urol 26, 295, 2008) and
maintained independently from core biopsies taken from five human
kidneys with advanced chronic kidney disease (CKD) and three
non-CKD human kidneys using standard biopsy procedures. After (2)
passages of UNFX ex vivo, cells were harvested and subjected to
density gradient methods (as described in Example 2 of Basu et al.,
WO 2012/064369) to generate subfractions, including subfractions
B2, B3, and/or B4.
[0287] Human kidney tissues were procured from non-CKD and CKD
human donors as summarized in Table 10.1 of Ilagan et al.
PCT/US2011/036347. FIG. 4 of Ilagan et al. PCT/US2011/036347 shows
histopathologic features of the HK17 and HK19 samples. Ex vivo
cultures were established from all non-CKD (3/3) and CKD (5/5)
kidneys. High content analysis (HCA) of albumin transport in human
NKA cells defining regions of interest (ROI) is shown in FIG. 5
(HCA of albumin transport in human NKA cells) of Ilagan et al.
PCT/US2011/036347. Quantitative comparison of albumin transport in
NKA cells derived from non-CKD and CKD kidney is shown in FIG. 6 of
Ilagan et al. PCT/US2011/036347. As shown in FIG. 6 of Ilagan et
al. PCT/US2011/036347, albumin transport is not compromised in
CKD-derived NKA cultures. Comparative analysis of marker expression
between tubular-enriched B2 and tubular cell-depleted B4
subfractions is shown in FIG. 7 (CK8/18/19) of Ilagan et al.
PCT/US2011/036347.
[0288] Comparative functional analysis of albumin transport between
tubular-enriched B2 and tubular cell-depleted B4 subfractions is
shown in FIG. 8 of Ilagan et al. PCT/US2011/036347. Subfraction B2
is enriched in proximal tubule cells and thus exhibits increased
albumin-transport function.
[0289] Albumin Uptake:
[0290] Culture media of cells grown to confluency in 24-well,
collagen IV plates (BD Biocoat.TM.) was replaced for 18-24 hours
with phenol red-free, serum-free, low-glucose DMEM (pr-/s-/Ig DMEM)
containing 1.times. antimycotic/antibiotic and 2 mM glutamine.
Immediately prior to assay, cells were washed and incubated for 30
minutes with pr-/s-/Ig DMEM+10 mM HEPES, 2 mM glutamine, 1.8 mM
CaCl2, and 1 mM MgCl2. Cells were exposed to 25 .mu.g/mL
rhodamine-conjugated bovine albumin (Invitrogen) for 30 min, washed
with ice cold PBS to stop endocytosis and fixed immediately with 2%
paraformaldehyde containing 25 .mu.g/mL Hoechst nuclear dye. For
inhibition experiments, 1 .mu.M receptor-associated protein (RAP)
(Ray Biotech, Inc., Norcross Ga.) was added 10 minutes prior to
albumin addition. Microscopic imaging and analysis was performed
with a BD Pathway.TM. 855 High-Content BioImager (Becton Dickinson)
(see Kelley et al. Am J Physiol Renal Physiol. 2010 November;
299(5):F1026-39. Epub Sep. 8, 2010).
[0291] In conclusion, HCA yields cellular level data and can reveal
populations dynamics that are undetectable by other assays, i.e.,
gene or protein expression. A quantifiable ex-vivo HCA assay for
measuring albumin transport (HCA-AT) function can be utilized to
characterize human renal tubular cells as components of human NKA
prototypes. HCA-AT enabled comparative evaluation of cellular
function, showing that albumin transport-competent cells were
retained in NKA cultures derived from human CKD kidneys. It was
also shown that specific subfractions of NKA cultures, B2 and B4,
were distinct in phenotype and function, with B2 representing a
tubular cell-enriched fraction with enhanced albumin transport
activity. The B2 cell subpopulation from human CKD are
phenotypically and functionally analogous to rodent B2 cells that
demonstrated efficacy in viva (as shown above).
Example 7
Low-Oxygen Culture Prior to Gradient Affects Band Distribution,
Composition, & Gene Expression
[0292] To determine the effect of oxygen conditions on distribution
and composition of prototypes B2 and B4, neokidney cell
preparations from different species were exposed to different
oxygen conditions prior to the gradient step. A rodent neo-kidney
augmentation (NKA) cell preparation (RK069) was established using
standard procedures for rat cell isolation and culture initiation,
as described in Kelley et al., 2010, supra. All flasks were
cultured for 2-3 days in 21% (atmospheric) oxygen conditions. Media
was changed and half of the flasks were then relocated to an
oxygen-controlled incubator set to 2% oxygen, while the remaining
flasks were kept at the 21% oxygen conditions, for an additional 24
hours. Cells were then harvested from each set of conditions using
standard enzymatic harvesting procedures described supra. Step
gradients were prepared according to standard procedures and the
"normoxic" (21% oxygen) and "hypoxic" (2% oxygen) cultures were
harvested separately and applied side-by-side to identical step
gradients. While 4 bands and a pellet were generated in both
conditions, the distribution of the cells throughout the gradient
was different in 21% and 2% oxygen-cultured batches (Table 7.1).
Specifically, the yield of B2 was increased with hypoxia, with a
concomitant decrease in 83. Furthermore, the expression of
B4-specific genes (such as erythropoietin) was enhanced in the
resulting gradient generated from the hypoxic-cultured cells (FIG.
73 of Presnell et al. WO/2010/056328).
[0293] A canine NKA cell preparation (DK008) was established using
standard procedures for dog cell isolation and culture (analogous
to rodent isolation and culture procedures), as described in Basu
et al., WO 2012/064369. All flasks were cultured for 4 days in 21%
(atmospheric) oxygen conditions, then a subset of flasks were
transferred to hypoxia (2%) for 24 hours while a subset of the
flasks were maintained at 21%. Subsequently, each set of flasks was
harvested and subjected to identical step gradients. Similar to the
rat results, the hypoxic-cultured dog cells distributed throughout
the gradient differently than the atmospheric oxygen-cultured dog
cells (Table 7.1). Again, the yield of B2 was increased with
hypoxic exposure prior to gradient, along with a concomitant
decrease in distribution into B3.
TABLE-US-00004 TABLE 7.1 Rat (RK069) Dog (DK008) 2% O2 21% O2 2% O2
21% O2 B1 0.77% 0.24% 1.20% 0.70% B2 88.50% 79.90% 64.80% 36.70% B3
10.50% 19.80% 29.10% 40.20% B4 0.23% 0.17% 4.40% 21.90%
[0294] The above data show that pre-gradient exposure to hypoxia
enhances composition of B2 as well as the distribution of specific
specialized cells (erythropoietin-producing cells, vascular cells,
and glomerular cells) into B4. Thus, hypoxic culture, followed by
density-gradient separation as described in Basu et al., supra, is
an effective way to generate `B2` and `B4` cell populations, across
species.
Example 8
Characterization of an Unfractionated Mixture of Renal Cells
Isolated from an Autoimmune Glomerulonephritis Patient Sample
[0295] An unfractionated mixture of renal cells was isolated, as
described above, from an autoimmune glomerulonephritis patient
sample. To determine the unbiased genotypic composition of specific
subpopulations of renal cells isolated and expanded from kidney
tissue, quantitative real time PCR (qRTPCR) analysis (Brunskill et
al., Dev. Cell. 2008 November; (5):781-791) was employed to
identify differential cell-type-specific and pathway-specific gene
expression patterns among the cell subfractions. As shown in Table
6.1 of Ilagan et al. PCT/US2011/036347, HK20 is an autoimmune
glomerulonephritis patient sample. As shown in Table 6.2 of Ilagan
et al. PCT/US2011/036347, cells generated from HK20 are lacking
glomerular cells, as determined by qRTPCR.
Example 9
Genetic Profiling of Therapeutically Relevant Renal Bioactive Cell
Populations Isolated from a Case of Focal Segmental
Glomerulosclerosis
[0296] To determine the unbiased genotypic composition of specific
subpopulations of renal cells isolated and expanded from kidney
tissue, quantitative real time PCR (qRTPCR) analysis (Brunskill et
al., supra 2008) was employed to identify differential
cell-type-specific and pathway-specific gene expression patterns
among the cell subfractions. Human preparation HK023, derived from
a case of focal segmental glomerulosclerosis (FSGS) in which a
large portion of glomeruli had been destroyed, was evaluated for
presence of glomerular cells in the B4 fraction at the time of
harvest. In brief, unfractionated (UNFX) cultures were generated
(Aboushwareb et al., World J Urol 26, 295, 2008) and maintained
independently from each of (4) core biopsies taken from the kidney
using standard biopsy procedures. After (2) passages of UNFX ex
vivo, cells were harvested and subjected to density gradient
methods according to Example 6 of Basu et al., WO2012/064369 to
generate subfractions, including subfraction B4, which is known to
be enriched for endocrine, vascular, and glomerular cells based on
work conducted in rodent, dog, and other human specimens.
[0297] The B4 fractions were collected separately from each
independent UNFX sample of HK023, appearing as distinct bands of
cells with buoyant density between 1.063-1.091 g/mL. RNA was
isolated from each sample and examined for expression of Podocin
(glomerular cell marker) and PECAM (endothelial cell marker) by
quantitative real-time PCR. As expected from a biopsy-generated
sample from a case of severe FSGS, the presence of podocin(+)
glomerular cells in 84 fractions was inconsistent, with podocin
undetectable in 2/4 of the samples. In contrast, PECAM+ vascular
cells were consistently present in the B4 fractions of 4/4 of the
biopsy-initiated cultures. Thus, the B4 fraction can be isolated at
the 1.063-1.091 g/mL density range, even from human kidneys with
severe disease states.
TABLE-US-00005 TABLE 9.1 Expression of Podocin and PECAM for
detection of glomerular and vascular cells in subfraction B4
isolated from a case of FSGS. HK02 RQ RQ 3/Biopsy (Podocin)/B4
(PECAM)/B4 #1/p2 0.188 0.003 #2/p2 ND 0.02 #3/p2 40.1 0.001 #4/p2
ND 0.003
[0298] Further, as shown in Table 7.2 of Ilagan et al.
PCT/US2011/036347, human sample (HK018) displayed undetected
Podocin (glomerular marker) by qRTPCR after density gradient
centrifugation.
Example 10
Enrichment/Depletion of Viable Kidney Cell Types Using Fluorescent
Activated Cell Sorting (FACS)
[0299] One or more isolated kidney cells may be enriched, and/or
one or more specific kidney cell types may be depleted from
isolated primary kidney tissue using fluorescent activated cell
sorting (FACS).
[0300] Reagents:
[0301] 70% ethanol; Wash buffer (PBS); 50:50 Kidney cell medium
(50% DMEM high glucose): 50% Keratinocyte-SFM; Trypan Blue 0.4%;
Primary antibodies to target kidney cell population such as CD31
for kidney endothelial cells and Nephrin for kidney glomerular
cells. Matched isotype specific fluorescent secondary antibodies;
Staining buffer (0.05% BSA in PBS).
[0302] Procedure:
[0303] Following standard procedures for cleaning the biological
safety cabinet (BSC), a single cell suspension of kidney cells from
either primary isolation or cultured cells may be obtained from a
T500 T/C treated flask and resuspend in kidney cell medium and
place on ice. Cell count and viability is then determined using
trypan blue exclusion method. For kidney cell enrichment/depletion
of, for example, glomerular cells or endothelial cells from a
heterogeneous population, between 10 and 50.times.10.sup.6 live
cells with a viability of at least 70% are obtained. The
heterogeneous population of kidney cells is then stained with
primary antibody specific for target cell type at a starting
concentration of 1 .mu.g/0.1 ml of staining buffer/1.times.10.sup.6
cells (titer if necessary). Target antibody can be conjugated such
as CD31 PE (specific for kidney endothelial cells) or un-conjugated
such as Nephrin (specific for kidney glomerular cells).
[0304] Cells are then stained for 30 minutes on ice or at 4.degree.
C. protected from light. After 30 minutes of incubation, cells are
washed by centrifugation at 300.times.g for 5 min. The pellet is
then resuspended in either PBS or staining buffer depending on
whether a conjugated isotype specific secondary antibody is
required. If cells are labeled with a fluorochrome conjugated
primary antibody, cells are resuspended in 2 mls of PBS per
10.sup.7 cells and proceed to FACS aria or equivalent cell sorter.
If cells are not labeled with a fluorochrome conjugated antibody,
then cells are labeled with an isotype specific fluorochrome
conjugated secondary antibody at a starting concentration of 1
.mu.g/0.1 ml/10.sup.6 cells.
[0305] Cells are then stained for 30 min. on ice or at 4.degree. C.
protected from light. After 30 minutes of incubation, cells are
washed by centrifugation at 300.times.g for 5 min. After
centrifugation, the pellet is resuspended in PBS at a concentration
of 5.times.10.sup.6/ml of PBS and then 4 mls per 12.times.75 mm is
transferred to a sterile tube.
[0306] FACs Aria is prepared for live cell sterile sorting per
manufacturer's instructions (BD FACs Aria User Manual). The sample
tube is loaded into the FACs Aria and PMT voltages are adjusted
after acquisition begins. The gates are drawn to select kidney
specific cells types using fluorescent intensity using a specific
wavelength. Another gate is drawn to select the negative
population. Once the desired gates have been drawn to encapsulate
the positive target population and the negative population, the
cells are sorted using manufacturer's instructions.
[0307] The positive target population is collected in one 15 ml
conical tube and the negative population in another 15 ml conical
tube filled with 1 ml of kidney cell medium. After collection, a
sample from each tube is analyzed by flow cytometry to determine
purity. Collected cells are washed by centrifugation at 300.times.g
for 5 min. and the pellet is resuspended in kidney cell medium for
further analysis and experimentation.
Example 11
Enrichment/Depletion of Kidney Cell Types Using Magnetic Cell
Sorting
[0308] One or more isolated kidney cells may be enriched and/or one
or more specific kidney cell types may be depleted from isolated
primary kidney tissue.
[0309] Reagents:
[0310] 70% ethanol, Wash buffer (PBS), 50:50 Kidney cell medium
(50% DMEM high glucose): 50% Keratinocyte-SFM, Trypan Blue 0.4%,
Running Buffer (PBS, 2 mM EDTA, 0.5% BSA), Rinsing Buffer (PBS, 2
mM EDTA), Cleaning Solution (70% v/v ethanol), Miltenyi FCR
Blocking reagent, Mlltenyi microbeads specific for either IgG
isotype, target antibody such as CD31(PECAM) or Nephrin, or
secondary antibody.
[0311] Procedure:
[0312] Following standard procedures for cleaning the biological
safety cabinet (BSC), a single cell suspension of kidney cells from
either primary isolation or culture is obtained and resuspended in
kidney cell medium. Cell count and viability is determined using
trypan blue exclusion method. For kidney cell enrichment/depletion
of, for example, glomerular cells or endothelial cells from a
heterogeneous population, at least 10.sup.6 up to 4.times.10.sup.9
live cells with a viability of at least 70% is obtained.
[0313] The best separation for enrichment/depletion approach is
determined based on target cell of interest. For enrichment of a
target frequency of less than 10%, for example, glomerular cells
using Nephrin antibody, the Miltenyi autoMACS, or equivalent,
instrument program POSSELDS (double positive selection in sensitive
mode) is used. For depletion of a target frequency of greater than
10%, the Miltenyi autoMACS, or equivalent, instrument program
DEPLETES (depletion in sensitive mode) is used.
[0314] Live cells are labeled with target specific primary
antibody, for example, Nephrin rb polyclonal antibody for
glomerular cells, by adding 1 .mu.g/10.sup.6 cells/0.1 ml of PBS
with 0.05% BSA in a 15 ml conical centrifuge tube, followed by
incubation for 15 minutes at 4.degree. C.
[0315] After labeling, cells are washed to remove unbound primary
antibody by adding 1-2 ml of buffer per 10.sup.7 cells followed by
centrifugation at 300.times.g for 5 min. After washing, isotype
specific secondary antibody, such as chicken anti-rabbit PE at 1
ug/10.sup.6/0.1 ml of PBS with 0.05% BSA, is added, followed by
incubation for 15 minutes at 4.degree. C.
[0316] After incubation, cells are washed to remove unbound
secondary antibody by adding 1-2 ml of buffer per 10.sup.7 cells
followed by centrifugation at 300.times.g for 5 min. The
supernatant is removed, and the cell pellet is resuspended in 60
.mu.l of buffer per 10.sup.7 total cells followed by addition of 20
.mu.l of FCR blocking reagent per 10.sup.7 total cells, which is
then mixed well.
[0317] Add 20 .mu.l of direct MACS microbeads (such as anti-PE
microbeads) and mix and then incubate for 15 min at 4.degree.
C.
[0318] After incubation, cells are washed by adding 10-20.times.
the labeling volume of buffer and centrifuging the cell suspension
at 300.times.g for 5 min. and resuspending the cell pellet in 500
.mu.l-2 mls of buffer per 10.sup.8 cells.
[0319] Per manufacturer's instructions, the autoMACS system is
cleaned and primed in preparation for magnetic cell separation
using autoMACS. New sterile collection tubes are placed under the
outlet ports. The autoMACS cell separation program is chosen. For
selection the POSSELDS program is chosen. For depletion the
DEPLETES program is chosen.
[0320] The labeled cells are inserted at uptake port, then
beginning the program. After cell selection or depletion, samples
are collected and placed on ice until use. Purity of the depleted
or selected sample is verified by flow cytometry.
Example 12
Phenotypic Characterization of the Enriched Heterogeneous Renal
Cell Population
[0321] The following example details the use of flow cytometry to
characterize the selected heterogeneous human renal cells of
Example 5. The heterogeneous renal cell population was composed
primarily of renal epithelial cells that are well known for their
regenerative potential. Other parenchymal (vascular) and stromal
(collecting duct) cells may be sparsely present in the autologous
cell population.
[0322] Cell phenotype is monitored by expression analysis of renal
cell markers using flow cytometry. Phenotypic analysis of cells is
based on the use of antigenic markers specific for the cell type
being analyzed. Flow cytometric analysis provides a quantitative
measure of cells in the sample population which express the
antigenic marker being analyzed.
[0323] A variety of markers have been reported in the literature as
being useful for phenotypic characterization of renal cells: (i)
cytokeratins; (ii) transport membrane proteins (aquaporins and
cubilin); (iii) cell binding molecules (adherins, cluster of
differentiation, and lectins); and (iv) metabolic enzymes
(glutathione). Since the majority of cells found in cultures
derived from whole kidney digests are epithelial and endothelial
cells, the markers examined focus on the expression of proteins
specific for these two groups.
[0324] Cytokeratins are a family of intermediate filament proteins
expressed by many types of epithelial cells to varying degrees. The
subset of cytokeratins expressed by an epithelial cell depends upon
the type of epithelium. For example, cytokeratins 7, 8, 18 and 19
are all expressed by normal simple epithelia of the kidney and
remaining urogenital tract as well as the digestive and respiratory
tracts. These cytokeratins in combination are responsible for the
structural integrity of epithelial cells. This combination
represents both the acidic (type I) and basic (type II) keratin
families and is found abundantly expressed in renal cells
(Oosterwijk et al., J Histochem Cytochem, 38(3):385-392, 1990).
Preferred cytokeratins for use herein are CK8, CK18, CK19 and
combinations thereof
[0325] Aquaporins are transport membrane proteins which allow the
passage of water into and out of the cell, while preventing the
passage of ions and other solutes. There are thirteen aquaporins
described in the literature, with six of these being found in the
kidney (Nielsen et al., J Histochem Cytochem, 38(3):385-392, 2002).
Aquaporin2, by exerting tight control in regulating water flow, is
responsible for the plasma membranes of renal collecting duct
epithelial cells having a high permeability to water, thus
permitting water to flow in the direction of an osmotic gradient
(Bedford et al., J Am Soc Nephrol, 14(10):2581-2587, 2003; Takata
et al., Histochem Cell Biol, 130(2):197-209, 2008; Tamma et al.,
Endocrinology, 148(3):1118-1130, 2007). Aquaporin1 is
characteristic of the proximal tubules (Baer et al., Cells Tissues
Organs: 184(1), 16-22, 2006; Nielsen et al., 2002, supra).
[0326] Cubilin is a transport membrane receptor protein. When it
co-localizes with the protein megalin, together they promote the
internalization of cubilin-bound ligands such as albumin. Cubilin
is located within the epithelium of the intestine and the kidney
(Christensen, Am J Physiol Renal Physiol, 280(4):F562-573,
2001).
[0327] CXCR4 is a transport membrane protein which serves as a
chemokine receptor for SDF1. Upon ligand binding, intracellular
calcium levels increase and MAPK1/MAPK3 activation is increased.
CXCR4 is constitutively expressed in the kidney and plays an
important role in kidney development and tubulogenesis (Ueland et
al., Dev Dyn, 238(5):1083-1091, 2009).
[0328] Cadherins are calcium-dependent cell adhesion proteins. They
are classified into four groups, with the E-cadherins being found
in epithelial tissue, and are involved in regulating mobility and
proliferation. E-cadherin is a transmembrane glycoprotein which has
been found to be localized in the adherins junctions of epithelial
cells which make up the distal tubules in the kidney (Prozialeck et
al., BMC Physiol, 4:10, 2004; Shen et al., Mod Pathol,
18(7):933-940, 2005).
[0329] DBA (Dolichos biflorus agglutinin) is an
.alpha.-N-acetylgalactosamine-binding lectin (cell binding protein)
carried on the surface of renal collecting duct structures, and is
regarded and used as a general marker of developing renal
collecting ducts and distal tubules (Michael et al., J Anat
210(1):89-97, 2007; Lazzeri et al., J Am Soc Nephrol 18
(12):3128-3138, 2007).
[0330] Cluster of differentiation 31 (CD31; also known as platelet
endothelial cell adhesion molecule, PECAM-1) is a cell adhesion
protein which is expressed by select populations of immune cells as
well as endothelial cells. In endothelial cells, this protein is
concentrated at the cell borders (DeLisser, 1997). Cluster of
differentiation 146 (CD146) is involved in cell adhesion and
cohesion of endothelial cells at intercellular junctions associated
with the actin cytoskeleton. Strongly expressed by blood vessel
endothelium and smooth muscle, CD146 is currently used as a marker
for endothelial cell lineage (Malyszko et al., J Clin Endocrinol
Metab, 89(9):4620-4627, 2004), and is the canine equivalent of
CD31.
[0331] Gamma-glutamyl transpeptidase (GGT) is a metabolic enzyme
that catalyzes the transfer of the gamma-glutamyl moiety of
glutathione to an acceptor that may be an amino acid, a peptide, or
water, to form glutamate. This enzyme also plays a role in the
synthesis and degradation of glutathione and the transfer of amino
acids across the cell membrane. GGT is present in the cell
membranes of many tissues, including the proximal tubule cells of
kidneys (Horiuchi et al., Eur J Biochem, 87(3):429-437, 1978;
Pretlow et al., J Histochem Cytochem, 35(4):483-487, 1987;
Welbourne et al., Am J Physiol, 277(4 Pt 2):F501-505, 1999). Table
12.1 provides a list of the specific types of renal cells
expressing these markers as detected by flow cytometry.
TABLE-US-00006 TABLE 12.1 Phenotypic Markers for SRC
Characterization Antigenic Marker Reactivity CK8/18/19 Epithelial
cells, proximal and distal tubules CK8 Epithelial cells, proximal
tubules CK18 Epithelial cells, proximal tubules CK19 Epithelial
cells, collecting ducts, distal tubules CK7 Epithelial cells,
collecting ducts, distal tubules CXCR4 Epithelial cells, proximal
and distal tubules E-cadherin Epithelial cells, distal tubules
Cubilin Epithelial cells, proximal tubules Aquaporin 1 Epithelial
cells, proximal tubules, descending thin limb GGT1 Fetal and adult
kidney cells, proximal tubules Aquaporin2 Renal collecting duct
cells, distal tubules DBA Renal collecting duct cells, distal
tubules CD31 Endothelial cells of the kidney (rat) CD146
Endothelial cells of the kidney (canine, human)
[0332] The SRC cells of Example 5 were investigated for phenotype
of specific biomarkers.
[0333] For immunophenotyping: The specific antibody was added to
100 microliters of cell suspension and the mixture was incubated in
the dark for 30-45 minutes at 4.degree. C. After incubation, cells
were washed with PBS and centrifuged to remove excess antibody.
Cells were resuspended to cells/microliters PBS and analyzed by
flow cytometry. Flow cytometry analysis was performed with a
FACSAria flow cytometer (Becton Dickinson) and FlowJo software
(Treestar, Inc.). Antibodies used to characterize the surface
marker phenotype are shown in Tables 12.2 and 12.3.
Isotype-specific primary antibody negative controls were used in
all experiments. Appropriate isotype-matched controls were used to
gate negative populations.
TABLE-US-00007 TABLE 12.2 Phenotype Panel for Human SRC in NKA
Reported in IND Antigenic Cell Commercial Marker Kidney
Distribution Component Description Primary Phenotypic Profile CK
Epithelial cells, proximal Cyto C-04 done, Ms mAb 18 tubules
plasmic IgG1, Abcam ab668 GG Proximal tubules Membrane Ms mAb
IgG2a, T1 Abcam ab55138 AQ Distal tubules, collecting Membrane Rb
pAb IgG, Abcam P2 ducts ab62628 Expanded Phenotypic Panel CK
Proximal and distal Cyto Ms mAb IgG1, 8, 18, 19 tubules plasmic
Abcam ab41825 CK Proximal tubules Cyto Ms mAb IgG1, 8 plasmic Abcam
ab9023 CK Distal tubules, collecting Cyto Clone BA-17, 19 ducts
plasmic Ms mAb Abcam ab7755 CK Distal tubules, collecting Cyto Ms
mAb IgG1, 7 ducts plasmic Abcam ab41825 CX Proximal and distal
Membrane Clone 12G5, Ms mAb CR4 tubules IgG2a, R&D Systems
(Fusin) MAB170 E- Distal tubules Membrane Ms mAb IgG2a, BD cadherin
610182 (CD324) Cu Proximal tubules Membrane Gt pAb IgG, Santa bilin
Cruz Biotechnology, sc-23644 AQ Proximal tubules, Membrane Clone
1/22, Ms mAb P1 descending thin limb IgG2a, Abcam ab9566 DB Distal
tubules, collecting Membrane Agglutinin, Vector A ducts Labs B-1035
CD Endothelial cells (rat) Membrane Ms mAb IgG1, BD 31 555444
(PECAM- 1) CD Endothelial cells Membrane Clone P1H12, Ms 146
(human, dog) mAb IgG1, Abcam ab24577 Abbreviations: Gt, goat; Ms,
mouse; mAb, monoclonal antibody; pAb, polyclonal antibody; Rb,
rabbit.
TABLE-US-00008 TABLE 12.3 Additional Phenotypic Markers for
Evaluation of Human SRC Kidney Antigenic Marker Distribution
Commercial Description AQP4 Collecting ducts Clone 8J301, Ms mAb
IgG1, US Biological A3000-14B CD24 Glomerulus and Rb pAb IgG, Abcam
ab110448 proximal tubules (progenitor/stem cell marker) CD54
Glomerulus Cone HA58, Ms mAb IgG1, BD 559771 (ICAM-1) (endothelial
cells, human) CD73 Renal Cortex Gt pAb IgG, Santa Cruz
Biotechnology, (interstitial fibroblasts) sc-14684 CD117 Proximal
tubules Clone 104D2, Ms mAb 104D2, BD (stem cell marker) 340867
CD133 Glomerulus and Rb pAb IgG, Abcam ab19898 proximal tubules
(progenitor/stem cell marker) CK8, 18 Proximal and Ms mAb IgG2a,
Abcam ab15224 distal tubules CK40 to 67 Proximal tubules Cone
AE1/AE3, Ms mAb IgG1, Dako M3515 N-cadherin Proximal tubules Ms mAb
IgG1, BD 610921 Pan-cadherin Proximal tubules Clone CH-19, Ms mAb
IgG1, Abcam ab6528 Calbindin Distal tubules Ms mAb IgG1, US
Biological C0113-15 Calponin Glomerulus Ms rnAb IgG1, Dako M3556
(interstitial fibroblasts) Connexin 43 Glomerulus Clone 4EG.2, Ms
mAb IgG1, Abcam ab79010 EPO Renal Cortex Ms mAb IgG2a, US
Biological E3455- (erythropoeitin) (interstitial fibroblasts) 13
GLEPP1 Glomerulus Gt pAb IgG, Santa Cruz Biotechnology (glomerular
epithelial sc-33415 protein 1) GST-1 Proximal tubules Rb pAb IgG,
Santa Cruz Biotechnology (alpha glutathione S- sc-459 transferase)
Haptoglobulin Glomerulus Chicken IgY, US Biological 03-003-02 Itgb1
Collecting ducts Clone JB1B, Ms mAb IgG2a, Abcam (Integrin 1)
ab30388 KIM-1/TIM-1 Proximal tubules Clone 212211, Ms mAb IgG2b,
(kidney injury molecule-1/ R&D Systems MAB1750 T-cell
immunoglobulin and mucin-containing molecule) Megalin Proximal and
Rb pAb IgG, Santa Cruz Biotechnology, distal tubules sc-25470 MAP-2
Proximal and Clone M13, Ms mAb IgG1, (microtubule-associated distal
tubules Zymed/Life Technologies 13-1500 protein 2) Nephrin
Glomerulus Ms mAb IgG1, US Biological N2028- 50D NKCC Distal
tubules Rb pAb IgG, Santa Cruz Biotechnology N-K-Cl-cotransporters)
sc-133823 OAT-1 Proximal tubules Rb pAb IgG, US Biological 041836
(organic anion transporter 1) Osteopontin Proximal and Clone 53, Ms
mAb IgG2a, Abcam distal tubules 69498 PCLP1 Glomerulus Gt pAb IgG,
Santa Cruz Biotechnology (podocalyxin-like 1 sc-10503 molecule)
Podocin Glomerulus Rb pAb IgG, Santa Cruz Biotechnology sc-21009
SMA Glomerulus Ms mAb IgG2a, Dako M0851 (smooth muscle alpha-
(interstitial fibroblasts) actin) Synaptopodin Glomerulus Rb pAb
IgG, Santa Cruz Biotechnology sc-50459 THP Distal tubules Ms mAb
IgG2a, Santa Cruz (tamm-horsfall protein) Biotechnology sc-20631
Vimentin Proximal tubules Rb pAb IgG, Atlas Antibodies
(progenitor/stem cell HPA001762 marker)
[0334] Cell suspensions were generated from initial tissue
dissociation or trypsinization of adherent cultures and analyzed by
flow cytometry to identify cellular components. Antibodies employed
are listed in Tables 12.2 and 12.3 (above). Isotype-specific
primary antibody negative controls were used in all experiments.
Labeled cells were analyzed with a FACSAria flow cytometer (Becton
Dickinson) and FlowJo software (Treestar, Inc.). Appropriate
isotype-matched controls were used to gate negative populations.
After an overnight incubation at 4.degree. C., the cells were
pelleted, washed twice with Triton Buffer (0.2% Triton X-100 in
PBS), resuspended in 1 mL of DBPS containing secondary antibody
goat anti-mouse IgG2A conjugated to the fluorochrome Alexa A647
(Invitrogen), and incubated for an additional 30 minutes. Cells
were then washed and resuspended in 1 mL of PBS for analysis as per
manufacturer instructions using FACSAria and FlowJo software. As a
negative control, cells were incubated in parallel with
isotype-matched monoclonal antibodies conjugated to the same
fluorochrome.
[0335] FIG. 5 (Phenotype Distribution) shows quantified expression
of these markers in SRC populations plotted as percentage values of
each phenotype in the population.
[0336] CK8/18/19 are the most consistently expressed renal cell
proteins detected across species. GGT1 and Aquaporin-1 (AQP1) are
expressed consistently but at varying levels. DBA, Aquaporin2
(AQP2), E-cadherin (CAD), CK7, and CXCR4 are also observed at
modest levels though with more variability, and CD31/146 and
Cubilin were lowest in expression. Table 12.4 provides the selected
markers, range and mean percentage values of phenotypic in SRC and
the rationale for their selection.
TABLE-US-00009 TABLE 12.4 Marker Selected for Phenotypic Analysis
of SRC Phenotypic Expression Expression Marker Range Average
Rationale Level CK18 81.1 to 96.7% Epithelial High 99.7% (n = 87)
marker GGT1 4.5 to 50.7% Functional Moderate 81.2% (n = 63) Tubular
marker AQP2 3.0 to 26.8% Collecting Low* 53.7 (n = 24) duct marker
*Collecting duct epithelial cells are expected to be low in SRC
based on their buoyant density
[0337] SRC Gene Expression
[0338] The gene expression profile of SRC isolated from human renal
cell cultures by quantitative real-time polymerase chain reaction
(qPCR), including those of aquaporin2, E-cadherin, cubulin, VEGF
and CD31 that were also tested for protein production. Genotypic
markers in Table 12.5 are representative of cell populations that
might be expected to be found in the renal cell cultures. NCAD,
Cubilin and CYP2R1 are markers of tubular epithelial cells, AQP2
and ECAD are markers of collecting duct and distal tubules. Podocin
and Nephrin are markers of podocytes. VEGF and CD31 are endothelial
markers. VEGF and EPO are oxygen responsive genes with related mRNA
present in a variety of different tissue and cell types.
[0339] Gene probes used were obtained from TaqMan. Passage 2 human
renal cells were harvested at 70-90% confluence. RNA was purified
from the cells using Qiagen's RNeasy Plus Mini Kit following the
protocol for Purification of Total RNA from Animal Cells. cDNA was
generated from a volume of RNA equal to 1.4 .mu.g using
Invitrogen's SuperScript.RTM. VILO.TM. cDNA Synthesis Kit following
the manufacturer's instructions. Averaged qPCR data for SRC
populations (n=3) is shown in Table 12.5.
[0340] The results suggest that a population of tubular epithelial
cells is present as evidenced by relatively higher level of
expression of NCAD, Cubilin and CYP2R1. Distal Collecting Duct
Tubule and Distal Tubule markers AQP2 and ECAD are relatively low
and CD31, an endothelial marker is even lower (Table 12.5).
TABLE-US-00010 TABLE 12.5 Gene Expression Analysis of Human SRC
Human Gene Average Std Gene Name Designation Marker RQ Error
Aquaporin2 AQP2 Distal 0.201 0.201 Tubule, Collecting Duct
E-cadherin/ ECAD/ Distal 0.318 0.191 Cadherin 1, Type 1 CDH1 Tubule
Neuronal NCAD/ Proximal 3.027 0.208 Cadherin/Cadherin CDH2 Tubule
2, Type 1 Cubilin CUBN Tubular 4.319 1.036 Nephrin NPHS1 Podocyte
0.768 0.422 Podocin NPHS2 Podocyte 0.000 0.000 Erythropoietin EPO
Cortical 2.795 0.426 Fibroblast Vitamin D CYP2R1 Tubular 1.562
0.028 24-Hydroxylase Vascular VEGFA Endothelial 2.232 0.121
Endothelial Growth Factor A Platelet/Endo- PECAM1/ Endothelial
0.005 0.005 thelial Cell CD31 Adhesion Molecule/CD31
[0341] Phenotypic and functional markers have been chosen based
upon early genotypic evaluation. VEGF gene expression levels were
high and aquaporin2 gene expression levels were low which is
consistent with the protein analysis data (Table 12.4 and Table
12.6).
[0342] SRC Enzymatic Activity
[0343] Presence of viable cells and SRC function was demonstrated
by metabolism of PrestoBlue and production of VEGF and KIM-1.
[0344] SRC actively secrete proteins that can be detected through
analysis of conditioned medium. Cell function is assessed by the
ability of cells to metabolize PrestoBlue and secrete VEGF
(Vascular Endothelial Growth Factor) and KIM-1 (Kidney Injury
Molecule-1).
[0345] Viable functioning cells can be monitored in NKA by their
ability to metabolize PrestoBlue. PrestoBlue Cell Viability Reagent
is a modified resazurin-based assay reagent that is a cell
permeable, non-fluorescent blue dye. Upon entry into cells which
are sufficiently viable to proliferate, the dye is reduced, via
natural cell processes involving dehydrogenase enzymes, to a bright
red fluorophore that can be measured by fluorescence or
absorbance.
[0346] Biomolecules VEGF and KIM-1 represent a selection of
molecules from those proposed as sensitive and specific analytical
nonclinical biomarkers of kidney injury and function (Sistare,
2010; Warnock, 2010). In vivo, both of these markers are indicative
of tubular function, injury and/or repair and in vitro are
recognized features of tubular epithelial cell cultures. KIM-1 is
an extracellular protein anchored in the membrane of renal proximal
tubule cells that serves to recognize and phagocytose apoptotic
cells which are shed during injury and cell turnover. VEGF,
constitutively expressed by kidney cells, is a pivotal angiogenic
and pro-survival factor that promotes cell division, migration,
endothelial cell survival and vascular sprouting. SRC have been
characterized as constitutively expressing VEGF mRNA (Table 12.5)
and actively produce the protein (Table 12.6). These proteins may
be detected in culture medium exposed to renal cells and SRC. Table
12.6 presents VEGF and KIM-1 quantities present in conditioned
medium from renal cells and SRC cultures. Renal cells were cultured
to near confluence. Conditioned medium from overnight exposure to
the renal cell cultures and SRC was tested for VEGF and KIM-1.
TABLE-US-00011 TABLE 12.6 Production of VEGF and KIM-1 by Human
Renal Cells and SRC Conditioned VEGF KIM-1 Medium ng/mL ng/million
cells ng/mL ng/million cells Renal Cell 0.50 to 2.42 2.98 to 14.6
0.20 to 3.41 1.14 to 15.2 Culture (n = 15) SRC 0.80 to 3.85 4.83 to
23.07 0.32 to 2.10 1.93 to 12.59 (n = 14)
[0347] Cell function of SRC, pre-formulation, was also evaluated by
measuring the activity of two specific enzymes; GGT
(.gamma.-glutamyl transpeptidase) and LAP (leucine aminopeptidase)
(Chung, 1982, J Cell Biol 95(1):118-126), found in kidney proximal
tubules. Methods to measure the activity of these enzymes in cells
utilize an enzyme-specific substrate in solution that, when added
to cells expressing active enzyme, are cleaved, releasing a
chromogenic product (Nachlas, 1960 J Biophys Biochem Cytol
7:261-264; Tate, 1974 Proc Natl Acad Sci USA 71(9):3329-3333). The
absorbance of the cell-exposed solution is measured and is relative
to the amount of cleavage product resulting from active enzyme. The
substrate utilized for GGT is L-glutamic acid
.gamma.-p-nitroanalide hydrochloride and for LAP is L-leucine
p-nitroanalide. FIG. 6 shows LAP and GGT activity in six SRC
samples produced from human donors (BP1-BP4).
[0348] Summary of SRC Characterization
[0349] Cell morphology was monitored during cell expansion by
comparison of culture observations with images in an Image Library.
Cell growth kinetics were monitored at each cell passage. Cell
growth is expected to be variable from patient to patient. SRC
counts and viability were monitored by Trypan Blue dye exclusion
and/or metabolism of PrestoBlue. SRC are characterized by
phenotypic expression of CK18, GGT1. In addition AQP2 expression
may be monitored. Metabolism of PrestoBlue and production of VEGF
and KIM-1 are used as markers for the presence of viable and
functional SRC. SRC function can be further elucidated with gene
expression profiling and measurement of enzymatic activity with LAP
and GGT.
Example 13
Biomaterial Preparation
[0350] The Biomaterial used in NKA (Gelatin Solution) is
characterized via two key parameters:
[0351] Concentration--Concentration of Gelatin Solution is measured
by absorbance at 280 nm using a spectrophotometer. The gelatin
concentration is determined from a calibration curve of absorbance
versus concentration.
[0352] Inversion Test--The inversion test provides a visual
assessment of the ability of the Gelatin Solution to form and
maintain a gel at a temperature of 2-8.degree. C. and for the gel
to liquefy at room temperature.
[0353] Elucidation of Other Biomaterial Characteristics
[0354] Biomaterials used in NKA can be further characterized for
the rheological properties and viscosity. Rheology and viscosity
testing will be performed for verification purposes only and are
intended to be used for expanded characterization of biomaterials
obtained from other vendors.
[0355] Rheological properties of the Biomaterial can be measured
first at 4.degree. C., then at 25.degree. C. through the use of a
Couette Cell style rheometer. The sample is equilibrated for at
least 30 minutes at each temperature. An acceptable storage modulus
(G'>10) at the lower temperature reflects the ability of the
solution to form and maintain a gel at NKA shipping and transport
temperature of 2-8.degree. C. An acceptable loss modulus
(G''<10) at the higher temperature reflects the ability of the
gel to liquefy at room temperature as required for delivery and
implantation of NKA.
[0356] Viscosity of the Biomaterial is measured using a cone and
plate viscometer at 37.degree. C. and a shear rate of 200-300 s-1.
Solutions with viscosities in range of 1.05-1.35 cP can be
efficiently delivered through 18-27 gauge needles.
[0357] In preparation for NKA formulation, gelatin is dissolved in
DPBS to a specified concentration (0.88%.+-.0.12% w/v) to form a
Gelatin Solution (FIG. 2D). Gelatin Solution was filter sterilized
through a 0.1 .mu.m filter and aliquoted into tubes. Samples were
taken for release of the Gelatin Solution prior to freezing or
formulation of NKA. The gelled hydrogel is stored refrigerated or
frozen as a bulk material ready for formulation (FIG. 2D).
Example 14
NKA Formulation
[0358] Washed SRC from Example 5 were counted using Trypan Blue dye
exclusion. Gelatin Solution was removed from cold storage and
liquefied by warming to 26-30.degree. C. A volume of SRC suspension
containing the required number of cells was centrifuged and
re-suspended in liquefied Gelatin Solution for a final wash step.
This suspension was centrifuged and the SRC pellet is re-suspended
in sufficient Gelatin Solution to achieve a resultant SRC
concentration of 100.times.10.sup.6 cells/mL in the formulated NKA
(FIG. 2D).
[0359] NKA is presented in a sterile, single-use 10 mL syringe. The
final volume was calculated from the concentration of
100.times.10.sup.6 SRC/mL of NKA and the target dose of
3.0.times.10.sup.6 SRC/g kidney weight (estimated by MRI).
Example 15
NKA Filling and Gelation
[0360] NKA product was aseptically filled into the syringe in the
NKA package in a BSC for tissue processing and cell culture
operations (FIG. 2D). Dynamic air sampling was performed for the
duration of the filling process, including viable and non-viable
sampling.
[0361] Formulated NKA was contained in a 50 mL sterile centrifuge
tube. A sterile cannula was attached to a 10 mL transfer syringe.
NKA was manually drawn into the transfer syringe from the 50 mL
tube via the cannula. The cannula was removed and the transfer
syringe was connected to the luer-lock fitting at the end of the
NKA package tubing. NKA was transferred to the syringe in the NKA
package by depressing the plunger on the transfer syringe. A
minimum of 8.5 mL of product was transferred to the syringe in the
NKA package. Air entrapped in the syringe was removed by inverting
the syringe and slowly depressing the plunger. After filling was
complete, the tubing on the NKA package was sealed with an RF
Sealer. Remaining product in the transfer syringe was returned to
the 50 mL tube. Quality control (release testing) samples were
taken from the 50 mL tube. The NKA package was rotated for a
minimum of 2 hours to keep the cells in suspension while cooling to
2-8.degree. C. to form the final gelled NKA (FIG. 2D).
[0362] Rapid cooling was required for gelation to take place so
that cells do not settle in the Gelatin Solution. The temperature
of the Gelatin Solution in a syringe was monitored as it was placed
into refrigerated conditions. Rapid temperature drop was observed.
After 1 hour, the temperature had dropped to within 0.3.degree. C.
of the final temperature 4.4.degree. C.
[0363] Cooling of the Gelatin Solution starts the gelation process
but a finite amount to time was required for the formed gel to
stabilize such that the SRC will remain suspended in the gel on
storage. Syringes containing formulated NKA were rotated either
overnight or for 1.25 hours and then held upright overnight.
Subsequently, the contents were removed and cell concentration was
measured in four different segments of the product. Analysis
indicates that there was no difference among the four segments,
thus no measurable cell settling occurs once NKA has rotated at
cold temperature for a minimum of 1.25 hours (data not shown).
Example 16
NKA Packaging and Shipping
[0364] NKA was packaged along with the appropriate documentation
into the NKA Shipper. The Shipper is designed to maintain a
temperature range of 2-8.degree. C. during transportation to the
clinical site. Cooled (gelled) formulated product has a shelf life
of 3 days.
[0365] A temperature recorder was included with the NKA package to
monitor the temperature during shipment. The Batch Number of NKA
was verified against the unique Patient ID recorded in the
shipping/receiving log book by the Quality group. NKA Shipper was
shipped to the clinical site via courier or similar secure
transportation.
Example 17
Implantation of the NKA (SRC Cell Population)
[0366] This example demonstrates the regenerative properties of the
selected heterogeneous human renal cells.
[0367] NKA Delivery System
[0368] NKA delivery system was composed of a cannula (needle)
compatible with cell delivery and a syringe. Different vendors use
the terms cannula or needle to describe cell delivery products. For
this description the term cannula and needle are used
interchangeably. The proposed clinical trial utilized the same
delivery system (cannula and syringe) used in the animal studies
adapted to human size and anatomy. A laparoscopic surgical
procedure was used.
[0369] The main component of NKA delivery system was the cannula. A
cannula that was compatible with NKA was used.
[0370] NKA Implantation
[0371] In preparation for implantation, NKA was allowed to warm to
room temperature just before injection into the kidney to liquefy
the product.
[0372] NKA was targeted for injection into the kidney cortex via a
needle or cannula and syringe compatible with cell delivery. The
use of a piercing needle (cannula) to penetrate the kidney capsule
allowed introduction of the delivery needle/cannula into the kidney
cortex. The syringe containing NKA was attached to the delivery
needle and NKA was injected into multiple sites into the kidney
cortex. The schematic in FIG. 7 illustrates the concept of
injecting NKA into a kidney using a needle compatible with cell
delivery and distribution into a solid organ.
[0373] NKA was delivered directly into the kidney cortex. NKA
delivery in patients used a standardized laparoscopic
procedure.
[0374] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *
References