U.S. patent application number 12/134813 was filed with the patent office on 2008-12-11 for selective cell therapy for the treatment of renal failure.
This patent application is currently assigned to Wake Forest University Health Sciences. Invention is credited to Anthony Atala, James J. Yoo.
Application Number | 20080305146 12/134813 |
Document ID | / |
Family ID | 39745597 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080305146 |
Kind Code |
A1 |
Atala; Anthony ; et
al. |
December 11, 2008 |
SELECTIVE CELL THERAPY FOR THE TREATMENT OF RENAL FAILURE
Abstract
Provided herein are isolated populations of kidney cells
harvested from differentiated cells of the kidney, wherein cells
have been expanded in vitro. The kidney cells may include
peritubular interstitial cells of the kidney, and preferably
produce erythropoietin (EPO). The kidney cells may also be selected
based upon EPO production. Methods of producing an isolated
population of EPO producing cells are also provided, and methods of
treating a kidney disease resulting in decreased EPO production in
a patient in need thereof are provided, including administering the
population to the patient, whereby the cells produce EPO in
vivo.
Inventors: |
Atala; Anthony;
(Winston-Salem, NC) ; Yoo; James J.;
(Winston-Salem, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
Wake Forest University Health
Sciences,
Winston-Salem
NC
|
Family ID: |
39745597 |
Appl. No.: |
12/134813 |
Filed: |
June 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60942716 |
Jun 8, 2007 |
|
|
|
Current U.S.
Class: |
424/423 ;
424/93.7; 435/369; 435/378 |
Current CPC
Class: |
G01N 33/56966 20130101;
A61K 35/12 20130101; G01N 2015/1081 20130101; C12N 2500/02
20130101; C12N 5/0686 20130101; A61P 13/12 20180101; A61P 5/00
20180101; G01N 15/1031 20130101; C12N 2502/256 20130101; A61K
9/0024 20130101; A61P 7/00 20180101; A61K 35/22 20130101; G01N
2015/0065 20130101; A61P 7/06 20180101 |
Class at
Publication: |
424/423 ;
435/369; 424/93.7; 435/378 |
International
Class: |
A61F 2/00 20060101
A61F002/00; C12N 5/06 20060101 C12N005/06; A61P 7/00 20060101
A61P007/00; A61K 35/12 20060101 A61K035/12 |
Claims
1. An isolated population of cells comprising differentiated
peritubular interstitial cells harvested from kidney tissue and
passaged in vitro.
2. The population of cells according to claim 1, wherein said
differentiated peritubular interstitial cells produce
erythropoietin (EPO).
3. The population of cells according to claim 1, wherein said
population consists essentially of differentiated peritubular
interstitial cells.
4. The population of cells according to claim 1, wherein said
population has been passaged from 1 to 4 times.
5. The population of cells according to claim 1, wherein said
population has been selected for EPO production.
6. The population of cells according to claim 1, subject to the
proviso that said cells are not transfected with an exogenous DNA
encoding a polypeptide.
7. A composition comprising the population of cells according to
claim 1 and a pharmaceutically acceptable carrier.
8. A method of producing an isolated population of EPO producing
cells, said method comprising the steps of: providing
differentiated kidney cells; and passaging said differentiated
kidney cells, wherein said cells produce EPO after said passaging;
thereby producing an isolated population of EPO producing
cells.
9. The method of claim 8 further comprising the step of selecting
said differentiated kidney cells for EPO production.
10. The method of claim 8, wherein said passaging step comprises
growth of differentiated kidney cells in a medium comprising
insulin transferrin selenium (ITS).
11. The method of claim 8, wherein said differentiated kidney cells
of said providing step consists essentially of differentiated
peritubular interstitial cells.
12. The method of claim 8, subject to the proviso that said
population of EPO producing cells are not transfected with an
exogenous DNA encoding a polypeptide.
13. A method of treating a kidney disease resulting in decreased
EPO production in a patient in need thereof, said method
comprising: providing a composition comprising an isolated
population of EPO producing cells in a pharmaceutically acceptable
carrier; and administering said composition to said patient,
whereby said EPO producing cells produce EPO in vivo.
14. The method of claim 13, wherein said pharmaceutically
acceptable carrier comprises a collagen gel.
15. The method of claim 13, wherein said administering step is
carried out by injecting said composition into the kidney or liver
of said patient.
16. The method of claim 13, wherein said administering step is
carried out by injecting or infusing said composition
intravascularly.
17. The method of claim 13, where said administering step is
carried out by infusing said composition into a portal vein of said
patient.
18. The method of claim 13, wherein said pharmaceutically
acceptable carrier comprises a biodegradable scaffold administering
step is carried out by implanting said composition into the kidney
of said patient.
19. The method of claims 13, wherein said population of EPO
producing cells consists essentially of differentiated peritubular
interstitial cells.
20. The method of claim 13, subject to the proviso that said EPO
producing cells are not transfected with an exogenous DNA encoding
a polypeptide.
21. The method of claim 13, wherein said kidney disease is an
anemia selected from the group consisting of: an anemia of renal
failure, an anemia of end-stage renal disease, an anemia of a
chemotherapy, an anemia of a radiation therapy, an anemia of
chronic infection, an anemia of an autoimmune disease, an anemia of
rheumatoid arthritis, an anemia of AIDS, an anemia of a malignancy,
an anemia of prematurity, an anemia of hypothyroidism, an anemia of
malnutrition, and an anemia of a blood disorder.
22. The method of claim 13, wherein said administering step is
carried out by injecting or implanting said composition.
23. The method of claim 13, wherein said EPO producing cells are
human.
24. An isolated population of cells comprising differentiated human
kidney cells harvested from human kidney tissue and passaged in
vitro.
25. The population of cells according to claim 24, wherein said
differentiated human kidney cells produce erythropoietin (EPO).
26. The population of cells according to claim 24, wherein said
population consists essentially of said differentiated human kidney
cells.
27. The population of cells according to claim 24, wherein said
human kidney cells have been passaged in vitro from 1 to 20
times.
28. The population of cells according to claim 24, wherein said
human kidney cells have been passaged in vitro at least 3
times.
29. The population of cells according to claim 24, wherein said
population has been selected for EPO production.
30. The population of cells according to claim 24, subject to the
proviso that said cells are not transfected with an exogenous DNA
encoding a polypeptide.
31. A composition comprising the population of cells according to
claim 24 and a pharmaceutically acceptable carrier.
32. The composition of claim 31, wherein said carrier comprises
collagen.
33. A method of treating anemia in a patient in need thereof, said
method comprising: providing a composition comprising the
population of cells according to claim 25; and administering said
composition to said patient in an amount effective to treat said
anemia.
34. The method of claim 33, wherein said administering step is
carried out by injecting or implanting said composition into said
patient.
35. The method of claim 33, wherein said anemia is selected from
the group consisting of: an anemia of renal failure, an anemia of
end-stage renal disease, an anemia of a chemotherapy, an anemia of
a radiation therapy, an anemia of chronic infection, an anemia of
an autoimmune disease, an anemia of rheumatoid arthritis, an anemia
of AIDS, an anemia of a malignancy, an anemia of prematurity, an
anemia of hypothyroidism, an anemia of malnutrition, and an anemia
of a blood disorder.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application Ser. No. 60/942,716,
filed Jun. 8, 2007, the disclosure of which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is in the field of selective cell
therapy for the restoration of organ function.
BACKGROUND OF THE INVENTION
[0003] Chronic renal failure is characterized by a gradual loss in
kidney function, and may eventually progress to end stage renal
failure, where the kidney no longer functions at a level to sustain
the body. End stage renal failure is a devastating disease that
involves multiple organs in affected individuals. The most common
cause of end stage renal disease in the U.S. is diabetes.
[0004] One of the functions performed by the kidney is the
production of erythropoietin (EPO). When the kidney is functioning
properly, low tissue oxygenation in the renal interstitium
stimulates the interstitial cells to produce EPO. The secreted EPO
in turn stimulates red blood cell production in the bone marrow,
which restores tissue oxygen tension to normal levels. Anemia
caused by ineffective hematopoiesis is one of the inevitable
outcomes of chronic renal failure due to the kidney's decreased
ability to produce EPO. EPO has also been reported to protect
against oxidative stress and apoptosis.
[0005] The kidney is the primary producer of EPO in the body and is
therefore a primary target of treatment for renal failure induced
anemia. Although dialysis can prolong survival for many patients
with end stage renal disease, only renal transplantation can
currently restore normal function. However, renal transplantation
is severely limited by a critical donor shortage.
[0006] Treatments used to alleviate anemia associated with renal
failure over the years include repeated transfusions of red blood
cells and administration of testosterone and other anabolic
steroids. However, none of these modalities has been entirely
satisfactory. Patients receiving repeated transfusions are subject
to iron overload, and may develop antibodies to major
histocompatibility antigens. Testosterone has a minimal effect on
erythropoeisis in the bone marrow, and it is associated with
undesirable, virilizing side effects.
[0007] Previous efforts to mitigate anemia associated with renal
failure have included the administration of purified recombinant
EPO (See, e.g., U.S. Pat. No. 6,747,002 to Cheung et al., U.S. Pat.
No. 6,784,154 to Westenfelder). However, the administration of
recombinant EPO only elevates EPO levels in the blood temporarily,
and may lead to iron deficiency. Gene therapy approaches have also
been pursued, in which EPO is produced using transfected host cells
(See, e.g., U.S. Pat. No. 5,994,127 to Selden et al., U.S. Pat. No.
5,952,226 to Aebischer et al., U.S. Pat. No. 6,777,205 to Carcagno
et al.; Rinsch et al. (2002) Kidney International 62:1395-1401).
However, these approaches involve the transfection of non-kidney
cells, and require techniques such as cell encapsulation to prevent
antigen recognition and immune rejection upon transplantation.
Also, transfection with exogenous DNA may be unstable, and the
cells may lose their ability to express EPO over time.
[0008] Renal cell-based approaches to the replacement of kidney
tissue is limited by the need to identify and expand renal cells in
sufficient quantities. In addition, the culturing of renal cells
for the purpose of kidney tissue engineering is particularly
difficult, owing to the kidney's unique structural and cellular
heterogeneity. The kidney is a complex organ with multiple
functions, including waste excretion, body homeostasis, electrolyte
balance, solute transport, as well as hormone production.
[0009] There remains a great need for alternative treatment options
to alleviate anemia caused by the failure of renal cells to produce
sufficient amounts of erythropoietin.
SUMMARY OF THE INVENTION
[0010] Provided herein in embodiments of the present invention are
isolated populations of kidney cells harvested from differentiated
cells of the kidney that have been passaged and/or expanded in
vitro. In some embodiments, the kidney cells include peritubular
interstitial and/or endothelial cells of the kidney. In some
embodiments, the kidney cells consist of or consist essentially of
peritubular interstitial and/or endothelial cells of the kidney
harvested from kidney tissue and passaged in vitro. In some
embodiments, cells produce erythropoietin (EPO). In further
embodiments, kidney cells are selected for EPO production.
[0011] Also provided are methods of producing an isolated
population of EPO producing cells, including the steps of: 1)
harvesting differentiated kidney cells; and 2) passaging the
differentiated kidney cells, wherein the cells produce EPO after
said passaging; thereby producing an isolated population of EPO
producing cells. In some embodiments the methods further include
the step of selecting the differentiated kidney cells for EPO
production. In some embodiments, the passaging step includes growth
of differentiated kidney cells in a medium comprising insulin
transferrin selenium (ITS).
[0012] Methods of treating a kidney disease or other ailment, which
disease or ailment results in decreased EPO production in a subject
(e.g., a patient) in need thereof are also provided, including the
steps of: 1) providing an isolated population of EPO producing
cells; and 2) administering the population to the subject (e.g., in
an amount effective to treat the kidney disease and/or the
decreased EPO production), whereby the EPO producing cells produce
EPO in vivo. In some embodiments, the providing step is performed
by harvesting differentiated kidney cells of the kidney and
passaging the cells in vitro. In some embodiments, the population
of EPO producing cells includes, consists of or consists
essentially of differentiated peritubular endothelial and/or
interstitial cells harvested from differentiated cells of the
kidney and passaged in vitro. In some embodiments, the population
is provided in a suitable carrier (e.g., a collagen gel) for
administration. In some embodiments, the administering step is
carried out by implanting the population of cells into the kidney
of the patient. In some embodiments, the administering step is
carried out by subcutaneously injecting or implanting said
composition. In some embodiments, the EPO producing cells are
human.
[0013] Further provided are isolated populations of cells including
differentiated human kidney cells harvested from human kidney
tissue and passaged in vitro. In some embodiments, the kidney cells
consist of or consist essentially of peritubular interstitial
and/or endothelial cells of the kidney harvested from kidney tissue
and passaged in vitro. In some embodiments, the differentiated
human kidney cells produce erythropoietin (EPO). In some
embodiments, the human kidney cells have been passaged from 1-20
times. In some embodiments, the human kidney cells have been
passaged at least 3 times. In some embodiments, the population has
been selected for EPO production. Some embodiments are subject to
the proviso that the cells are not transfected with an exogenous
DNA encoding a polypeptide.
[0014] Compositions comprising the population of human kidney cells
as described herein and a pharmaceutically acceptable carrier are
also provided. In some embodiments, the carrier comprises
collagen.
[0015] Another aspect of the present invention is the use of the
methods as described herein for the preparation of a composition or
medicament for use in treatment or for carrying out a method of
treatment as described herein (e.g., for treating a kidney disease
or other ailment resulting in decreased EPO production), or for
making an article of manufacture as described herein.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0016] FIG. 1. Mechanism of erythropoietin (EPO) production. Renal
interstitial peritubular cells of the kidney detect low blood
oxygen levels, and EPO is secreted into the blood. EPO stimulates
the proliferation and differentiation of erythroid progenitors into
reticulocytes, and prevents apoptosis, causing more reticulocytes
to enter the circulating blood. The reticulocytes differentiate
into erythrocytes, increasing the erythron size. Oxygen delivery to
the tissues is thereby increased.
[0017] FIG. 2. Intracellular erythropoietin immunoreactivity was
confirmed in the primary culture of renal cells at passage 1 (P1),
passage 2 (P2) and passage 3 (P3), compared to the negative control
(.times.400).
[0018] FIG. 3. Microscopy images of erythropoietin expressing cells
in kidney tissue (left panel) and in cultured kidney cells (right
panel).
[0019] FIG. 4. Quantification of erythropoietin (EPO) producing
cells. The number of cells expressing EPO decreased with the
subsequent passages (*p<0.05).
[0020] FIG. 5. Western blot analysis of detergent-solubilized cell
extracts detected EPO protein (34 kDa) of early passage primary
cultured renal cells (P0-P3).
[0021] FIG. 6. EPO expression analysis using FACS. Top Row: Mouse
cells, passages 0-3. Bottom Row: Rat cells, passages 0-3.
[0022] FIG. 7A-7B. Mouse renal cell characterization. EPO
expression is confirmed by immunofluorescence (FIG. 7A) (KNRK cells
were used as positive control). GLEPP1 and Tamm Horsfall kidney
markers were also detected (FIG. 7B).
[0023] FIG. 8. Rat renal cell characterization. Cultured rat kidney
cells have various cell morphologies shown by phase contrast
microscope (left panels), and express GLEPP1 and Tamm Horsfall
kidney markers (right panels).
[0024] FIG. 9. EPO expression in HepG2 cells was shown by western
blot and compared with EPO expression in kidney tissue.
[0025] FIG. 10. EPO protein expression of cultured cells under
hypoxic conditions. Lewis rat kidney cells and HepG2 cells were
cultured under normal and hypoxic conditions, and EPO production
was assessed by western blot of cells. 34 kDa=EPO; 43
kDa=.beta.-Actin.
[0026] FIG. 11. EPO protein expression in the culture medium under
hypoxic conditions. EPO in the culture medium of Lewis rat kidney
cells and HepG2 cells was assessed by western blot. 34 kDa=EPO; 43
kDa=.beta.-Actin.
[0027] FIG. 12. Total protein lysates were prepared from rat renal
primary cells at passages 1 and 2. Plates from normoxic samples
(NC), samples in 3% O2 and 7% O2 were processed and run on 10%
SDS-PAGE. KNRK cell line was used as positive control.
[0028] FIG. 13. Measuring EPO in media concentrates by western
blot. Primary cultured cells from Lewis rats were raised close to
confluency at each passage on 10 cm plates. The cells were starved
with KSFM for 24 hrs and then placed in a hypoxic chamber (1% O2)
for 24, 48 or 72 hrs. Following hypoxia incubation, the media was
collected and concentrated with a 10K mwco amicon ultra centrifugal
device (Millipore). 40 ug of total protein was then loaded on a 10%
polyacrylamide gel. KNRK cells were used as positive control.
[0029] FIG. 14. Histological analysis of the retrieved implants
showed that the kidney cells survived and formed tissue in vivo.
Presence of EPO producing cells were confirmed
immunohistochemically using EPO specific antibodies (.times.400).
Left panel: Initial cell density of 1.times.10.sup.6
cells/injection. Right panel: Initial cell density of
1.times.10.sup.6 cells/injection. Top row of each panel: 2 weeks.
Bottom row of each panel: 4 weeks.
[0030] FIG. 15. Effect of culture media and hypoxia on renal
primary cells measured by real time PCR. Renal primary cells (p0)
were grown to 80% confluency in 10 cm plates. Three plates of cells
were grown with either serum free KSFM or DMEM and placed in a
hypoxic chamber at 3% O2. After 24 hrs, samples were processed for
total RNA and cDNA synthesis. Real time PCR was done in triplicate,
and samples were quantified relative to normoxic sample.
[0031] FIG. 16. Effect of hypoxia on renal primary cells measured
by real time PCR. Renal primary cells (passages 0 and 2) were grown
to 80% confluency in 10 cm plates. Cells were then grown in serum
free KSFM and placed in a hypoxic chamber at 1% O2. After 24, 48 or
72 hrs, samples were processed for total RNA and cDNA synthesis.
Real time PCR was done in triplicate, and samples were quantified
relative to normoxic sample.
[0032] FIG. 17. Effect of hypoxia on renal primary cells measured
by real time PCR. Renal primary cells (passage 0) were grown to 80%
confluency in 10 cm plates. Cells were then placed in a hypoxic
chamber at 1% O2 for up to 24 hrs. Samples were then processed for
total RNA and cDNA synthesis. Real time PCR was done in triplicate,
and samples were quantified relative to normoxic sample
[0033] FIG. 18. Primary human kidney cells were expanded. Shown are
cells of passages 2, 4, 7 and 9.
[0034] FIG. 19. Human primary renal cells were maintained through
20 doublings.
[0035] FIG. 20. Human kidney cell characterization. GLEPP1 and EPO
positive cells are present in the population.
[0036] FIG. 21. Human kidney cell delivery in vivo with a 20 mg/ml
collagen carrier. At retrieval, 3 weeks after injection, the
injection volume had been maintained, and neovascularization was
present.
[0037] FIG. 22. Injection of collagen with cultured human kidney
cells resulted in EPO expressing tissue formation in vivo.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] Cell based therapy for renal failure can be approached in
two directions: total and selective. Described herein is the
selective cell therapy approach for achieving restoration of
specific functional organ components.
[0039] The disclosures of all United States patent references cited
herein are hereby incorporated by reference to the extent they are
consistent with the disclosure set forth herein. As used herein in
the description of the invention and the appended claims, the
singular forms "a," "an" and "the" are intended to include the
plural forms as well, unless the context clearly indicates
otherwise. Furthermore, the terms "about" and "approximately" as
used herein when referring to a measurable value such as an amount
of a compound, dose, time, temperature, and the like, is meant to
encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the
specified amount. Also, as used herein, "and/or" or "/" refers to
and encompasses any and all possible combinations of one or more of
the associated listed items, as well as the lack of combinations
when interpreted in the alternative ("or").
[0040] "Kidney tissue" is tissue isolated or harvested from the
kidney, which tissue contains kidney cells. In some embodiments,
kidney cells are positive for one or more known kidney markers,
e.g., GLEPP1, Tamm Horsfall, etc. "Cell" or "cells" may be of any
suitable species, and in some embodiments are of the same species
as the subject into which tissues produced by the processes herein
are implanted. Mammalian cells (including mouse, rat, dog, cat,
monkey and human cells) are in some embodiments particularly
preferred. "Isolated" as used herein signifies that the cells are
placed into conditions other than their natural environment. Tissue
or cells are "harvested" when initially isolated from a subject,
e.g., a primary explant.
[0041] "Subjects" are generally human subjects and include, but are
not limited to, "patients." The subjects may be male or female and
may be of any race or ethnicity, including, but not limited to,
Caucasian, African-American, African, Asian, Hispanic, Indian, etc.
The subjects may be of any age, including newborn, neonate, infant,
child, adolescent, adult, and geriatric.
[0042] Subjects may also include animal subjects, particularly
mammalian subjects such as canines, felines, bovines, caprines,
equines, ovines, porcines, rodents (e.g., rats and mice),
lagomorphs, non-human primates, etc., for, e.g., veterinary
medicine and/or pharmaceutical drug development purposes.
[0043] Cells may be syngeneic (i.e., genetically identical or
closely related, so as to minimize tissue transplant rejection),
allogeneic (i.e., from a non-genetically identical member of the
same species) or xenogeneic (i.e., from a member of a different
species). Syngeneic cells include those that are autogeneic (i.e.,
from the patient to be treated) and isogeneic (i.e., a genetically
identical but different subject, e.g., from an identical twin).
Cells may be obtained from, e.g., a donor (either living or
cadaveric) or derived from an established cell strain or cell line.
Cells may be harvested from a donor, e.g., using standard biopsy
techniques known in the art.
[0044] The "primary culture" is the first culture to become
established after seeding disaggregated cells or primary explants
into a culture vessel. "Expanding" as used herein refers to an
increase in number of viable cells. Expanding may be accomplished
by, e.g., "growing" the cells through one or more cell cycles,
wherein at least a portion of the cells divide to produce
additional cells.
[0045] "Passaged in vitro" or "passaged" refers to the transfer or
subculture of a cell culture to a second culture vessel, usually
implying mechanical or enzymatic disaggregation, reseeding, and
often division into two or more daughter cultures, depending upon
the rate of proliferation. If the population is selected for a
particular genotype or phenotype, the culture becomes a "cell
strain" upon subculture, i.e., the culture is homogeneous and
possesses desirable characteristics (e.g., the ability to express
EPO).
[0046] "Express" or "expression" of EPO means that a gene encoding
EPO is transcribed, and preferably, translated. Typically,
according to the present invention, expression of an EPO coding
region will result in production of the encoded polypeptide, such
that the cell is an "EPO producing cell." In some embodiments,
cells produce EPO without further manipulation such as the
introduction of an exogenous gene. In some embodiments, the
invention is subject to the proviso that the EPO producing cells
are not manipulated by the introduction of an exogenous gene and/or
by an exogenous chemical that stimulates the production of EPO.
[0047] In some embodiments, harvested cells are not passaged. In
other embodiments, cells are passaged once, twice, or three times.
In still other embodiments, cells are passaged more than 3 times.
In some embodiments, cells are passaged 0-1, 0-2 or 0-3 times. In
some embodiments, cells are passaged 1-2, 1-3, or 1-4 or more
times. In some embodiments, cells are passaged 2-3 or 2-4 or more
times. In further embodiments, cells are passaged 5, 8, 10, 12 or
15 or more times. In some embodiments, cells are passaged 0, 1, 2,
3 or 4 to 8, 10, 15 or 20 or more times. The number of passages
used may be selected by, e.g., the relative EPO production measured
in the cell population after each passage.
[0048] Growing and expansion of kidney cells is particularly
challenging because these cells are prone to the cessation of
growth and early differentiation. This challenge is overcome in
some embodiments of the present invention by using kidney cell
specific media that contains additives that promote their growth.
Accordingly, in some embodiments kidney cells are grown in media
that includes additives such as growth factors and other
supplements that promote their growth. Further, in some
embodiments, EPO producing cells are grown in co-culture with other
renal cell types.
[0049] In some embodiments, kidney cells are grown in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) or fetal calf serum (FCS) and, optionally,
penicillin-streptomycin (P/S). In other embodiments, kidney cells
are grown in keratinocyte serum-free medium (KSFM). In further
embodiments, kidney cells are grown in KSFM with one or more of the
following additives: bovine pituitary extract (BPE) (e.g., 50
g/mL), epidermal growth factor (EGF) (e.g., 5 ng/mL),
antibiotic-antimycotic solution (GIBCO) (e.g., 5 mL), fetal bovine
serum (FBS) (Gemini Bio-Product) (e.g., 12.5 mL of 2.5%), and
insulin transferrin selenium (ITS) (Roche) (e.g., 50 mg for 5 L
medium). As understood by those of skill in the art, in some
embodiments of the above media, penicillin-streptomycin (P/S) and
antibiotic-antimycotic solution are interchangeable.
[0050] Passaging of kidney cells according to some embodiments may
be accomplished using standard procedures known in the art. For
example, the cells may be detached using trypsin/EDTA and
transferred to other plates. This is a standard procedure for many
cell types. Briefly, in some embodiments this may be accomplished
with the following steps: 1) Remove medium. 2) Add 10 ml PBS/EDTA
(0.5 M) for 4 minutes. Confirm the separation of cell junctions
under a phase contrast microscope. 3) Remove PBS/EDTA and add 7 ml
Trypsin/EDTA. 4) Add 5 ml medium when 80-90% of the cells lift
under microscope. 5) Aspirate the cell suspension into a 15 ml test
tube. 6) Centrifuge the cells at 1000 rpm for 4 minutes. 7) Remove
the supernatant. 8) Resuspend cells in 5 ml of medium. 9) Pipet out
100 .mu.l of the cell suspension and perform trypan blue stain for
viability assay. 10) Count the number of cells on hemocytometer.
11) Aliquot the desired number of cells on the plate and make the
volume of medium to a total of 10 ml. 12) Place the cells in the
incubator.
[0051] "Selection" can be based upon any unique properties that
distinguish one cell type from another, e.g., density, size, unique
markers, unique metabolic pathways, nutritional requirements,
protein expression, protein excretion, etc. For example, cells may
be selected based on density and size with the use of centrifugal
gradients. Unique markers may be selected with fluorescent
activated cell sorting (FASC), immunomagnetic bead sorting,
magnetic activated cell sorting (MASC), panning, etc. Unique
metabolic pathways and nutritional requirements may be exploited by
varying the makeup and/or quantity of nutritional ingredients of
the medium on which cells are grown, particularly in a serum-free
environment. Protein expression and/or excretion may be detected
with various assays, e.g., ELISA.
[0052] "EPO producing cell" refers to differentiated cells, of
which at least a portion produce EPO (e.g., at least 20, 30, 40, or
50% or more, or more preferably 60, 70, 80, or 90% or more of the
cells produce EPO). In some embodiments, cells produce EPO without
further manipulation such as the introduction of an exogenous gene.
In some embodiments, the invention is subject to the proviso that
the EPO producing cells are not manipulated by the introduction of
an exogenous gene and/or by an exogenous chemical that stimulates
the production of EPO. The cells may be harvested from, e.g., the
peritubular interstitial cells of the kidney. In some embodiments,
the cells are selected for their ability to produce EPO. In other
embodiments, the cells are expanded in number by cell culture
techniques, e.g., passaging. Cells with the specific function of
EPO production can be used from the kidney and from other sources.
For example, EPO is also normally produced in the liver.
[0053] In the kidney, EPO is generally known to be produced by the
interstitial peritubular cells (FIG. 1). In some embodiments, an
isolated population of differentiated kidney cells comprises,
consists of or consists essentially of interstitial peritubular
cells of the kidney, consisting of or consisting essentially of 80,
90, 95, or 99 percent or more, or not more than 20, 10, 5 or 1
percent or less, by number of other cell types. In other
embodiments, the isolated population of differentiated kidney cells
includes other cell types, e.g., endothelial peritubular cells.
[0054] In some embodiments, the isolated population of
differentiated kidney cells comprises, consists of or consists
essentially of kidney cells that are selected for EPO production,
consisting of or consisting essentially of 80, 90, 95, or 99
percent or more, or not more than 20, 10, 5 or 1 percent or less,
by number of cells not expressing EPO. Selection may be
accomplished by selecting the cells that express EPO using specific
markers. In some embodiments, cells may include various types of
kidney cells, so long as the cells express EPO. In further
embodiments, the entire renal cell colony may be used for expansion
and treatment.
[0055] In some embodiments, the isolated population of
differentiated kidney cells have a "longevity" such that they are
capable of growing through at least 5, 10, 15, 20, 25 or 30 or more
population doublings when grown in vitro. In some embodiments, the
cells are capable of proliferating through 40, 50 or 60 population
doublings or more when grown in vitro.
[0056] "Differentiated" refers to cells or a population containing
cells that have specialized functions, e.g., EPO production and/or
expression of known markers of differentiated cells (e.g., GLEPP1
and/or Tamm Horsfall kidney cell markers). In this sense they are
not progenitor or stem cells. Some embodiments of the present
invention are subject to the proviso that harvested differentiated
cells are not passaged under conditions to create a population of
less specialized cells.
[0057] Alternatively, in other embodiments, cells are cultured to
produce cell lines, which may later be differentiated to produce
more specialized cells. The establishment of "cell lines," as
opposed to cell strains, are by and large undifferentiated, though
they may be committed to a particular lineage. Propagation
naturally favors the proliferative phenotype, and in some
embodiments cells may require a reinduction of differentiation by,
e.g., alteration of the culture conditions. There are a number of
differentiation factors known in the art that may induce
differentiation in cell lines (e.g., cytokines such as epimorphin
and HGF, vitamins, etc.).
Methods of Treatment.
[0058] In some embodiments, EPO producing cells are administered to
a subject in need thereof (e.g., by injection) to the kidney (e.g.,
into the cortex and/or medulla). In other embodiments, EPO
producing cells are administered to other areas of the body, e.g.,
the liver, peritoneum, etc. In some embodiments, the EPO producing
cells are administered subcutaneously, subcapsular, etc. In further
embodiments, EPO producing cells are administered by implantation
of a substrate (e.g., a collagen gel scaffold) containing said EPO
producing cells described herein. In still other embodiments, EPO
producing cells are administered through vascular access (e.g.,
systemically or locally).
[0059] Diseases that may be treated with the methods disclosed
herein include, but are not limited to, anemias. Anemias include,
but are not limited to, those associated with renal failure or
end-stage renal disease, anemias caused by chemotherapies or
radiation, anemias of chronic disorders, e.g., chronic infections,
autoimmune diseases, rheumatoid arthritis, AIDS, malignancies,
anemia of prematurity, anemia of hypothyroidism, anemia of
malnutrition (e.g., iron deficiency), and anemias associated with
blood disorders.
[0060] "Treat" refers to any type of treatment that imparts a
benefit to a patient, e.g., a patient afflicted with or at risk for
developing a disease (e.g., kidney disease, anemia, etc.). Treating
includes actions taken and actions refrained from being taken for
the purpose of improving the condition of the patient (e.g., the
relief of one or more symptoms), delay in the onset or progression
of the disease, etc.
[0061] Other endocrine systems may benefit from the therapies
disclosed herein, for example, vitamin D producing cell therapy or
the angiotensin system. See, e.g., U.S. Patent Application
Publication No. 2005/0002915 to Atala et al., which is incorporated
herein by reference. Cells with a specific function can be used
from the kidney and other sources, i.e., cells that would produce
target functions. For example, EPO is also normally produced in the
liver.
[0062] Preferably the cells are mixed with or seeded onto a
pharmaceutically acceptable carrier prior to administration.
"Pharmaceutically acceptable" means that the compound or
composition is suitable for administration to a subject to achieve
the treatments described herein, without unduly deleterious side
effects in light of the severity of the disease and necessity of
the treatment. Such formulations can be prepared using techniques
well known in the art. See, e.g., U.S. Patent Application
2003/0180289; Remington: The Science and Practice of Pharmacy,
Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams &
Wilkins: Philadelphia, Pa., 2000. The carrier may be a solid or a
liquid, or both (e.g., hydrogels), and can be formulated with the
cells as a unit-dose formulation. In some embodiments the cells are
provided as a suspension in the carrier to reduce clumping of the
cells. In other embodiments cells are seeded onto a biodegradable
scaffold or matrix.
[0063] In some embodiments, cells are mixed with a suitable gel for
administration. Suitable gels that may be used in the present
invention include, but are not limited to, agars, collagen, fibrin,
hydrogels, etc. Besides gels, other support compounds may also be
utilized in the present invention. Extracellular matrix analogs,
for example, may be combined with support gels to optimize or
functionalize the gel. One or more growth factors may also be
introduced into the cell suspensions.
[0064] Formulations of the invention include those for parenteral
administration (e.g., subcutaneous, intramuscular, intradermal,
intravenous, intraarterial, intraperitoneal injection) by injection
or implantation. In one embodiment, administration is carried out
intravascularly, either by simple injection, or by injection
through a catheter positioned in a suitable blood vessel, such as a
renal artery. In some embodiments, administration is carried out by
"infusion," whereby compositions are introduced into the body
through a vein (e.g., the portal vein). In another embodiment,
administration is carried out as a graft to an organ or tissue to
be augmented as discussed above, e.g., kidney and/or liver.
[0065] A "biodegradable scaffold or matrix" is any substance not
having toxic or injurious effects on biological function and is
capable of being broken down into is elemental components by a
host. Preferably, the scaffold or matrix is porous to allow for
cell deposition both on and in the pores of the matrix. Such
formulations can be prepared by supplying at least one cell
population to a biodegradable scaffold to seed the cell population
on and/or into the scaffold. The seeded scaffold may then implanted
in the body of a recipient subject.
[0066] In some embodiments, cells are administered by injection of
the cells (e.g., in a suitable carrier) directly into the tissue of
a subject. For example, cells may be injected into the kidney
(e.g., the subcapsular space of the kidney). Because the functional
effects of EPO production will be systemic, cells may also be
administered by injection into other tissues (e.g., the liver,
subcutaneously, etc.).
[0067] Cells may also be delivered systemically. In further
embodiments, cells are delivered to tissue outside of the kidney
(e.g., the liver), as the outcome of the functional effects of EPO
production will be systemic. See, e.g., the "Edmonton protocol," an
established delivery method, where cells are infused into a
patient's portal vein (Shapiro et al. (2000) N Engl J Med
343:230-238).
[0068] According to some embodiments, the cells administered to the
subject may be syngeneic (i.e., genetically identical or closely
related, so as to minimize tissue transplant rejection), allogeneic
(i.e., from a non-genetically identical member of the same species)
or xenogeneic (i.e., from a member of a different species), as
above, with respect to the subject being treated, depending upon
other steps such as the presence or absence of encapsulation or the
administration of immune suppression therapy of the cells.
Syngeneic cells include those that are autogeneic (i.e., from the
subject to be treated) and isogeneic (i.e., a genetically identical
but different subject, e.g., from an identical twin). Cells may be
obtained from, e.g., a donor (either living or cadaveric) or
derived from an established cell strain or cell line. As an example
of a method that can be used to obtain cells from a donor (e.g., a
potential recipient of a bioscaffold graft), standard biopsy
techniques known in the art may be employed. Alternatively, cells
may be harvested from the subject, expanded/selected in vitro, and
reintroduced into the same subject (i.e., autogeneic).
[0069] In some embodiments, cells are administered in a
therapeutically effective amount. The therapeutically effective
dosage of cells will vary somewhat from subject to subject, and
will depend upon factors such as the age, weight, and condition of
the subject and the route of delivery. Such dosages can be
determined in accordance with procedures known to those skilled in
the art. In general, in some embodiments, a dosage of
1.times.10.sup.5, 1.times.10.sup.6 or 5.times.10.sup.6 up to
1.times.10.sup.7, 1.times.10.sup.8 or 1.times.10.sup.9 cells or
more per subject may be given, administered together at a single
time or given as several subdivided administrations. In other
embodiments, a dosage of between 1-100.times.10.sup.8 cells per
kilogram subject body weight can be given, administered together at
a single time or given as several subdivided administration. Of
course, follow-up administrations may be given if necessary.
[0070] Cells may be administered according to some embodiments to
achieve a target hematocrit range. The ideal or target hematocrit
range may vary from subject to subject, depending upon, e.g.,
specific comorbidities. In some embodiments the target hematocrit
is from 30-40%, in some embodiments the target hematocrit is from
33-38%, and in some embodiments the target hematocrit is from
33-36%. Upon administration of cells according to the present
invention, hematocrit may be measured and, if desired or necessary,
corrected by, e.g., further implantation of cells and/or other
methods known in the art (e.g., supplementing with recombinant
EPO). Other methods of treatment for anemia and/or renal disease
may be used in conjunction with the methods of treatment provided
herein, for example, an adapted protein-caloric intake diet.
[0071] In further embodiments, if desired or necessary, the subject
may be administered an agent for inhibiting transplant rejection of
the administered cells, such as rapamycin, azathioprine,
corticosteroids, cyclosporin and/or FK506, in accordance with known
techniques. See, e.g., R. Calne, U.S. Pat. Nos. 5,461,058,
5,403,833 and 5,100,899; see also U.S. Pat. Nos. 6,455,518,
6,346,243 and 5,321,043. Some embodiments use a combination of
implantation and immunosuppression, which minimizes graft
rejection. The implantation may be repeated as needed to create an
adequate mass of transplanted tissue.
[0072] The present invention is explained in greater detail in the
following non-limiting Examples.
EXAMPLES
[0073] Anemia is an inevitable outcome of chronic renal failure due
to the kidney's decreased ability to produce erythropoietin (EPO)
by peritubular interstitial cells. We investigated whether
supplementation of erythropoietin producing cells would be a
possible treatment option for renal failure-induced anemia by
examining the feasibility of selecting and expanding erythropoietin
producing cells for cell-based therapy.
[0074] The following examples demonstrate that EPO producing cells
are present in renal cells harvested from mouse and rat kidneys. In
addition, cells isolated and expanded using the methods described
below include cells expressing EPO at every culture stage examined.
Further, the actual percentage of cells expressing the EPO marker
in culture was consistent with the cell population present in
normal kidney tissues (see Yamaguchi-Yamada et al., J Vet Med Sci,
67: 891, 2005; Sasaki et al., Biosci Biotechnol Biochem, 64: 1775,
2000; Krantz, Blood, 77: 419, 1991).
Example 1
Expansion of Renal Cell Primary Cultures
[0075] Renal cells from 7-10 day old mice C57BL/6 were culture
expanded. Minced kidney (1 kidney of mouse) was placed into a 50 cc
tube with 15 ml of collagenase/dispase (0.2 mg/ml). The kidney
tissue fragments were incubated in a 37.degree. C. shaker for 30
min with collagenase/dispase mix (0.2 mg/ml; 15 ml). Sterile PBS
with Gelatin (20 ml), was added (with Gelatin (DIFCO) 2 mg/ml) to
the digestion solution. The mixture was filtered thorough a 70
micron filter to remove undigested tissue fragments. The collected
solution was mixed well (being careful not to make air bubbles),
and divided into two 50 cc tubes. The tubes were centrifuged at
1000 (-1500) RPM for 5 min. The supernatant was discarded and the
pellet of each tube was resuspended in 3 ml of KSFM medium. DMEM
medium (10% FBS, 5 ml P/S) is used for stromal cells, and KSFM with
BPE, EGF, 5 ml antibiotic-antimycotic, 12.5 ml FBS (Gemini
Bio-Product, 2.5%), Insulin Transferrin Selenium (Roche) (50 mg for
5 L medium) with BPE and EGF for epithelial components. P/S or
antibiotic-antimycotic (GIBCO) may also be added. Each tissue was
seeded on to a 25 mm plate and medium was added (total 3 ml).
[0076] Cells were maintained by changing the medium the next day,
and then every 2 days depending on the cell density. Cells were
passaged when they were 80-90% confluent by detachment using
trypsin/EDTA and transferred to other plates with the following
steps: 1) Remove medium. 2) Add 10 ml PBS/EDTA (0.5 M) for 4
minutes. Confirm the separation of cell junctions under a phase
contrast microscope. 3) Remove PBS/EDTA and add 7 ml Trypsin/EDTA.
4) Add 5 ml medium when 80-90% of the cells lift under microscope.
5) Aspirate the cell suspension into a 15 ml test tube. 6)
Centrifuge the cells at 1000 rpm for 4 minutes. 7) Remove the
supernatant. 8) Resuspend cells in 5 ml of medium. 9) Pipet out 100
.mu.l of the cell suspension and perform trypan blue stain for
viability assay. 10) Count the number of cells on hemocytometer.
11) Aliquot the desired number of cells on the plate and make the
volume of medium to a total of 10 ml. 12) Place the cells in the
incubator.
[0077] Alternatively, the following protocol was used. Kidneys from
10 day old male C57BL/6 mice were collected in Krebs buffer
solution (Sigma Aldrich, St. Louis, Mo. USA) containing 10%
antibiotic/antimycotic (Gibco Invitrogen, Carlsbad, Calif. USA) to
avoid risk of contamination. The kidneys were immediately
transported to a culture hood where the capsule was removed. The
medullary region of the kidney was removed, and only the cortical
tissue was used to isolate cells that had been previously
identified as EPO producing cells (Maxwell et al., Kidney
International, 44: 1149, 1993). The kidney tissue was minced and
enzymatically digested using Liberase Blendzyme (Roche, Mannheim,
Germany) for 25 minutes at 37 degrees Celsius in a shaking water
bath. The supernatant was removed and the cell pellet was passed
through a 100 .mu.m cell strainer to obtain a single cell
suspension for culture.
[0078] Subsequently, the cells were plated at a density of
5.times.10.sup.5 cells/ml in 10 cm tissue culture treated plates
filled with culture media. The culture media consisted of a mixture
of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's
Modified Eagle's Medium (DMEM) at a ratio of 1:1. The premixed DMEM
media contained 3/4 DMEM and 1/4 HAM's F12 nutrient mixture
supplemented with 10% fetal bovine serum (FBS), 1%
Penicillin/Streptomycin, 1% glutamine 100.times. (Gibco), 1 ml of
0.4 .mu.g/ml hydrocortisone, 0.5 ml of a 10.sup.-10 M cholera toxin
solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a
1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136
mg/ml triiodothyronine mixture, and 0.5 ml of a 10 .mu.g/ml
epidermal growth factor (EGF) solution. All tissue culture reagents
were purchased from Sigma-Aldrich (St. Louis, Mo. USA) unless
otherwise stated. The cells were incubated at 37.degree. C. under
5% CO.sub.2 with medium change every 3 days, and the cells were
subcultured for expansion at a ratio of 1:3 when confluent.
Example 2
Characterization for EPO Production
[0079] The cells from early passages (1, 2 and 3) were
characterized for EPO expression using immunocytochemistry and
western blot analysis with specific antibodies (rabbit polyclonal
anti-EPO antibodies, sc-7956, Santa Cruz Technologies, Santa Cruz,
Calif.).
[0080] Renal cells were plated in 8-well chamber slides at a
density of 3000 cells per well. The cells were incubated at
37.degree. C. under 5% CO.sub.2 for 24 h to allow attachment. This
was followed by fixation with 4% paraformaldehyde for 10 minutes at
room temperature. Permeabilization of cell membranes was performed
by adding 0.1% Triton-X 100 in PBS for 3 minutes at room
temperature. Cells were then incubated in goat serum for 30 minutes
at room temperature. After washing, cells were incubated with the
primary antibodies for 1 h (1:50) at room temperature. Cells were
washed a second time and biotinylated goat polyclonal anti-rabbit
antibodies (polyclonal anti rabbit IgG, Vector Laboratories, Inc.,
Burlingame, Calif.) (1:200) were added, followed by incubation at
room temperature for 45 minutes. Chromogenic detection of EPO
followed a final washing step and was performed using the Vector
ABC kit according to the manufacturer's instructions (Vector
Laboratories, Inc., Burlingame, Calif.). Slides without the primary
antibodies served as internal negative controls, and normal mouse
renal tissue served as the positive control.
[0081] Renal cells in culture showed multiple phenotypes under the
microscope. The cells reached confluency within 7 to 10 days of
plating. Many of the cells observed in the first 3 passages after
isolation from the kidney stained positively for EPO, as compared
to the negative controls, which showed no background or nonspecific
staining (FIG. 2), which indicated that the observed staining was
likely due to the presence of EPO in the cultures. The number of
cells that stained positively for EPO remained constant throughout
the 3 passages studied, even when phenotypic changes were observed
in the culture during the same time period. Immunohistochemical
staining of kidney tissue indicated a similar amount of EPO
expression as that found in cultured cells (FIG. 3).
[0082] The number of cells expressing EPO decreased slightly with
subsequent passages (FIG. 4). This is most likely due to the
increased number of passages and loss of cells/function over time
and manipulation. However, the relative percentage appears to
remain stable after the first passage.
[0083] EPO expression was also confirmed by western blot, shown in
FIG. 5.
Example 3
Mouse and Rat Renal Cell Characterization
[0084] FACS analysis was used to quantify the number of
EPO-producing cells in the established renal cell cultures at each
passage (1-3 passages). The cells were collected by trypsinization
and centrifugation, resuspended in media, and passed through a 70
.mu.m cell strainer to ensure a single cell suspension. After
counting the cells, they were spun down and resuspended in PBS at
5-7.5.times.10.sup.5 cells/ tube to remove FBS from the cells. The
cells were fixed with 2% formaldehyde for 10 minutes at 4.degree.
C. and permeabilized using 100% methanol for 10 minutes at room
temperature. Subsequently, the cells were resuspended in 3% goat
serum in PBS followed by incubation with the rabbit anti-EPO
primary antibody sc-7956 (Santa Cruz Biotechnology, Santa Cruz,
Calif.) for 45 minutes on ice. Cells were washed twice with 3% goat
serum in PBS prior to incubation with fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit secondary antibodies for 1 hour.
The cells were then washed thoroughly with 3% serum in PBS and
transferred to the FACS machine (FACS Calibur E6204,
Becton-Dickinson, Franklin Lakes, N.J.).
[0085] Fluorescent activated cell sorting experiments demonstrated
that 44% of passage 1 (P1) cells were EPO positive. This percentage
increased to 82% at passage 2 (P2), and then dropped back to 42% at
passage 3 (P3). This may indicate that, during the first few days
of culture, proliferation of EPO-producing cells and/or
upregulation of EPO gene expression occurs in response to the lower
oxygen concentration in the media compared to normal living tissue.
These responses could then normalize over the next few days,
resulting in numbers of EPO-producing cells that are close to those
found in renal tissue (FIG. 6, top row).
[0086] The FACS data demonstrate the maintenance of EPO expression
over several passages. It should be noted that there was a surge in
the number of cells expressing EPO (82%) in the passage 2 culture,
which was confirmed by several repeat experiments. Though not
wishing to be bound to any particular theory, one possible
explanation for this phenomenon could be that EPO expression is an
inherent trait of all renal cells that can be turned on and off as
needed. In this case, following the abrupt change in survival
conditions between the body and the culture plate, the cells may
have been driven to express EPO momentarily until stabilization of
the culture occurred. Consistent with this, the EPO surge was
quickly reversed and passage 3 analyses showed a lower percentage
of EPO producing cells (42%).
[0087] Mouse cell characterization by immunofluorescence confirmed
EPO expression (FIG. 7A). The population of cells was positive for
the kidney cell markers GLEPP1 and Tamm Horsfall (FIG. 7B).
[0088] Rat cell passages 0, 1 and 2 were also analyzed for EPO
production using fluorescence activated cell sorting (FACS) (FIG.
6, bottom row). Cultured rat cells had various cell morphologies
and were positive for GLEPP1 and Tamm Horsfall kidney cell markers
(FIG. 8).
Example 4
Exposure of EPO Producing Cultures to Hypoxic Conditions
[0089] While maintenance of phenotypic characteristics is essential
during cell expansion stages, a critical component that ensures the
success of cell therapy is the ability of EPO producing cells to
regulate and maintain normal EPO levels. EPO belongs to the
hematopoietic cytokine family, and it controls erythropoiesis in
bone marrow, and regulates the proliferation, differentiation and
survival of erythroid progenitor cells through EPO receptor
(EPOR)-mediated signal transduction. EPO is largely produced in the
kidney, and when this organ fails, EPO production falls, leading to
anemia. EPO expression in the body depends largely on the oxygen
tension in the environment surrounding the cells capable of
producing EPO. Factors influencing oxygen levels include lack of
oxygen in the ambient air and decreased renal blood flow.
[0090] To determine whether the EPO expressing cells in culture
could respond to changing oxygen levels, an experiment was
performed in which the cells were serum-starved for 24 hours
followed by exposing them to various levels of oxygen in vitro.
Lewis rat kidney cells and HepG2 (human hepatocellular liver
carcinoma cell line) cells were cultured under normal and hypoxic
conditions, and EPO production was assessed and confirmed by
western blot of cells. EPO presence in the culture medium was also
measured and confirmed by analyzing the supernatants from cultured
renal cells under normoxic and hypoxic conditions with the double
antibody sandwich enzyme-linked immunosorberbent assay using a
Quantikine.RTM. IVD.RTM. Erythropoietin ELISA kit (R&D
Systems.RTM., Minneapolis, Minn.).
[0091] The cells were placed in serum free media for 24 hours prior
to the experiment. The plates were then transferred to a hypoxic
chamber and exposed to different hypoxic conditions (1%, 3%, 5%,
and 7% oxygen). HepG2 cells were used as positive controls, as they
have been previously reported to produce high levels of EPO in
culture (Horiguchi et al., Blood, 96: 3743). EPO expression by
HepG2 was confirmed by western blot (FIG. 9). All cells were
harvested in lysis buffer containing NP-40. Protein concentration
in each sample was measured using a Bio-Rad protein assay. 40 .mu.g
total protein was run out on a 10% acrylamide gel using SDS-PAGE.
Proteins were then transferred onto a PVDF membrane (Millipore
Corp.). Detection of .beta.-actin expression in the lysates was
used as the loading control. EPO antibody (rabbit polyclonal
sc-7956, Santa Cruz Biotechnology) was used at 1:200 and the
secondary antibody (goat anti-rabbit 7074, Cell Signaling
Technology, Beverly, Mass.) was used at 1:2000. To measure the
amount of EPO secreted into the media by the primary renal
cultures, the media was collected and concentrated down to 500 ul
using an Amicon Ultra centrifugal filter device (Millipore
Corporation, Billerica, Mass.). Samples of this media were run on a
10% polyacrylamide gel. EPO antibody (rabbit polyclonal sc-7956,
Santa Cruz Biotechnology) was used at 1:100 and the secondary
antibody (goat anti-rabbit 7074, Cell Signaling Technology,
Beverly, Mass., USA) was used at 1:2000.
[0092] Western blotting showed a slight increase in the EPO
expression in the cell lysate after hypoxia (FIG. 10). These
results, however, were not seen when media concentrates were used
to measure EPO (FIG. 11). The media testing indicated that all
media concentrates (hypoxic and normoxic conditions) contained the
same low amount of EPO.
[0093] Alternatively, total protein lysates were prepared from rat
renal primary cells at passages 1 and 2. Plates from normoxic
samples (NC), samples in 3% O2 and 7% O2 were processed and Run on
10% SDS-PAGE. The KNRK cell line was used as positive control.
Results are shown in FIG. 12.
[0094] Without wishing to be bound by any particular theory, this
may indicate that 24 hours might not be enough time for secreted
EPO levels to rise to a level that is detectable by western blot.
It is likely that a longer exposure time would be required for the
cells to begin to secrete EPO, as de novo protein production may
take several hours to become apparent. Therefore the following
experiment was performed, in which cells were placed in hypoxic
conditions for 24, 48 and 72 hours.
[0095] Primary cultured cells from Lewis rats were raised close to
confluency at each passage on 10 cm plates. The cells were placed
in a hypoxic chamber (1% O.sub.2) for 24, 48 or 72 hrs. Following
hypoxia incubation, the media was collected and concentrated with a
10K molecular weight cutoff Amicon Ultra centrifugal device
(Millipore). 40 .mu.g of total protein was then loaded on a 10%
Polyacrylamide gel. KNRK cells were used as a positive control.
Results are shown in FIG. 13.
[0096] In summary, all experiments indicated that the EPO levels in
primary culture cells were greater than or equal to those measured
in the HepG2 positive controls, and the EPO producing cells are
able to respond to changing environment.
Example 5
Administration of EPO Producing Cells In Vivo
[0097] To determine whether EPO producing cells survive and form
the tissues in vivo, renal cells mixed in collagen gel were
implanted subcutaneously in athymic mice at concentrations of
1.times.10.sup.6 and 5.times.10.sup.6 followed by retrieval at 14
and 28 days after implantation for analysis. Cells at different
passages from 1-5 were used. The cells were suspended in a collagen
gel for easy injection (concentration: 0.1 mg/ml).
[0098] Histologically, the retrieved implants showed that surviving
renal cells continue expressing EPO proteins, confirmed
immunohistochemically using EPO specific antibodies (FIG. 14).
[0099] These results demonstrate that EPO producing renal cells
grown and expanded in culture stably expressed EPO in vivo. Thus,
EPO producing cells may be used as a treatment option for anemia
caused by chronic renal failure.
Example 6
Analysis of EPO Expression with Real Time PCR
[0100] Real time PCR was performed to assess rat cell expression of
EPO in response to hypoxic conditions.
[0101] To test the effect of culture media, cells grown in either
KSFM and DMEM were exposed to hypoxic conditions (3% O.sub.2).
Renal primary cells (passage 0) were grown to 80% confluency in 10
cm plates. Three plates of cells were grown with either serum free
KSFM or DMEM and placed in a hypoxic chamber at 3% O.sub.2. After
24 hrs, samples were processed for total RNA and cDNA synthesis.
Real time PCR was done in triplicate, and samples were quantified
relative to normoxic sample. Results are shown in FIG. 15.
[0102] Rat kidney culture EPO expression was compared with real
time PCR across 24, 48 and 72 hours. Renal primary cells (passages
0 and 2) were grown to 80% confluency in 10 cm plates. Cells were
then grown in serum free KSFM and placed in a hypoxic chamber at 1%
O2. After 24, 48 or 72 hours, samples were processed for total RNA
and cDNA synthesis. Real time PCR was done in triplicate, and
samples were quantified relative to normoxic sample. Results are
shown in FIG. 16.
[0103] Testing timepoints for up to 24 hours, renal primary cells
(passage 0) were grown to 80% confluency in 10 cm plates. Cells
were then placed in a hypoxic chamber at 1% O2 for up to 24 hours.
Samples were then processed for total RNA and cDNA synthesis. Real
time PCR was run in triplicate, and samples were quantified
relative to normoxic sample. Results are shown in FIG. 17.
Example 7
Expansion of Human Kidney Cells
[0104] The growth and expandability of primary human kidney cells
were also demonstrated using the media and conditions described
above. Cultures from passages 2, 4, 7 and 9 are shown in FIG. 18.
It was demonstrated that human primary renal cells can be
maintained through twenty doublings (FIG. 19). Human kidney cell
cultures were characterized for EPO and GLEPP1 expression (FIG.
20).
Example 8
Human Kidney Cell Delivery Via Collagen Injection
[0105] Human renal cells mixed in collagen gel were implanted
subcutaneously in athymic mice as described above in Example 5.
Collagen concentrations of 1 mg/ml, 2 mg/ml and 20 mg/ml were
compared. At 1 and 2 mg/ml, the in vivo volume disappeared after
administration. At 20 mg/ml, the in vivo injection volume was
maintained, and neo-vascularization was seen FIG. 21. Histology
confirmed that EPO expressing tissue was formed in vivo (FIG.
22).
Example 9
EPO Producing Cell Selection with Magnetic Cell Sorting
[0106] Cells are selected for EPO production using magnetic cell
sorting. A single-cell suspension is isolated using a standard
preparation method. After preparation of single-cell suspension,
count the total number of the cells and centrifuge cell samples to
obtain a pellet. Block the cells with 10% of goat serum (of animal
where the secondary antibody is made) for 10 minutes. Add 1 or 2 mL
of the blocking solution. After 10 minutes of centrifugation,
resuspend the cells in the primary antibody for EPO (use 1 .mu.g of
the primary antibody/ million of cells). Typically, label for 15
minutes at 4-8.degree. C. is sufficient. Wash the cells twice to
remove any unbound primary antibody with 1-2 mL of buffer per
10.sup.7 cells and centrifuge at 300.times.g for 10 minutes. After
two successive washes, the pellet is resuspended in 80 .mu.L of PBS
(0.5% of BSA and 2 mM of EDTA, pH 7.2) per 10.sup.7 cells. Add 20
.mu.L of Goat Anti-Rabbit MicroBeads per 10.sup.7 cells. Mix well
and incubate for 15 minutes at 4-8.degree. C. Wash the cells twice
by adding 1-2 mL of buffer per 10.sup.7 cells and centrifuge at
300.times.g for 10 minutes. Pipette off supernatant completely.
Resuspend up to 10.sup.8 cells in 500 .mu.L of buffer (Note: For
higher cell numbers, scale up buffer volume accordingly; for
depletion with LD Columns, resuspend cell pellet in 500 .mu.L of
buffer for up to 1.25.times.108 cells). Proceed to magnetic cell
separation
[0107] Note: Work fast, keep cells cold, and use pre-cooled
solutions. This will prevent capping of antibodies on the cell
surface and non-specific cell labeling. Volumes for magnetic
labeling given below are for up to 10.sup.7 total cells. When
working with fewer than 10.sup.7 cells, use the same volumes as
indicated. When working with higher cell numbers, scale up all
reagent volumes and total volumes accordingly (e.g. for
2.times.10.sup.7 total cells, use twice the volume of all indicated
reagent volumes and total volumes). Working on ice may require
increased incubation times. Higher temperatures and/or longer
incubation times lead to non-specific cell labeling.
[0108] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
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