U.S. patent application number 13/911755 was filed with the patent office on 2014-01-09 for methods of tissue generation and tissue engineered compositions.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Sanjay Kumar Nigam, Eran Rosines.
Application Number | 20140011278 13/911755 |
Document ID | / |
Family ID | 40094190 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011278 |
Kind Code |
A1 |
Nigam; Sanjay Kumar ; et
al. |
January 9, 2014 |
METHODS OF TISSUE GENERATION AND TISSUE ENGINEERED COMPOSITIONS
Abstract
Provided are methods and compositions for constructing stable
mammalian embryonic epithelial tissues and organs as well as
constructing kidney tissue, and treating renal failure. Disclosed
are methods of using an active epithelial growth factor having the
capability of effectuating induction of growth and morphogenesis is
cells.
Inventors: |
Nigam; Sanjay Kumar; (Del
Mar, CA) ; Rosines; Eran; (Virginia Beach,
VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
40094190 |
Appl. No.: |
13/911755 |
Filed: |
June 6, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12663170 |
Sep 29, 2010 |
8460929 |
|
|
PCT/US08/65818 |
Jun 4, 2008 |
|
|
|
13911755 |
|
|
|
|
60941934 |
Jun 4, 2007 |
|
|
|
Current U.S.
Class: |
435/377 |
Current CPC
Class: |
C12N 5/0686 20130101;
C12N 2502/253 20130101; C12N 2501/13 20130101; C12N 2501/91
20130101; C12N 2501/115 20130101; C12N 2502/243 20130101 |
Class at
Publication: |
435/377 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. A method of generating a tubular tissue structure, comprising:
(a) contacting a stem cell with a cell survival agent or biological
active agent to stimulate growth and proliferation; (b) contacting
the cells with a branching agent that promotes formation of tubular
tissue branches and/or globular morphology to generate ureteric bud
or Wolffian duct cell tissue; (c) combining the ureteric bud tissue
with metenephric mesenchyme in a biocompatible matrix; and (d)
culturing the combination to form a tubular tissue or kidney tissue
in vitro.
2. A method of generating a tubular tissue structure, comprising:
(a) contacting a stem cell with a cell survival agent or biological
active agent to stimulate growth and proliferation; (b) contacting
the cells with an agent that promotes formation of metenephric
mesenchyme cells to generate a metenephric mesenchyme; (c)
combining the metenephric mesenchyme tissue with ureteric bud
tissue in a biocompatible matrix; and (d) culturing the combination
to form a tubular tissue or kidney tissue.
3. A method of generating a tubular tissue structure, comprising:
(a) contacting a metenephric mesenchyme progenitor cell with a
bioactive agent that induces growth and proliferation of the MM
progenitor; (b) contacting a ureteric bud or Wolffian duct
progenitor cell with a bioactive agent that induces growth and
proliferation of the UB or WD progenitor; (c) growing the
progenitor cells to form a population; (d) inducing branching or
the formation of globular structures in the UB or WD progenitor
cells to generate a population of UBs or WDs by contacting the
cells with a branching factor; (e) inducing formation of MM from
the MM progenitors by culturing the cells with a differentiation
factor; (f) combining the MM and UB and/or WD cells in a
biocompatible matrix or gel; and (d) culturing the combination to
form a tubular tissue or kidney tissue.
4. The method of claim 1, 2, or 3, wherein the cell survival agent
is selected from the group consisting of FGF1, FGF7 and a
combination thereof either alone or in combination with one or more
of GDNF, PTN, HRG, or BSN-CM, wherein the growth agent is selected
from the group consisting of FGF1, FGF7, FGF1 and FGF7, PTN and
GDNF, FGF1 and GDNF, FGF7 and GDNF, BSN-CM and FGF1, HRG and FGF1,
PTN and FGF1, BSN and FGF7, HRG and FGF7, PTN and FGF7, BSN and
FGF1 and GDNF, HRG and FGF1 and GDNF, PTN and FGF1 and GDNF, BSN
and FGF7 and GDNF, HRG and FGF7 and GDNF, and PTN and FGF7 and
GDNF.
5. The method of claim 1, 2, or 3, wherein the branching agent is
selected from the group consisting of FGF1, FGF7, FGF1 and FGF7,
PTN and GDNF, FGF1 and GDNF, FGF7 and GDNF, BSN-CM and FGF1, HRG
and FGF1, PTN and FGF1, BSN and FGF7, HRG and FGF7, PTN and FGF7,
BSN and FGF1 and GDNF, HRG and FGF1 and GDNF, PTN and FGF1 and
GDNF, BSN and FGF7 and GDNF, HRG and FGF7 and GDNF, and PTN and
FGF7 and GDNF.
6. The method of claim 1, 2, or 3, further comprising: culturing
stem or progenitor cells in vitro under conditions that induce
branching morphogenesis to generate a population of UBs, WDs, or
UBs and WDs comprising tubular branches; and subdividing the UB,
WD, OR UB AND WD population; and resuspending each subpopulation in
culture media and repeating.
7. A method for in vitro culturing and propagating ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud tissue,
comprising: isolating ureteric bud, Wolffian duct bud, or ureteric
and Wolffian duct bud tissue from mesenchyme tissue obtained from
embryonic kidney rudiments; culturing the isolated ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud tissue in a
biocompatible matrix in the presence of a culture medium comprising
a growth factor, growth factor combination as set forth in Table 2
for a sufficient time and under sufficient conditions to produce
tubular branches within the biocompatible matrix; separating the
plurality of branch tips to generate bud fragments; and culturing
each of the bud fragments in a biocompatible matrix with a culture
medium comprising pleiotrophin and/or heregulin or an active
fragment thereof.
8. The method of claim 7, wherein the biocompatible matrix
comprises a material selected from the group consisting of a
cotton, a collagen, a polyglycolic acid, a cat gut suture, a
cellulose, a gelatin, a dextran, a polyamide, a polyester, a
polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a
polycarbonate, a polytetrafluorethylene, a nitrocellulose compound,
and a Matrigel.
9. The method of claim 8, wherein the material is treated to
contain proteoglycans, Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, or combinations thereof.
10. A method comprising: differentiating stem cells to form
metenephric mesenchyme tissue; differentiating stem cells to form
ureteric bud cells; and combining the UB cells and MM cells in a
biocompatible matrix or gel; and culturing the combination tissue
to form a rudimentary kidney tissue.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/663,170, filed Sep. 29, 2010 (now U.S. Pat. No. 8,460,929),
which is a U.S. National Stage Application filed under 35 U.S.C.
371 and claims priority to International Application No.
PCT/US08/65818, filed Jun. 4, 2008, which application claims
priority under 35 U.S.C. .sctn.119 to U.S. Provisional Application
Ser. No. 60/941,934, filed Jun. 4, 2007, the disclosures of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention generally concerns methods of tissue
engineering, and more particularly relates to methods and
compositions for tubular tissue generation and more particularly to
kidney tissue generation and replacement.
BACKGROUND
[0003] End-stage renal disease (ESRD) affects almost 350,000 people
living in the United States with an incidence that has increased by
over 50% in the past decade. Total Medicare expenditures on
patients with ESRD exceed $11.3 billion (U.S. Renal Data Service:
2001 Annual Data Report: Atlas of End-Stage Renal Disease in the
United States. Bethesda NIH, NIDDKD, 2001). The two current
treatment modalities for ESRD, dialysis and transplantation, both
have significant limitations. Patients on dialysis have an
extremely high mortality rate, approaching 20% per year. Patient
survival is markedly improved with renal transplantation; however,
the number of renal transplants is severely limited by the short
supply of available organs and many patients die while awaiting
transplantation of a kidney allograft.
SUMMARY
[0004] The disclosure provides a stepwise in vitro method of
engineering kidney-like tissues capable of being implanted,
recruiting a vasculature and developing glomeruli. Using a
reconstitution approach based, in part, upon the fact that stages
of kidney development are separable in vitro, the disclosure
provides an approach to sequentially induce an epithelial tubule
(e.g., the Wolffian duct (WD)), to undergo in vitro budding,
combining bud/metanephric mesenchyme and implantation of the
combined tissue that results in vascularization. The disclosure
demonstrates that tubular tissues can be stimulated in vitro to
reproduce kidney formation with appropriate spatial relationships
of nephrons in a stepwise fashion resulting in an in vitro tissue
that can be implanted in host recipients, thereby recruiting
vascularized glomeruli. Exclusive optimization studies of growth
factor and matrix (natural and artificial) conditions indicate
multiple suitable combinations and suggest both a robust and a
minimal system. A whole genome microarray analysis was performed on
the recombined tissue to verify that the tissue was recapitulating
gene expression changes that occur in vivo during later stages of
kidney development.
[0005] The disclosure provides a method for in vitro engineering
and constructing a mammalian kidney. The method includes separating
ureteric bud, Wolffian duct bud, or ureteric and Wolffian duct bud
(UB, WD, or UB and WD) tissue from mesenchyme tissue obtained from
an embryonic kidney rudiment; culturing the isolated ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud tissue in a
biocompatible matrix in the presence of a culture medium comprising
pleiotrophin and/or heregulin or an active fragment thereof for a
sufficient time and under sufficient conditions to produce tubular
branches within the biocompatible matrix; separating the tubular
branches to obtain a plurality of bud fragments; culturing each of
the bud fragments in a biocompatible matrix with a culture medium
comprising pleiotrophin and/or heregulin or an active fragment
thereof to generate a plurality of tissues comprising tubular
branches; combining the plurality of tissues comprising tubular
branches with metanephric mesenchyme (MM) tissue in the presence of
nutrient medium comprising pleiotrophin, heregulin, a combination
of pleotrophin and heregulin or an active fragment of the foregoing
alone or in combination; and culturing the UB, WD, or UB and WD and
MM under conditions sufficient to cause the MM to differentiate and
form nephron structures thereby forming a kidney.
[0006] The disclosure provides a functional mammalian kidney
engineered and constructed in vitro, comprising: a ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud (UB, WD, or UB
and WD) tissue propagated in culture in the presence of a
composition comprising pleiotrophin, heregulin, a combination of
pleotrophin and heregulin or an active fragment of the foregoing
alone or in combination to produce a functioning tubular
structures; and a metanephric mesenchyme (MM) tissue propagated
from cultured embryonic mesenchymal tissue fragments or cells to
produce functioning nephrons wherein the ureteric bud, Wolffian
duct bud, or ureteric and Wolffian duct bud tissue and the
metanephric mesenchyme are co-cultured and wherein the ureteric
bud, Wolffian duct bud, or ureteric and Wolffian duct bud tissue
induces the metanephric mesenchyme to form nephrons, thereby
forming a functional mammalian kidney.
[0007] The disclosure provides a method of propagating ureteric bud
(UB), Wolffian duct bud (WD), or ureteric and Wolffian duct bud
cells in culture. The method includes culturing a UB, WD, or UB and
WD in vitro under conditions that induce the UB, WD, or UB and WD
to undergo branching morphogenesis to generate a population of UBs,
WDs, or UBs and WDs comprising tubular branches; subdividing the
UB, WD, or UB and WD population; and resuspending each
subpopulation in culture media.
[0008] The disclosure also provides a method for in vitro culturing
and propagating ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct bud tissue. The method includes isolating ureteric
bud, Wolffian duct bud, or ureteric and Wolffian duct bud tissue
from mesenchyme tissue obtained from embryonic kidney rudiments;
culturing the isolated ureteric bud, Wolffian duct bud, or ureteric
and Wolffian duct bud tissue in a biocompatible matrix in the
presence of a culture medium comprising pleiotrophin, heregulin, a
combination of pleotrophin and heregulin or an active fragment of
the foregoing alone or in combination for a sufficient time and
under sufficient conditions to product tubular branches within the
biocompatible matrix; separating the plurality of branch tips to
generate bud fragments; and culturing each of the bud fragments in
a biocompatible matrix with a culture medium comprising
pleiotrophin, heregulin, a combination of pleotrophin and heregulin
or an active fragment of the foregoing alone or in combination.
[0009] The disclosure also provides a method for growing renal
tubule cells in vitro, comprising culturing kidney cells in a
growth medium comprising pleiotrophin, heregulin, a combination of
pleotrophin and heregulin or an active fragment of the foregoing
alone or in combination in an amount effective for achieving
tubulogenesis.
[0010] The disclosure further provides a method for stimulating
epithelial organogenesis, by contacting an epithelial tissue with
an effective amount of a composition comprising one or more
mesenchymally derived growth factor(s) secreted by mesenchymal
tissue in culture; and culturing the epithelial tissue and the
composition for a sufficient period of time and under conditions to
allow the tissue and the composition to interact, wherein the
composition stimulates epithelial organogenesis.
[0011] The disclosure also provides in vitro tissue generated by
the foregoing method.
[0012] The disclosure includes a method of stimulating branching
morphogenesis in an epithelial tissue comprising contacting the
epithelial tissue with a composition comprising pleiotrophin and/or
heregulin.
[0013] The disclosure provides a method for in vitro tissue
engineering of a functional mammalian epithelial tissue, organ or a
fragment thereof by culturing and propagating embryonic epithelial
explant, tissues, and/or cells by isolating the explant, tissue,
and/or cells and growing the explant, tissue, and/or cells in a
culture medium comprising pleiotrophin, heregulin, a combination of
pleotrophin and heregulin or an active fragment of the foregoing
alone or in combination, permitting the culture to form multiple
branches, dissecting out individual tips of the branches;
reculturing the branch tips in the culture medium comprising a
heparin binding molecule (e.g., pleiotrophin, heregulin, a
combination of pleotrophin and heregulin or an active fragment of
the foregoing alone or in combination); combining the branch tips
with embryonic or fetal mesenchymal tissue and/or cells, in the
presence of the mixture of a culture medium in or on a
biocompatible substrate; and culturing the combination in culture
medium conditions suitable for tissue growth and tubulogenesis.
[0014] The disclosure further provides a method for stimulating
branching morphogenesis in a kidney cell culture. The method
includes contacting the kidney cell culture with an effective
amount of a composition comprising one or more mesenchymally
derived growth factor(s) secreted by a mesenchyme tissue in
culture; and culturing the kidney cell culture and the composition
for a sufficient period of time and under conditions to allow the
cells and the composition to interact, wherein the composition
stimulates branching tubular morphogenesis.
[0015] The disclosure also provides in vitro engineered kidney
tissue. In one aspect of the disclosure the in vitro engineered
kidney tissue is generated by the methods as described herein.
[0016] Also provided by the disclosure is a method of in vitro
culturing and propagating metanephric mesenchyme tissue,
comprising: isolating mesenchyme tissue at the time of induction;
culturing the mesenchymal tissue in a composition comprising serum,
nutrient rich medium, and mesenchymal and/or ureteric bud, Wolffian
duct bud, or ureteric and Wolffian duct bud cell conditioned
medium; and partitioning the cultured mesenchyme into multiple
pieces and growing each piece separately in culture.
[0017] The disclosure also provide a genetically engineered
mammalian kidney produced by culturing a population of cells
comprising ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct bud (UB, WD, or UB and WD) cells in a biocompatible
matrix in the presence of a culture medium comprising pleiotrophin
and/or heregulin or an active fragment thereof for a sufficient
time and under sufficient conditions to produce tubular branches
within the biocompatible matrix, and wherein at least one ureteric
bud, Wolffian duct bud, or ureteric and Wolffian duct bud cell of
the population of cells is transfected with an exogenous
polynucleotide such that the exogenous polynucleotide expresses a
product; separating the tubular branches to obtain a plurality of
bud fragments; culturing each of the bud fragments in a
biocompatible matrix with a culture medium comprising pleiotrophin
and/or heregulin or an active fragment thereof to generate a
plurality of tissues comprising tubular branches; combining the
plurality of tissues comprising tubular branches with metanephric
mesenchyme (MM) tissue in the presence of nutrient medium
comprising pleiotrophin and/or heregulin; and culturing the UB, WD,
or UB and WD and MM under conditions sufficient to cause the MM to
differentiate and form nephron structures thereby forming a
kidney.
[0018] The disclosure provides a method of treating a subject
suffering from kidney failure comprising transplanting a
tissue-engineered kidney of the invention into a subject.
[0019] The disclosure also includes a method for treating acute
renal failure (ARF) comprising administering to a subject suffering
from ARF with a pharmaceutically effective amount of a composition
comprising pleiotrophin, heregulin, a combination of pleotrophin
and heregulin or an active fragment of the foregoing alone or in
combination such that a symptom of ARF is ameliorated.
[0020] The disclosure also includes a renal tubule cell produced by
culturing ureteric bud, Wolffian duct bud, or ureteric and Wolffian
duct bud cells in a culture medium comprising pleiotrophin and/or
heregulin in an amount effective for achieving tubulogenesis.
[0021] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0022] FIG. 1A-F shows a Wolffian duct budding systems. The whole
mesonephros (A, B), the Wolffian duct with a thin layer of
intermediate mesoderm (C, D), and the Wolffian duct void of all
other cell layers (E, F) can all be induced to bud according to the
conditions outlined in Table 1. Scale bar corresponds to 500
.mu.m.
[0023] FIG. 2A-C shows a method of Wolffian duct budding to
isolated in vitro-formed UB branching. One bud from a budded
Wolffian duct after 4 days in culture (A) can be excised, suspended
in a gel (B), and induced to branch (C). This demonstrates how one
Wolffian duct progenitor tissue can lead to the creation of
multiple branched in vitro collecting ducts.
[0024] FIG. 3 shows an effect of soluble factors on isolated
ureteric bud growth and shape and growth in 3D. Rat E13 ureteric
buds were suspended in extracellular matrix gels comprising growth
factor reduced Matrigel.TM. and Type 1 Collagen in the presence of
the growth factors indicated. Pictures were taken at 4-6 days of
culture. Pleiotrophin (PTN) was purified from BSN-CM.
Concentrations used were GDNF 125 ng/ml, HRG 500 ng/ml, FGF1 250
ng/ml, FGF7 50 ng/ml. Scale bar corresponds to 500 .mu.m.
[0025] FIG. 4A-I depicts the effects of extracellular matrix
conditions on ureteric bud branching. (A) 100%, (B) 75%, (C) 50%,
(D) 25%, (E) 15% Matrigel.TM. solutions. 3.0 mg/mL type I collagen
(F) and the inert gels, 1% Alginate (G) and Puramtrix.TM. (H), did
not support branching. Type IV collagen (0.75 mg/mL) alone could
support branching morphogenesis (J) with no increases by the
addition of laminin (0.33 mg/mL type IV collagen, 0.67 mg/mL
laminin) (K), but were both less branched the 50% Matrigel.TM.
control (I). All pictures taken after 7 days in culture. Scale bar
corresponds to 500 .mu.m.
[0026] FIG. 5A-F shows recombination of branched in vitro-formed UB
with metanephric mesenchyme. The branched in vitro-formed UB (A)
was mechanically separated from the matrix and recombined with
freshly dissected undifferentiated MM (B). Recombined tissues were
grown for approximately 4 to 7 days. During this time, the in
vitro-formed UB continued to branch and invade the MM. The MM
simultaneously was induced to undergo epithelial transformation
(C). A 10.times. dual fluorescent micrograph of the recombined
tissues stained with FITC-labeled D. biflorus and
rhodamine-conjugated peanut agglutinin (PNA) lectin shows the
mesenchymal to epithelial transition occurring around the UB
branches (D,E-4.times.). A higher magnification (40.times.) at the
fusion of the WD- and MM-derived epithelial cells demonstrates a
contiguous tubule lumen (F).
[0027] FIG. 6 shows grouping of the genes based on developmental
expression patterns. Before the gene expression of the recombined
tissue was analyzed, the genes on the chip were grouped according
to their kidney developmental expression pattern. Up-regulated
genes were defined as genes with 3 fold expression increases from
E13 to Wk4. The genes were further grouped based on E18 expression
level; if E18 was within 1.5 fold of E13 or Wk 4 then, the gene was
defined as Group I or Group II, respectively. If E18 was in between
those criteria then the gene was defined as Group III. An analogous
grouping was performed for down-regulated genes. Developmentally
regulated genes were defined as genes whose E18 expression was at
least 3 fold higher or lower than E13 or Wk4 and 1.5 higher or
lower than the other. Recombination regulated genes represent genes
that were 3 fold higher or lower in the recombined tissue than all
three developmental time points.
[0028] FIG. 7A-D shows recombined tissue 14 days after implantation
in to host rat. The recombined tissue has acquired blood vessels
and developed glomeruli. (A) 4.times. (B) 20.times.. The presence
of erythrocytes (arrow) in the glomeruli confirms blood flow in the
recombined tissue -40.times. (C). The cells of the glomerulus
express the endothelial marker, P-CAM1, and type IV collagen along
its basement membrane (D)-60.times.. Scale bar corresponds to 50
.mu.m.
[0029] FIG. 8A-E shows a general in vitro kidney engineering
strategy. Overview of the steps to recapitulate kidney development
in vitro. The Wolffian Duct is isolated (B) and induced to bud.
Then, each bud can be isolated (D) and induced to undergo branching
(E). The branched in vitro-formed UB is then recombined with
metanephric mesenchyme (F) and after 4-6 days of mutual induction
the recombined tissue resembles a late-stage embryonic kidney (G).
The recombined tissue is then implanted into a host animal where it
is vascularized and forms glomeruli (H).
[0030] FIG. 9A-B shows another general in vitro kidney engineering
strategy. (B) Schematic of the procedure followed in engineering
kidney tissue from the Wolffian duct and MM. (B-G) Photomicrographs
of the engineered tissues. (B) Whole embryonic day 13 rat kidney.
(C) Kidney which has been separated into isolated UB and isolated
MM. (D) Isolated MM from c, in which a piece of WD has been used to
replace the UB. (E) After 7 days, the WD/MM co-culture grew similar
to traditional in vitro kidney culture. (F-G) Confocal fluorescent
micrographs of the engineered kidney tissue after 7 (f) and 12 (g)
days of culture. (f; bright=DB lectin, UB derived tissues;
faint=E-Cadherin, UB and MM derived epithelial tissues; g: PNA
lectin staining (faint) revealed differentiation of glomerular
podocytes). (300 .mu.m scale bar).
[0031] FIG. 10A-D shows UB cell aggregate co-culture with MM.
Photomicrographs showing the recombination of cultured UB cells
with freshly isolated MM. (A) Phase contrast of a 3D culture of UB
cells. UB cells will form branching tubular structures when
cultured in the appropriate matrix and with the appropriate growth
factors. (B) Hanging drop aggregate of UB cells (outlined in
yellow) surrounded by numerous freshly isolated MMs. (C-D) Confocal
fluorescent photomicrograph of recombined tissue after 7 days of
growth in culture. UBs are stained (D. biflorus) and both UB and MM
derived polarized epithelial cells were stained (E-Cadherin) (C)
(400 .mu.m scale bar--A-C). d. Higher magnification examination of
the recombined tissue showing that the MM derived tubule is
continuous with the green UB cells -25 .mu.m scale bar.
[0032] FIG. 11A-D shows IMCD cell aggregate co-culture with MM.
After 7 days, the IMCD cells organized into epithelial tubules,
however MM induction did not appear very widespread (a).
Cytokeratin staining (green) demonstrates that the IMCD cell
aggregate formed tubular structures with lumens (b,c,d). While MM
induction did not occur as strongly as with the UB cell aggregate,
occasional comma shaped bodies (evident by PAX-2 staining, red)
indicated mesenchymal-to epithelial-transformation could be induced
by the IMCD cell aggregate (e) (400 .mu.m scale bar--a,b; 50 .mu.m
scale bar--c,d).
[0033] FIG. 12A-H shows three MM derived cell lines were tested for
the ability to induce isolated UB branching. The BSN (a), RIMM-18
(b), and MM primary (c) cell lines are all MM derived cell lines
that are vimentin positive, cytokeratin negative. 3T3 fibroblasts
(d) are also vimentin positive, cytokeratin negative cells, but are
not MM derived. The conditioned medium from BSN cells strongly
induced isolated UB branching (e) whereas the conditioned medium
from primary MM cells slightly induced branching (g). The
conditioned medium from RIMM-18 and 3T3 cells did not induce
branching morphogenesis (f, h, respectively). Plot of tip number
vs. cell-conditioned medium used (i) (ANOVA, P.ltoreq.0.00001);
*=P.ltoreq.0.05, **=P.ltoreq.0.00005. (50 .mu.m--a,b,c,d; 250 .mu.m
scale bar--e,f,g,h).
DETAILED DESCRIPTION
[0034] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a protein" includes a plurality of such proteins and reference to
"the cell" includes reference to one or more cells known to those
skilled in the art, and so forth.
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0036] Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
[0037] It is to be further understood that where descriptions of
various embodiments use the term "comprising," those skilled in the
art would understand that in some specific instances, an embodiment
can be alternatively described using language "consisting
essentially of" or "consisting of:"
[0038] The publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
[0039] As used herein, the abbreviation UB, WD, or UB and WD
includes ureteric bud, Wolffian duct bud, or ureteric and Wolffian
duct bud cells obtained from UB, WD, or UB and WD tissue, as well
as UB, WD, or UB and WD tissue fragments, whole UB, WD, or UB and
WD tissue, and UB, WD, or UB and WD cell lines, unless clearly
indicated otherwise in the specification. The UB, WD, or UB and WD
cells may be primary cells obtained from embryonic kidney tissue by
various techniques known in the art. Such primary UB, WD, or UB and
WD cells are not immortalized (e.g., by SV40), but may be
transfected and/or transformed to express a desired product, as
discussed in more detail herein.
[0040] The culture system and methods of the disclosure provide the
ability to propagate the isolated UB, WD, or UB and WD in vitro
through several generations. For example, isolated stem cells, UB,
WD, or any combination thereof are cultured in vitro and induced to
undergo branching morphogenesis in the presence of BSN-CM or
pleiotrophin and GDNF or pleiotrophin, FGF1, and GDNF. Following
propagation of the buds, the propagated buds can be recombined with
metanephric mesenchyme (MM) to induce nephron tissue formation. The
tissue can then undergo vascularization in vivo upon implantation
within the kidney of a subject. Using the various component pieces
a functional kidney can be obtained. The disclosure provides a
cell-based kidney development strategy. Unlike prior strategies,
which have used tissue segments and recombination, the present
disclosure demonstrates the ability to develop tissue components
from substantially homogenous cell types followed by recombination
of the tissue components. In one aspect, stem cells are useful as
the initial cell type used in the methods of the disclosure.
[0041] The term "precursor cell," "progenitor cell," and "stem
cell" are used interchangeably in the art and herein and refer
either to a pluripotent, or lineage-uncommitted, progenitor cell,
which is potentially capable of an unlimited number of mitotic
divisions to either renew its line or to produce progeny cells
which will differentiate into a desired cell type; or a
lineage-committed progenitor cell and its progeny, which is capable
of self-renewal and is capable of differentiating into a further
lineage defined cell type. Unlike pluripotent stem cells,
lineage-committed progenitor cells are generally considered to be
incapable of giving rise to numerous cell types that phenotypically
differ from each other. Instead, they give rise to one or possibly
two lineage-committed cell types. As a further description, stem
cells are cells capable of differentiation into other cell types,
including those having a particular, specialized function (e.g.,
tissue specific cells, parenchymal cells and progenitors thereof).
Progenitor cells (i.e., "multipotent") are cells that can give rise
to different terminally differentiated cell types, and cells that
are capable of giving rise to various progenitor cells. Cells that
give rise to some or many, but not all, of the cell types of an
organism are often termed "pluripotent" stem cells, which are able
to differentiate into any cell type in the body of a mature
organism, although without reprogramming they are unable to
de-differentiate into the cells from which they were derived. As
will be appreciated, "multipotent" stem/progenitor cells have a
more narrow differentiation potential than do pluripotent stem
cells. Another class of cells even more primitive (i.e.,
uncommitted to a particular differentiation fate) than pluripotent
stem cells are the so-called "totipotent" stem cells (e.g.,
fertilized oocytes, cells of embryos at the two and four cell
stages of development), which have the ability to differentiate
into any type of cell of the particular species. For example, a
single totipotent stem cell could give rise to a complete animal,
as well as to any of the myriad of cell types found in the
particular species (e.g., humans).
[0042] Embryonic stem cells are generated and maintained using
methods well known to the skilled artisan such as those described
by Doetschman et al. (1985) J. Embryol. Exp. Mol. Biol. 87:27-45).
Any line of ES cells can be used. One mouse strain that is
typically used for production of ES cells, is the 129J strain.
Another ES cell line is murine cell line D3 (American Type Culture
Collection, catalog no. CKL 1934). Still another ES cell line is
the WW6 cell line (Ioffe et al. (1995) PNAS 92:7357-7361). Human
embryonic stem cells (hESCs) can be isolated, for example, from
human blastocysts obtained from human in vivo preimplantation
embryos, in vitro fertilized embryos, or one-cell human embryos
expanded to the blastocyst stage (Bongso, et al. (1989), Hum.
Reprod., vol. 4: 706). Human embryos can be cultured to the
blastocyst stage in G1.2 and G2.2 medium (Gardner, et al. (1998),
Fertil. Steril., vol. 69:84). The zona pellucida is removed from
blastocysts by brief exposure to pronase (Sigma). The inner cell
masses can be isolated by immunosurgery or by mechanical
separation, and are plated on mouse embryonic feeder layers, or in
the defined culture system as described herein. After nine to
fifteen days, inner cell mass-derived outgrowths are dissociated
into clumps either by exposure to calcium and magnesium-free
phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to
dispase, collagenase, or trypsin, or by mechanical dissociation
with a micropipette. The dissociated cells are then replated as
before in fresh medium and observed for colony formation. Colonies
demonstrating undifferentiated morphology are individually selected
by micropipette, mechanically dissociated into clumps, and
replated. Embryonic stem cell-like morphology is characterized as
compact colonies with apparently high nucleus to cytoplasm ratio
and prominent nucleoli. Resulting embryonic stem cells are then
routinely split every 1-2 weeks by brief trypsinization, exposure
to Dulbecco's PBS (without calcium or magnesium and with 2 mM
EDTA), exposure to type IV collagenase (about 200 U/mL), or by
selection of individual colonies by mechanical dissociation, for
example, using a micropipette.
[0043] Once isolated, the stem cells, can be cultured in a culture
medium according to the invention that supports the substantially
undifferentiated growth of stem cells using any suitable cell
culturing technique. For example, a matrix laid down prior to lysis
of primate feeder cells (preferably allogeneic feeder cells) or a
synthetic or purified matrix can be prepared using standard
methods. The stem cells to be cultured are then added atop the
matrix along with the culture medium. In other embodiments, once
isolated, undifferentiated stem cells can be directly added to an
extracellular matrix that contains laminin or a growth-arrested
human feeder cell layer (e.g., a human foreskin fibroblast cell
layer) and maintained in a serum-free growth environment according
to the culture methods of invention. In yet another embodiment, the
stem cells can be directly added to a biocompatible cell culture
plate in the absence of an extracellular matrix material (e.g.,
directly on polystrene, glass or the like). Unlike existing
embryonic stem cell lines cultured using conventional techniques,
embryonic stem cells and their derivatives prepared and cultured in
accordance with the methods of the invention avoid or have reduced
exposure to xenogeneic antigens that may be present in feeder
layers. This is due in part to the media compositions promoting
growth in the absence of feeder layers or directly on a cell
culture substrate. This avoids the risks of contaminating human
cells, for example, with non-human animal cells, transmitting
pathogens from non-human animal cells to human cells, forming
heterogeneous fusion cells, and exposing human cells to toxic
xenogeneic factors.
[0044] In yet another aspect, mesenchymal stem cells are used.
Mesenchymal stem cells are multipotent stem cells. Mesenchyme is
embryonic connective tissue that is derived from the mesoderm and
that differentiates into hematopoietic and connective tissue. MSCs
can be obtained from both marrow and non-marrow tissues, such as
adult muscle side-population cells or the Wharton's jelly present
in the umbilical cord.
[0045] Substantially homogenous populations of cells (e.g., 80, 90,
95, 98, 99 or 100% homogenous) can be used in the development of
metanephric mesenchyme tissue or ureteric bud and Wolffian duct
cells. For example, many epithelial organs such as kidney, lung,
and prostate under go branching morphogenesis in the course of
development. The kidney is formed by mutual induction between two
tissues derived from the intermediate mesoderm, the metanephric
mesenchyme (MM), and the ureteric bud, Wolffian duct bud, or
ureteric and Wolffian duct bud (UB, WD, UB and WD). The UB, WD, or
UB and WD induces the MM to differentiate and form the proximal
nephron, while the UB, WD, or UB and WD undergoes dichotomous
branching and elongation as it invades the MM, ultimately forming
the kidney collecting system.
[0046] A number of factors are known to cause differentiation of
stem cells or progenitor cells along a directed lineage specific
for various tissues. Non-limiting examples of bioactive molecules
include activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF,
angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3,
angiopoietin-4, angiostatin, angiotropin, angiotensin-2,
AtT20-ECGF, betacellulin, bFGF, B61, bFGF inducing activity,
cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen,
collagen receptors .alpha..sub.1.beta..sub.1 and
.alpha..sub.1.beta..sub.1, connexins, Cox-2, ECDGF (endothelial
cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin,
endothelins, endostatin, endothelial cell growth inhibitor,
endothelial cell-viability maintaining factor, endothelial
differentiation shpingolipid G-protein coupled receptor-1 (EDG1),
ephrins, Epo, HGF, TNF-.alpha., TGF-.beta., PD-ECGF, PDGF, IGF,
IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin,
fibronectin receptor .alpha..sub.1.beta..sub.1, Factor X, HB-EGF,
HBNF, HGF, HUAF, heart derived inhibitor of vascular cell
proliferation, IFN-gamma, IL1, IGF-2 IFN-gamma, integrin receptors,
K-FGF, LIF, leiomyoma-derived growth factor, MCP-1,
macrophage-derived growth factor, monocyte-derived growth factor,
MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokiase plasminogen activator,
neuropilin (NRP1, NRP2), neurothelin, nitric oxide donors, nitric
oxide synthases (NOSs), notch, occludins, zona occludins,
oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-.beta., PD-ECGF,
PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR.gamma., PPAR.gamma.
ligands, phosphodiesterase, prolactin, prostacyclin, protein S,
smooth muscle cell-derived growth factor, smooth muscle
cell-derived migration factor, sphingosine-1-phosphate-1 (S1P1),
Syk, SLP76, tachykinins, TGF-beta, Tie 1, Tie2, TGF-.beta., and
TGF-.beta. receptors, TIMPs, TNF-alpha, TNF-beta, transferrin,
thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,
VEGF, VEGF.sub.164, VEGI, EG-VEGF, VEGF receptors, PF4, 16 kDa
fragment of prolactin, prostaglandins E1 and E2, steroids, heparin,
1-butyryl glycerol (monobutyrin), and nicotinic amide.
[0047] Soluble factors that have been thought to play a role in
morphogenetic capacity include hepatocytes growth factor (HGF) and
epidermal growth factor (EGF) receptor ligands, which have been
shown to induce branching tubular structures in epithelial cells
cultured in collagen gels.
[0048] The cells used in the generation of the engineered kidney
tissue as described herein can be induced to proliferate and/or
differentiate using any number or combination of factor such as
those described above. In one aspect, the disclosure provides
culturing a stem cell in a culture medium comprising a bioactive
molecule to expand the stem cells and/or cause differentiation of
the stem cell into a ureteric bud or Wolffian duct cell. Such
culture conditions can generate a population of ureteric bud cells
that can be used as a component for generation of a functioning
kidney tissue. In another aspect, the same stem cell type or a
different stem cell type can be culture in a culture medium
comprising a bioactive molecule to expand the stem cells and/or
cause differentiation of the stem cell into a metanephric
mesenchyme. Such culture conditions can generate a population of
metanephric mesenchyme cells that can be used as a component for
generation of a functioning kidney tissue.
[0049] Accordingly, the identification of specific soluble factors
(e.g., MM-derived soluble factors) mediating UB, WD, or UB and WD
branching morphogenesis and culture conditions are useful for
proper in vitro development. Hepatocyte growth factor (HGF) has
been shown to induce the formation of branching tubular structures
with lumens in three-dimensional cultures of epithelial cell lines
derived from adult kidneys (i.e., MDCK and mIMCD cells) (Barros et
al., 1995; Cantley et al., 1994; Montesano et al., 1991; Santos et
al., 1993).
[0050] Another group of soluble factors implicated in branching
morphogenesis of epithelial cells are the family of epidermal
growth factor (EGF) receptor ligands. EGF receptor ligands are
capable of inducing the formation of branching tubular structures
with lumens in three-dimensional cultures of mIMCD cells, a kidney
cell line derived from adult collecting duct cells (Barros et al.,
1995; Sakurai et al., 1997).
[0051] Tubulogenesis is a phenotypic transformation of the cells
such that condensed aggregates of tubule cells form about a central
lumen wherein the lumen is bordered by cells possessing a polarized
epithelial phenotype and tight junctional complexes along the
lumenal border. Conditioned medium elaborated by MM-derived cell
lines, BSN-CM, induced UBs, WDs, or UBs and WDs in
three-dimensional culture to form branching tubular structures with
clearly distinguishable lumens.
[0052] A role for GDNF in UB, WD, or UB and WD (from buds or stem
cells) development is demonstrated by the disclosure. GDNF plays a
role in branching morphogenesis of the isolated UB, WD, or UB and
WD and can be used with stem cells.
[0053] Studies in the developing mammalian lung and Drosophila
trachea indicate that members of the FGF family function in
branching morphogenesis of epithelial tissues (Hogan, 1999; Metzger
and Krasnow, 1999; Zelzer and Shilo, 2000). Furthermore, null
mutations of either fgf7 or fgf10 have also been reported to affect
kidney development (Obuchi et al., 2000; Qiao et al., 1999),
although in both cases the kidneys appear to be modestly affected.
For example, in fgf7-null kidneys, there is a 30% reduction in the
number of nephrons, and the kidneys appear to function normally
(Qiao et al., 1999). Moreover, since FGF7 is detected in the
developing kidney after several iterations of UB, WD, or UB and WD
branching has already occurred, it is likely that other factors are
necessary for the early steps of the branching program. In the case
of FGF 10, the defect appears similar. Nevertheless, by
potentiating the effect of an essential branching morphogen
produced by the MM, certain FGFs are demonstrated herein to play a
role in stem cell, UB, WD, or UB and WD branching
morphogenesis.
[0054] The disclosure demonstrates that UBs, WDs, or UBs and WDs
undergo branching tubulogenesis in the presence of a conditioned
medium elaborated by a cell line derived from the MM or isolated
from an E11.5 mouse (BSN cells). Soluble factors present in BSN-CM
are important for UB, WD, stem cells, or UB and WD morphogenesis.
Factors that are secreted by the MM are important for the
development of the collecting system in artificial systems as well
as in vivo.
[0055] The MM-derived cell conditioned medium (BSN-CM), when
supplemented with GDNF, also induces the isolated UB, WD, stem
cells, or UB and WD (in the absence of MM) to undergo dichotomous
branching reminiscent of that seen in the developing kidney. This
indicates that the MM-derived cell line, reflecting the MM itself,
secretes soluble factors capable of inducing branching
morphogenesis of the UB, WD, stem cells, or UB and WD. This
isolated cell culture system can serve as a powerful assay system
since it directly assesses the effect of soluble factors on cell
morphogenesis and tubulogenesis.
[0056] The disclosure demonstrates that serial liquid column
chromatographic fractionation of BSN-CM contain an active
morphogenetic fraction comprising a polypeptide (capable of
inducing branching morphogenesis comparable to whole BSN-CM). One
such polypeptide is pleiotrophin. Pleiotrophin was originally
discovered as a fibroblast proliferative factor (Milner et al,
BBRC, 165:1096-1103, 1989) and a neurite outgrowth-promoting factor
(Rauvala, EMBO J, 8:2933-41, 1989). Outside the nervous system
pleiotrophin is generally detected in those embryonic organs in
which mesenchymal-epithelial interactions are thought to play an
important role, such as salivary glands, lung, pancreas, and kidney
(Mitsiadis et al., Development 121:37-51, 1995; Vanderwinden et
al., Anat Ebryol (Berl) 186:387-406, 1992). Although pleiotrophin
has been shown to be mitogenic for certain epithelial cells (Li et
al., Science 250:1690-4, 1990; Sato et al., Exp Cell Res
246:152-64, 1999), there has been no compelling pleiotrophin during
epithelial organogenesis.
[0057] Immunoblot analysis of BSN-CM as well as in situ
hybridization data of developing kidney (Vanderwinden et al.,
1992), demonstrate that the embryonic MM is a source of
pleiotrophin. In addition to its ability to induce branching
morphogenesis in the isolated stem cell, UB, WD, or a combination
thereof, pleiotrophin also induced branching tubular structures
with lumens, and is thus a soluble factor with this capability. The
disclosure provides methods and compositions for use in vitro and
in vivo to induce morphogenesis and tubular formation of tissues
(e.g., kidney tissue).
[0058] The disclosure provides culture techniques and factors, and
combination of factors capable of inducing stem cell, UB, WD, or
any combination thereof (e.g., UB and WD) to undergo branching
morphogenetic activity. In one aspect, the disclosure provides an
18 kDa heparin binding protein, pleiotrophin, obtained from the
BSN-CM.
[0059] The disclosure demonstrates that pleiotrophin can induce
branching morphogenesis of the isolated stem cell, UB, WD, or any
combination thereof (e.g., UB and WD) in vitro. Thus, pleiotrophin
and compositions comprising pleiotrophin can be used to induce
morphogenesis of cells to develop into kidney cells in vitro and in
vivo.
[0060] A wide range of concentrations of pleiotrophin has been
reported to exhibit biological activity (up to 50 .mu.g/ml) in
various systems (Imai et al., 1998; Li et al., 1990; Rauvala et
al., 1994; Souttou et al., 1997). Pleiotrophin binds to the
extracellular matrix, which may explain why concentrations of
200-600 ng/ml were useful for morphogenetic activity in the systems
employed in Examples below. In the examples below the stem cell,
UB, WD, or any combination thereof (e.g., UB and WD) cell-line and
isolated UBs, WDs, or UBs and WDs were cultured in collagen IV or
within basement membrane Matrigel.TM., which could conceivably bind
a large fraction of pleiotrophin. In one aspect of the disclosure,
similar artificial matrix systems, e.g., cell-free extracellular
matrices (e.g., obtained by decellularizing a desired tissue), or
synthesized matrices (e.g., lactic acid, glycolic acid, or
combinations thereof) can be used and may similarly be modified to
bind pleiotrophin.
[0061] To date, several glycoproteins, including brain-specific
proteoglycans, the receptor type tyrosine phosphatase beta (Maeda
and Noda, 1998; Meng et al., 2000) and syndecan-3 (Raulo et al.,
1994) have been postulated to function as receptors for
pleiotrophin. The UB, WD, or any combination thereof (e.g., UB and
WD) has been shown to express syndecan-1 (Vainio et al., 1989), and
pleiotrophin is capable of binding to syndecan-1 (Mitsiadis et al.,
1995).
[0062] The involvement of proteoglycans in pleiotrophin-mediated
branching morphogenesis of stem cells, UBs, WDs, or any combination
thereof (e.g., UBs and WDs) is particularly interesting in light of
data demonstrating the importance of proteoglycans in UB and WD
development (Bullock et al., 1998; Davies et al., 1995; Kispert et
al., 1996). In these studies, chemical or genetic depletion of
sulfated proteoglycans inhibits branching morphogenesis, and this
is accompanied by decreased GDNF expression, and loss of c-ret at
the UB, WD, or UB and WD tips (Bullock et al., 1998; Kispert et
al., 1996). Even at early time points, when c-ret expression is
still preserved, addition of exogenous GDNF alone does not
completely restore UB, WD, or UB and WD branching morphogenesis
(Sainio et al., 1997). One possibility is that depletion of
sulfated proteoglycans also affects pleiotrophin-mediated signaling
or binding. Accordingly, the disclosure provides that pleiotrophin
functions as a MM-derived morphogen acting upon the stem cell, UB,
WD, or any combination thereof (e.g., UB and WD). Moreover, the
results support the idea that stem cell, UB, WD, or any combination
thereof (e.g., UB and WD) branching morphogenesis can be induced by
more than a single factor. At least two soluble factors, GDNF and
pleiotrophin can be used to induce morphogenetic changes. Other
heparin-binding agents including heregulin can also be used in the
methods of the disclosure as a substitute for pleiotrophin, for
example. GDNF can initiate the stem cell, UB, WD, or any
combination thereof (e.g., UB and WD) outgrowth, and pleiotrophin
(or other heparin binding agent such as heregulin) can induce
proliferation and/or facilitate branching (see Table 2). In
addition, the disclosure provides a combination of factors such as
pleiotrophin and GDNF and may include FGF. FGF and related members
play a facilitory role, since FGF1 potentiates the effects of
pleiotrophin on stem cells, UB, WD, or any combination thereof
(e.g., UB and WD) on branching morphogenesis, though by itself
(with GDNF present) exerts little if any morphogenetic
activity.
[0063] Inhibitory factors can also play a role in morphogenesis
regulation and can include members of the transforming growth
factor-beta family (Sakurai and Nigam, 1997). For example,
gradients of positive and negative factors, most of which are
matrix-bound, may exist in the mesenchyme and stroma. By regulating
proliferation, apoptosis and the expression of morphogenetic
molecules at branch tips, branch points and stalks, the global and
local balance of these stimulatory and inhibitory factors provide
determinants of branching patterns during collecting system
development. In addition, sulfated proteoglycans should also be
present either to maintain expression of these soluble factors or
to secure their binding sites. At later stages, other soluble
factors such as HGF and/or EGF receptor ligands may play
supplementary roles, either during branching (particularly in the
later stages) or shaping/maturation of tubular structures.
[0064] It should also be noted that the concentration-dependent
morphogenetic changes induced by pleiotrophin in the stem cell, UB,
WD, or any combination thereof (e.g., UB and WD), provides evidence
that pleiotrophin represents a "classical morphogen," in the sense
of activin in early Xenopus development (Green and Smith, 1990).
Such a molecule is expected to produce different phenotypic changes
in the responding tissue depending upon the concentration of the
molecule to which it is exposed. In this regard, the basement
membrane of the developing stem cell, UB, WD, or any combination
thereof (e.g., UB and WD) to which pleiotrophin is localized, can
act as a "reservoir." Release of pleiotrophin from the basement
membrane at the UB, WD, or UB and WD tips, perhaps through
digestion by matrix degrading proteases, can produce a local
concentration gradient, resulting in increased growth and
proliferation of tips, while lower amounts of pleiotrophin along
the length of the stalk would induce elongation of the forming
tubule. Such a concentration gradient of pleiotrophin provides a
basis for modulating the shape and directionality of the developing
stem cell, UB, WD, or any combination thereof (e.g., UB and
WD).
[0065] Populations of UB, WD or UB and WD cells developed by the
methods and compositions of the disclosure can be culture in
biocompatible matrices or gels used in tissue engineering.
Similarly, metanephric mesenchyme cells can be cultured in
biocompatible matrices or gels. Furthermore, co-culture of MM and
UB, WD or UB and WD cells can be performed in biocompatible
matrices or gels. The biocompatible matrix or gel may be designed
to promote branching (e.g., by photolithography techniques,
printing techniques and the like; see, e.g., Nelson et al., Science
314, 298 (2006), incorporated herein by reference)
[0066] Branching Morphogenesis in Organotypic Cultures
[0067] Alternatively, the stem cells or UB, WD, or MM progenitor
cells of the disclosure may be seeded onto or into a
three-dimensional framework or scaffold alone (e.g., as a
homogenous population) or in combination (e.g., a heterogeneous
population) and cultured to, allow the cells to grow and fill the
matrix or immediately implanted in vivo, where the seeded cells
will proliferate. Such a framework can be implanted in combination
with any one or more growth factors, drugs, additional cell types,
or other components that stimulate tissue (e.g., kidney tissue)
formation or otherwise enhance or improve the practice of the
disclosure.
[0068] The cell compositions of the disclosure can be used to
produce new kidney tissue in vitro, which can then be implanted,
transplanted or otherwise inserted to replace or augment a
subject's tissue wherein the kidney tissue becomes vascularized. In
a non-limiting embodiment, the stem cells of the disclosure are
used to produce a three-dimensional kidney tissue construct in
vitro by combining the UB, WD or UB and WD cells with MM cells
derived from the stem cells, which are then implanted in vivo.
[0069] A biocompatible matrix or gel may be of any material and/or
shape that allows cells to attach to it (or can be modified to
allow cells to attach to it) and allows cells to grow in more than
one layer. A number of different materials may be used to form the
matrix, including but not limited to: nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
compounds (e.g., polyvinylchloride), polycarbonate (PVC),
polytetrafluorethylene (PTFE, teflon), thermanox (TPX),
nitrocellulose, cotton, polyglycolic acid (PGA), collagen (in the
form of sponges, braids, or woven threads, and the like), cat gut
sutures, cellulose, gelatin, or other naturally occurring
biodegradable materials or synthetic materials, including, for
example, a variety of polyhydroxyalkanoates. Any of these materials
may be woven into a mesh, for example, to form the
three-dimensional framework or scaffold. The pores or spaces in the
matrix can be adjusted by one of skill in the art to allow or
prevent migration of cells into or through the matrix material.
[0070] The three-dimensional framework, matrix, hydrogel, and the
like, can be molded into a form suitable for the tissue to be
replaced or repaired. For example, various techniques are known
wherein a biocompatible matrix can be molded to form tubes,
channels, islands, wells, and various shapes.
[0071] The stem cells, their progeny, and generated tissue of the
disclosure can be used in a variety of applications. These include,
but are not limited to, transplantation or implantation of the
cells either in unattached form or as attached, for example, to a
three-dimensional framework or gel, as described herein.
[0072] The cells or tissue developed according to the disclosure
can be administered prior to, concurrently with, or following
injection of the angiogenic factor. In addition, the cells of the
invention may be administered immediately adjacent to, at the same
site, or remotely from the site of administration of the angiogenic
factor. By angiogenic factor is meant a growth factor, protein or
agent that promotes or induces angiogenesis in a subject.
[0073] In another aspect of the disclosure, artificial matrices
comprising biocompatible material may be used as a support for cell
growth. Such matrices may be designed such that concentrations of
pleiotrophin may change at desired branch points within the matrix
material. In this manner, kidney cells may grow and proliferate
through the matrix and branch at locations where pleiotrophin
concentrations are at a level to induce branching
morphogenesis.
[0074] In another embodiment, the disclosure provides clonal
subcolonies of specifically engineered, functional kidneys that are
suitable for use in screening of drugs and agents to measure
effects on specific kidney functions as well as for use in
transplantation. Using the compositions and methods of the
disclosure, it is possible to culture kidney components derived
from a single stem cell, UB, WD, or any combination thereof (e.g.
UB and WD) in order to develop a kidney tissue in vitro. Normal
kidney development comprises reciprocal interaction between the
stem cell, UB, WD, or any combination thereof (e.g. UB and WD) and
the metanephric mesenchyme (MM) as described herein. The methods of
the disclosure provide the ability to reduce the amount of tissue
that must be sacrificed from cadaver tissue or through invasive
biopsy techniques in order to obtain sufficient tissue for in vitro
generation of kidney tissue for screening and transplantation. The
disclosure provides methods and compositions whereby a single
progenitor cell or tissue is capable of generating multiple kidney
tissues in vitro.
[0075] Using the methods of the disclosure and compositions of the
disclosure it is possible to stimulate UB, WD, or UB and WD
morphogenesis and MM epithelialization. The methods of and
compositions provide for kidney development through co-culturing of
MM and stem cells, UBs, WDs, or UBs and WDs in culture systems.
[0076] Normal kidney development is initiated when the metanephric
mesenchyme (MM) induces an epithelial outgrowth of the Wolffian
duct, termed the ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct bud (UB, WD, or UB and WD). The disclosure provides
methods of developing an in vitro kidney tissue comprising
contacting a stem cell, WD, or UB with factors to induce branching
morphogenesis and culturing the cells on a biocompatible matrix
comprising collagen IV (e.g., Matrigel.TM.). The cells can then be
co-cultured with MM. The MM further induces the UB, WD, or UB and
WD to elongate and branch, and through multiple iterations of this
branching program, the UB, WD, or UB and WD subsequently develops
into the renal collecting system. In turn, the branching stem
cells, UB, WD, or UB and WD initiates the reciprocal induction of
the MM and stimulates it to epithelialize and to form the tubular
nephron. These nephrons then connect with the UB, WD, or UB and WD
derived collecting system allowing drainage of fluid (e.g., urine)
into the bladder in vivo. This process is repeated through
successive iterations to achieve the approximately 1 million
nephrons present in the adult human kidney. The disclosure also
provides for in vivo vascularization of the artificial kidney
tissue. For example, once the in vitro generated tissue is
developed, implantation into the kidney of a subject results in
vascularization of the tissue.
[0077] As described herein, the disclosure demonstrates that
isolated stem cells, UBs, WDs, or any combination thereof undergoes
branching morphogenesis in vitro when exposed to several growth
factors including pleiotrophin (PTN) alone or in combination with
other factors including glial cell-derived neurotrophic factor
(GDNF), fibroblast growth factor-1 or -7 (FGF1, FGF7) and proteins
secreted by a mesenchymally derived cell line or any combination
thereof. In addition, the disclosure provides methods for
regulating processes that govern stem cell, UB, and WD branching
morphogenesis, such as the matrix-binding requirements vis-a-vis
integrin expression, the dependence of branching morphogenesis on
heparin sulfate proteoglycans, and the roles of positive and
negative modulators of branching. Other growth factors present in
media conditioned by ureteric bud, Wolffian duct bud, or ureteric
and Wolffian duct bud cells that can induce differentiation of
isolated mesenchyme cultured in vitro include, for example,
leukemia-inhibitory factor (LIF) and FGF2.
[0078] Subcultures of each of the components of the kidney--the
ureteric bud, Wolffian duct bud, or ureteric and Wolffian duct bud
and the mesenchyme allow for "staged" development of an artificial
kidney tissue. Using the methods and compositions of the disclosure
the isolated UB, WD, or UB and WD and mesenchyme can be recombined
in vitro and grown in an autonomous fashion. The resultant kidney
is morphologically and architecturally indistinguishable from a
"normal" kidney and can be used for transplantation, as a source
for the study of kidney function, and as a resource for determining
drug-effects upon kidney function. Furthermore, the disclosure
provides methods for partitioning/propagating the kidney or the
cultured isolated ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct bud into smaller fragments and support the in vitro
development of these subfractions through several "generations."
The methods of the disclosure further allow for these subfractions
to be recombined with fresh mesenchyme to develop additional kidney
tissue through the induction of the mesenchyme. Furthermore, these
nascent nephrons formed contiguous connections with limbs of the
branched UB, WD, or UB and WD. Consequently, the disclosure
provides in vitro engineered kidney tissue comprising a population
of renal primordia suitable for transplantation and derived from a
single progenitor.
[0079] The methods provided by the disclosure utilize an in vitro,
approach to renal engineering that provides an ability to create
colonies of kidney tissue (in some cases comprising genetically
engineered cells) suitable for transplantation. In one aspect of
the disclosure, a stem cell population, an embryonic ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud is obtained or
separated from the surrounding metanephric mesenchyme and each
component (e.g., the MM and UB, WD, or any combination thereof) is
cultured in isolation. The stem cell, UB, WD, or combination
thereof and/or the MM can modified in vitro (as described herein)
in a tailored fashion to express a specific polynucleotide (e.g., a
heterologous polynucleotide) or reduce expression of a specific
polynucleotide to obtain a desired function (e.g., to reduce
expression of immunogenic proteins). The components are then
recombined to allow the morphogenesis and development of kidney
tissue in vitro (e.g., to generate an in vitro engineered kidney).
The in vitro engineered kidney can then be used in transplantation,
to screen for desired biological function, and/or to screen for
agents, which modulate kidney function.
[0080] For example, stem cell, embryonic UB, WD, or UB and WD are
dissected and separated from the surrounding tissue or metanephric
mesenchyme (MM). The dissected cells are then used to grow an
arborized structure, which can be subdivided into smaller fractions
and used to induce additional generations of UBs, WDs, or UBs and
WDs that grow and branch in vitro. The continued growth and
branching is maintained in the culture by culturing and
subculturing the stem cells, UBs, WDs, or UBs and WDs in the
presence MMs in a culture medium comprising pleiotrophin (e.g. PTN
and GDNF or PGN, GNDF and an FGF). The subfraction of UBs, WDs, or
UBs and WDs can then be used through multiple generations to renew
kidney tissue development. For example, UB, WD, or UB and WD
generations can be dissected and recombined with freshly isolated
metanephric mesenchyme. The cells retained the ability to induce
dramatic tubular epithelial differentiation of the mesenchyme.
Furthermore, there appeared to be connections between induced
tubules of the mesenchyme and terminal portions of the UB, WD, or
UB and WD thereby providing a conduit between the tubule and
urinary collecting system. The generated kidney opens up the
possibility of uniquely tailoring specific components of either the
nephron (derived from the mesenchyme) or tie collecting system
(derived from the UB, WD, or UB and WD) in vitro in a potentially
functional and transplantable organ.
[0081] The source of cells used to ultimately engineer kidney
tissues need not be derived from the kidney per se (see, e.g., Kim
and Dressler, J Am Soc Nephrol 16: 3527-3534, 2005; incorporated
herein by reference). Pluripotent embryonic stem (ES) cells and
pluripotent embryonic germ (EG) cells can serve as progenitor cells
for a variety of differentiated cell types and recent work with
human ES and EG cells has opened the doors to some potential
beneficial therapeutics. When cultivated in vitro, human ES and EG
cells form 3-dimensional aggregates called embryoid bodies (EB)
that can then differentiate into derivatives of all three primary
germ cell layers. Furthermore, these EB can be induced to
differentiate into specific but different cellular components such
as UBs, WDs, or UBs and WD based on conditioning by certain growth
factors, such as FGF and TGF-beta. Cells derived from ES and EG
cells can organize and can display a diverse set of functional
properties. Finally, multipotent adult bone marrow-derived
mesenchymal stem cells (MSC) may serve as an adult source of stem
cells readily available for engineering of tissues derived from
mesenchyme. Within the context of the kidney, cells derived from
the bone marrow were found to repopulate or regenerate a variety of
renal territories, including the glomerular podocyte and mesangium,
interstitium, and renal epithelial tubule. Recent work suggests
that there may exist one or more self-renewing "renal stem cells"
found within the MM that can differentiate into the myofibroblasts
of the renal stroma and/or endothelium. In addition, renal tubular
progenitor cells can be obtained using the techniques as described
by Maeshima et al., J Am Soc Nephrol 17: 188-198, 2006
(incorporated herein by reference).
[0082] As discussed herein, the disclosure provide methods and
compositions whereby isolated UBs, WDs, or UBs and WDs can be
co-cultured and stimulated by extrinsic factors to induce branching
and kidney development. For example, whole isolated intact UB, WD,
or UB and WD (cleanly separated from surrounding MM) can be induced
to undergo branching morphogenesis in vitro in a manner similar to
UB, WD, or UB and WD culture. Suspension of the isolated UBs, WDs,
or UBs and WDs within, or on, a natural or artificial biocompatible
substrate (e.g., Matrigel.TM./collagen gel) and when exposed to a
mixture of mesenchyme-cultured media augmented with GDNF, results
in the isolated unbranched UB, WD, or UB and WD rapidly forming a
polarized, extensively branched structure with an internal lumen.
As described further herein, pleiotrophin, which induced branching
of stem cells, UB, WD, or any combination thereof, also induces
branching morphogenesis of the whole ureteric bud, Wolffian duct
bud, or ureteric and Wolffian duct bud. This modulation is
typically branch-promoting, elongation promoting, or
branch-inhibiting. For example, FGF1 induced the formation of
elongated stem cells, UB, WD, or any combination thereof branching
stalks whereas FGF7 induced amorphous buds displaying nonselective
proliferation with little distinction between stalks and ampullae.
TGF-beta, which inhibits branching in several cell-culture model
systems, also appears to inhibit the branching of the isolated stem
cells, UB, WD, or any combination thereof. Endostatin, which is a
cleavage product of collagen XVIII normally found in the UB, WD, or
UB and WD basement membrane, also selectively inhibits branching of
the UB, WD, or UB and WD. Growth factors, such as LIF, have been
isolated from UB, WD, or UB and WD conditioned media and induce
mesenchymal-to-epithelial transformation of cultured mesenchyme.
Other factors, such as FGF2, appear to promote survival but not
differentiation of mesenchyme.
[0083] The branching isolated ureteric bud, Wolffian duct bud, or
ureteric and Wolffian duct bud retains the ability to induce
freshly isolated mesenchyme when recombined in vitro without
exogenous growth factors. By removing the surrounding biocompatible
matrix from the cultured UB, WD, or UB and WD and placing
mesenchyme in close proximity, the UB, WD, or UB and WD continues
to grow and extend branches into the surrounding mesenchyme.
Furthermore, the mesenchyme condenses in areas where the UB, WD, or
UB and WD has extended branches, and then epithelializes in a
manner similar to normal kidney development. This has wide-ranging
implications for in vitro kidney engineering, including the ability
to independently culture ureteric bud, Wolffian duct bud, or
ureteric and Wolffian duct bud and metanephric mesenchyme, modify
their phenotypes in vitro, and then recombine them. For example, it
may be possible to develop engineer kidneys with properties such as
enhanced drug or toxin secretion by in vitro modification of
organic anion transporters, improved immune tolerance by
suppression of costimulatory molecules, as discussed herein. The
disclosure demonstrates that these recombined "in vitro engineered
kidneys," comprised of cultured isolated UB, WD, or UB and WD and
freshly isolated mesenchyme, form cohesive intact tubular conduits.
That is, the nascent tubular nephron, derived from MM, has a
tubular lumen in direct connection with the tubular lumen of the
collecting system, derived from the UB, WD, or UB and WD.
[0084] The culture system and methods of the disclosure provide the
ability to propagate the isolated UB, WD, or UB and WD in vitro
through several generations. For example, isolated stem cells, UB,
WD, or any combination thereof are cultured in vitro and induced to
undergo branching morphogenesis in the presence of BSN-CM or
pleiotrophin and GDNF or pleiotrophin, FGF1, and GDNF. After 8
days, the cultured bud is subdivided into approximate 3rds and
resuspended within a suitable biocompatible matrix
(Matrigel.TM./collagen gel). This 2nd generation bud is further
subdivided after another 8 days of culture, and the 3rd generation
bud is cultured for 8 days (thus yielding at least 9 subdivided
buds from one progenitor bud). These subsequent clonal generations
of cultured UB, WD, or UB and WD retain the ability of the
progenitor bud to induce mesenchyme upon in vitro recombination.
The buds also retained the capacity to form cohesive conduits with
the mesenchymally-derived tubules that they induced. Thus, the
disclosure provides the ability to develop and propagate a clonal,
expanded, and long-lived colony of ureteric bud, Wolffian duct bud,
or ureteric and Wolffian duct buds, derived from a single
progenitor bud that retains the properties of the progenitor. Using
similar techniques with the MM, it is possible to develop colonies
of mesenchyme derived from a single progenitor mesenchyme that can
then be recombined with a propagated UB, WD, or UB and WD.
[0085] Whole embryonic kidneys can be propagated in a similar
manner in vitro. After culturing these kidneys for 3 days, it is
possible, using the methods of the disclosure, to subdivide into
approximate 3rds the whole cultured kidney and then propagate the
subsequent generations in vitro. At least 3 generations, yielding 9
kidneys, were generated from a single progenitor kidney using the
methods and compositions of the disclosure. Thus, the methods and
compositions of the disclosure provide the ability for expansion of
syngenic rudiments in vitro prior to transplantation into suitable
hosts.
[0086] In many tissue-engineering technologies, an extrinsic
biocompatible scaffold is required to provide orientation and
support to the developing tissue. In one sense, the native
polymeric basement membrane (BM) is a bioactive scaffold directing
the normal development of the kidney. BM constituents such as
endostatin can directly influence branching of the UB, WD, or UB
and WD, and other components, such as HSPGs, can indirectly
regulate growth by binding and releasing growth factors. The
bioartificial scaffolds used in tissue engineering can be synthetic
or biologic and contain or can be coated with ECM constituents,
such as collagen or proteoglycans. Exciting new techniques in
materials science are emerging that allow these scaffolds to be
impregnated with drugs, proteins, or even DNA, and thus may be more
biologically relevant. By combining a truly bioactive scaffold with
cultured pluripotent cells, such as ES cells, or multipotent cells,
such as MSCs or other progenitor cells derived from the mesenchyme,
it may be possible to coordinate inductive signals required to
derive/engineer an organ such as the kidney. For example, by
varying the concentration of factors at points where branching is
desired, it is possible to design a tissue having a predicted
number of branch point.
[0087] Biocompatible support materials (biocompatible scaffolds)
for culturing kidney cells include any material and/or shape that:
(a) allows cells to attach to it (or can be modified to allow cells
to attach to it); and (b) allows cells to grow in more than one
layer. A number of different materials may be used as a culture
support, including, but not limited to, nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
compounds (e.g., polyvinylchloride), polycarbonate (PVC),
polytetrafluorethylene (PTFE; teflon), thermanox (TPX),
nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures,
cellulose, gelatin, dextran, collagen, decellulularized tissue
(both allogenic and xenogeneic), and like.
[0088] In one aspect of the disclosure a ureteric bud, Wolffian
duct bud, or ureteric and Wolffian duct bud is used as a bioactive
scaffold, which could then serve as a biologically-relevant
orchestrator (conductor) of the complex inductive signals that
underlie "normal" renal development. In an organ such as the
kidney, where development is dependent on coordinated interactions
between epithelium and mesenchyme, utilization of a biologically
active epithelial scaffold to induce proper differentiation,
maturation and integration of surrounding multipotent cells may
provide a unique opportunity to modify specific cellular functions
in vitro but yet to retain the complex organizational direction
required to develop a mature kidney. This principle is applicable
to engineering of other organs, such as lung, liver, pancreas,
salivary gland or breast, which are also dependent upon
mesenchymal-epithelial interactions within the context of a
branching epithelial derivative. In one experiment, the stem cells,
UB, WD, or any combination thereof, are co-cultured with lung
mesenchyme, can begin to express surfactant protein. Accordingly,
the methods and compositions serve as a scaffold for a number of
novel "chimeric" organs. The ability to independently culture and
then combine mesenchymally-derived elements with epithelial-derived
elements allow for the integration of cellular and organ-based
approaches to tissue engineering. This approach would allow one to
modify cell-based elements in vitro to possess certain desirable
properties but still take advantage of an organ-based approach to
tissue engineering.
[0089] By culturing kidney tissue derived from stem cells, UB, WD,
or UB and WD and MM in vitro, the disclosure provides the unique
opportunity to modulate each of their component functions in a
site-specific manner. For example, transfection of the mesenchyme
with constructs expressing organic ion transporters would lead to
increased capability to handle drugs and toxins, insertion of genes
coding for growth factors, such as insulin-like growth factor
(IGF), would lead to markedly enhanced in vitro engineered kidney
development and improved functionality, insertion of
immunomodulatory elements, such as repressors of co-stimulatory
molecules, could be used to improved immune tolerance; stimulation
of branching in the UB, WD, or UB and WD can lead to an increased
number of resultant nephrons and improved renal functionality.
Thus, there are numerous of ways to design an in vitro engineered
kidney with tailored function. Furthermore, by subcloning UBs, WDs,
or UBs and WDs, the disclosure provides the potential to develop a
large number of kidneys derived from a single progenitor, thus
removing concerns surrounding limited supply of transplantable
tissue. Third, it is possible to create a chimeric kidney using the
UB, WD, or UB and WD as a scaffold and recombining the UB, WD, or
UB and WD with heterologous mesenchymal cells. These mesenchymal
cells could be derived from embryonic stem cells that, when exposed
to kidney derived signals from the UB, WD, or UB and WD induce
differentiation of the renal mesenchymal cells into epithelial
tissues. In normal adults, stem cells originating in the bone
marrow repopulate portions of the kidney and differentiate into
renal cells, and it is likely that embryonic stem cells also posses
this ability.
[0090] The approach provided by the methods and compositions of the
disclosure, whereby in vitro engineered kidneys developed and/or
are designed to possess specific functions, such as improved immune
tolerance or enhanced tubular secretion of substrate, offer
original approaches to transplantation and kidney therapy.
Furthermore, creating clonal populations of in vitro engineered
kidneys creates the potential for development of organ propagation
from a single tissue. This approach is potentially applicable to
other epithelial tissues such as lung and pancreas.
[0091] The methods provided by the disclosure allow for the
development of colonies of subcloned in vitro engineered kidneys
that have been specifically tailored to express certain functions,
and are immune-naive, particularly where the tissue is derived from
stem cells. Immune naive means that the cells lack "self"
identification as the cells were fetally or stem cell derived and
therefore should be immune tolerant.
[0092] Methods of transfecting and transforming cells are known in
the art. For example, methods of transfecting/transforming kidney
cell are known and include the following methods. Tomita et al.
(Biochem. and Biophys. Res. Comm. 186:129-134, 1992) report a
method for in vivo gene transfer into the rat kidney. They utilize
HVJ (Sendai virus) and liposome methodology. In this protocol,
plasmid DNA and a nuclear protein are coencapsulated in liposomes
and later cointroduced into cells. The reporter gene utilized in
these studies was the SV40 large T antigen. The gene transfer can
be performed by inserting a cell or culture of kidney tissue with a
liposome suspension. Transfection/transformation of the kidney
cells can be assay by detecting SV40 large T antigen
immunohistochemically. A study by Zhu et al. (Science 261:209-11,
1993), reports the use of a particular cationic liposome DNA
mixture to deliver genes with high efficiency into a vast number of
endothelial cells in a rat. Moullier et al. (Kidney International
45:1220-1225, 1994) provides a first report of an
adenoviral-mediated gene transfer into a kidney.
[0093] As used herein, the term "transfect" or "transform" refers
to the transfer of genetic material (e.g., DNA or RNA) of interest
via a vector into cells of a mammalian organ or tissue (e.g.,
kidney/renal tissue). The vector will typically be designed to
infect mammalian kidney cell. The genetic material of interest
encodes a product (e.g., a protein polypeptide, peptide or
functional RNA) whose production by kidney cells is desired. For
example, the genetic material of interest can encode a hormone,
receptor, enzyme or (poly)peptide of therapeutic value. Examples of
genetic material of interest include but are not limited to DNA
encoding cytokines, growth factors and other molecules which
function extracellularly such as chimeric toxins, e.g., a growth
factor such as interleukin-2 (IL-2) fused to a toxin, e.g., the
pseudomonas exotoxin, dominant negative receptors (soluble or
transmembrane forms), truncated cell adhesion or cell surface
molecules with or without fusions to immunoglobulin domains to
increase their half-life (e.g., CTLA4-Ig). For example, cells of an
organ or a tissue do not express a gene product encoded by the
genetic material prior to transfection or transformation.
Alternatively, infection of the cells of an organ or a tissue may
result in an increased production of a gene product already
expressed by those cells or result in production of a gene product
(e.g., an antisense RNA molecule) which decreases production of
another, undesirable gene product normally expressed by the cells
of that organ or tissue. Generally, the genetic material encodes a
gene product, which is the desired gene product to be supplied to
the cells of that organ or tissue. Alternatively, the genetic
material encodes a gene product, which induces the expression of
the desired gene product by the cells of that organ or tissue
(e.g., introduced genetic material encodes a transcription factor
which induces the transcription of the gene product to be supplied
to the subject). Furthermore, the genetic material could simply
contain a polynucleotide, e.g., in the form of single stranded DNA
to act as an antisense nucleotide. A genetic material infected into
a cell of an organ or a tissue via a vector is in a form suitable
for expression in the cell of the gene product encoded by that
genetic material. Accordingly, the genetic material includes coding
and regulatory sequences required for transcription of a gene (or
portion thereof) and, when the gene product is a protein or
peptide, translation of the gene product encoded by the genetic
material. Regulatory sequences which can be included in the genetic
material include promoters, enhancers and polyadenylation signals,
as well as sequences necessary for transport of an encoded protein
or peptide, for example N-terminal signal sequences for transport
of proteins or peptides to the surface of the cell or for
secretion, or for cell surface expression or secretion
preferentially to the luminal or basal side. Enhancers might be
ubiquitous or tissue or cell specific or inducible by factors in
the local environment, e.g., inflammatory cytokines.
[0094] As used herein, the term an "effective amount" refers to a
level of expression of a heterologous polynucleotide transfected or
transformed into a kidney cell, which brings about at least
partially a desired therapeutic or prophylactic effect in an organ
or tissue infected by the method of the invention. For example,
expression of genetic material of interest can then result in the
modification of the cellular activities, e.g., a change in
phenotype, in an organ or a tissue that has been infected by the
method of the disclosure. In one embodiment, an effective amount of
the expression of a heterologous genetic material of interest
results in modulation of cellular activity in a significant number
of cells of an organ transfected or transformed with the
heterologous polynucleotide. A "significant number" refers to the
ability of the vector to infect at least about 0.1% to at least
about 15% of the renal endothelial cells or UBs, WDs, or UBs and
WDs. Typically, at least about 5% to at least about 15% of the
cells are transfected/transformed. Most commonly, at least about
10% of the cells are transfected/transformed.
[0095] A vector refers to a polynucleotide molecule capable of
transporting another nucleic acid to which it has been linked into
cells. Examples of vectors that exist in the art include: plasmids,
yeast artificial chromosomes (YACs) and viral vectors. However, the
invention is intended to include such other forms of vectors which
serve equivalent functions and which become known in the art
subsequently hereto.
[0096] The efficacy of a particular expression vector system and
method of introducing genetic material into a cell can be assessed
by standard approaches routinely used in the art. For example, DNA
introduced into a cell can be detected by a filter hybridization
technique (e.g., Southern blotting) and RNA produced by
transcription of introduced DNA can be detected, for example, by
Northern blotting, RNase protection or reverse transcriptase
polymerase chain reaction (RT-PCR). The gene product can be
detected by an appropriate assay, for example by immunological
detection of a produced protein, such as with a specific antibody,
or by a functional assay to detect a functional activity of the
gene product, such as an enzymatic assay. If the gene product of
interest to be expressed by a cell is not readily assayable, an
expression system can first be optimized using a reporter gene
linked to the regulatory elements and vector to be used. The
reporter gene encodes a gene product, which is easily detectable
and, thus, can be used to evaluate the efficacy of the system.
Standard reporter genes used in the art include genes encoding
.beta.-galactosidase, chloramphenicol acetyl transferase,
luciferase and human growth hormone.
[0097] The method of the disclosure can be used to infect kidney
cells to obtain designer kidneys (e.g., genetically engineered
kidney cells). As used herein, the term "kidney cells" is intended
to including UB, WD, or UB and WD and MM cell types as well as the
other 15 different cell types, e.g., glomerular cells, mesangial
cells, interstitial cells, tubular cell, endothelial cells, are
intended to be encompassed by the term "kidney cells".
[0098] The method of the disclosure can also be used to infect a
kidney tissue generated ex vivo. For example, in a transplant
setting, a kidney is engineered by the methods of the disclosure,
the "in vitro engineered kidney" is then perfused (e.g., the
collecting ducts) with a vector carrying genetic material of
interest.
[0099] One potential application of the disclosure is in renal
allograft or xenograft tissue transplantation. In this aspect,
kidney tissue generated by the methods and compositions of the
disclosure are transfected/transformed with an agent (e.g.,
delivered to MM cells and/or UBs, WDs, or UBs and WDs) that results
in organ tolerance or might help in the post-operative period for
decreasing the incidence of early transplant rejection or function
(e.g., due to acute tubular necrosis). Either the organ can be made
less immunogenic so as to reduce the number of host T cells
generated and/or the endothelial cells (e.g., endothelial cells
derived from MM) can be altered so as to prevent the
adhesion/transmigration of primed immune T-cells or killer effector
T-cells (e.g., by use of IL-2-toxin fusion proteins). Moreover,
genes transfected/transformed into in vitro engineered kidney
tissue, such as nitric oxide synthetase (NOS), prior to
transplantation could also serve to protect the organ post
transplantation. These strategies make clinical sense since it is
well known that early rejection episodes and malfunction lead to a
worse long-term graft survival. Therefore, prevention of acute
rejection and preservation of function immediately post transplant
are of particular importance. Delivery of the genetic material
(i.e., the heterologous polynucleotides) for this purpose can be
done using methods known in the art including utilizing an
adenovirus vector, lipofection, or other techniques known in the
art. In addition to the heterologous polynucleotides mentioned
above, these vectors can carry additional sequences comprising
anti-sense constructs to one or more cell adhesion molecules
(involved in lymphocyte homing) or dominant negative constructs to
these molecules, or antisense constructs to MHC antigens in the
transplant or locally immune suppressive lymphokines such as
interleukin-10 (IL-10) or viral IL-10 or chimeric toxins which
would preferentially kill T-cells, e.g., IL-2 toxin fusion protein.
It is also possible that one could interfere with the recognition
part of the immune system by, for example, the local secretion of
CTLA4-IgG fusion proteins. This list of candidate polynucleotides
is not exhaustive. Those skilled in the art of transplantation know
of others. The genes could be delivered with constitutive promoters
or with appropriate inducible enhancers.
[0100] In another aspect of the disclosure, the kidney cultures
(UB, WD, or UB and WD alone, MM alone, and co-cultures thereof) may
be used in vitro to screen a wide variety of compounds, such as
cytotoxic compounds, growth/regulatory factors, pharmaceutical
agents, and the like to identify agents that modify kidney function
and/or cause cytotoxicity and/or kidney cell death or modify kidney
proliferative activity. Examples of such agents or compounds
include growth factors, peptides, and small organic molecules. In
another aspect, the cells can be genetically engineered and the
kidney culture implanted in vivo, whereby screening can be measured
by detecting changes in the kidney culture using a genetically
engineered label. In this aspect, vascularization can assist in
providing information on the effect an agent has on kidney
tissue.
[0101] To this end, the cultures (e.g., stem cells, UB, WD, or UB
and WD primary cells, UB, WD, or UB and WD cell lines, MM cells,
whole organ cultures, MM/spinal cord co-cultures, and UB, WD, OR UB
AND WD/MM co-cultures) are maintained in vitro and exposed to the
compound to be tested. The activity of a cytotoxic compound can be
measured by its ability to damage or kill cells in culture or by
its ability to modify the function of kidney cells (e.g., UB, WD,
or UB and WD proliferative capacity, branching capacity, MM
epithelialization capacity, particular gene expression, cell size,
cell morphology, protein expression, and the like). This may
readily be assessed by vital staining techniques, ELISA assays,
immunohistochemistry, PCR, microarray analysis, and the like. The
effect of growth/regulatory factors on the kidney cells (e.g., UBs,
WDs, or UBs and WDs, MMs) may be assessed by analyzing the cellular
content of the culture, e.g., by total cell counts, and
differential cell counts, including the number of branch points.
This may be accomplished using standard cytological and/or
histological techniques including the use of immunocytochemical
techniques employing antibodies that define type-specific cellular
antigens. The effect of various drugs on normal cells cultured in
the culture system may be assessed. For example, UB, WD, or UB and
WD primary cells or cell lines may be cultured in vitro under
conditions that stimulate branching morphogenesis/tubulogenesis
(e.g., in the presence of BSN-CM, pleiotrophin, or
pleiotrophin+other factors). A test compound is then contacted with
the culture and the effect the test compound has on branching
morphogenesis/tubulogenesis can be compared to a control, wherein a
difference is indicative of an compound that increases or decreases
branching morphogenesis.
[0102] The cytotoxicity to kidney cells (e.g., human UBs, WDs, or
UBs and WDs and co-cultures of MM and UBs, WDs, or UBs and WDs) of
pharmaceuticals, anti-neoplastic agents, carcinogens, food
additives, and other substances may be tested by utilizing the
culture system of the invention.
[0103] First, a stable, growing kidney culture comprising UB, WD,
OR UB AND WD and/or MM cells is established. Then, the culture is
exposed to varying concentrations of a test agent. After incubation
with a test agent, the culture is examined by phase microscopy to
determine the highest tolerated dose--the concentration of test
agent at which the earliest morphological abnormalities appear.
Cytotoxicity testing can be performed using a variety of supravital
dyes to assess cell viability in the culture system, using
techniques known to those skilled in the art.
[0104] Once a testing range is established, varying concentrations
of the test agent can be examined for their effect on viability,
growth, and/or morphology of the different cell types constituting
the kidney culture by means well known to those skilled in the
art.
[0105] Similarly, the beneficial effects of drugs may be assessed
using the culture system in vitro; for example, growth factors,
hormones, drugs which enhance kidney formation, or activity (e.g.,
branching activity) can be tested. In this case, stable cultures
may be exposed to a test agent. After incubation, the cultures may
be examined for viability, growth, morphology, cell typing, and the
like as an indication of the efficacy of the test subtance. Varying
concentrations of the drug may be tested to derive a dose-response
curve.
[0106] The culture systems of the disclosure may be used as model
systems for the study of physiologic or pathologic conditions. For
example, in a specific embodiment of the invention, the culture
system can be optimized to act in a specific functional manner as
described herein by modifying genome of the cells.
[0107] The kidney culture system of the disclosure may also be used
to aid in the diagnosis and treatment of malignancies and diseases.
For example, a biopsy of a kidney tissue may be taken from a
subject suspected of having a malignancy or other disease or
disorder of the kidney. The biopsy cells can then be separated
(e.g., UBs, WDs, or UBs and WDs from MM cells etc.) and cultured in
the according to the methods of the invention. UBs, WDs, or UBs and
WDs from the subject can be co-cultured with normal (e.g.,
heterologous MM cells) to determine biological function of the UBs,
WDs, or UBs and WDs compared to UBs, WDs, or UBs and WDs derived
from a normal kidney. Similarly MM cells from the subject can be
cultured with normal UBs, WDs, or UBs and WDs to examine MM
function and activity. In addition, such cultures obtained from
biopsies can be used to screen agent that modify the activity in
order to identify a therapeutic regimen to treat the subject. For
example, the subject's culture could be used in vitro to screen
cytotoxic and/or pharmaceutical compounds in order to identify
those that are most efficacious; i.e. those that kill the malignant
or diseased cells, yet spare the normal cells. These agents could
then be used to therapeutically treat the subject.
[0108] Where in vitro engineered kidney tissue is generated
according to the methods and compositions of the disclosure
transplantation of the tissue can be performed as follows. Surgery
is performed on the recipient subject to expose one or both
kidneys. The in vitro engineered kidney tissue is implanted
directly into/adjacent to the recipient subject's kidney to result
in the formation of chimeric kidney, or into a fold of the omentum
where it forms a chimeric kidney that functions independently of
the recipient's kidney. The omentum, which is a membranous
structure that connects the bowels, is a highly vascularized tissue
sufficient for the transplantation of the in vitro engineered
kidney. The in vitro engineered kidney can be placed adjacent to
any portion of the omentum, however, in one aspect the in vitro
engineered kidney is transplanted at or near an omental fold. In
another aspect, the in vitro engineered kidney is transplanted at
an omental fold located near one of the recipient's kidneys,
particularly near the ureter, so that the developing ureter of the
metanephros can be readily connected to the recipient's excretory
system.
[0109] When implanted into the recipient's kidney, an incision,
large enough to receive the in vitro engineered kidney tissue is
made in the fibrous renal capsule that surrounds the recipient's
kidney. The location of the incision can be anywhere in a viable
portion of the recipient's kidney, but most conveniently will be at
an external border of the kidney that is easily accessible during
surgery. The in vitro engineered kidney tissue is placed between
the capsule and the cortex of the recipient kidney.
[0110] The implanted in vitro engineered kidney tissue is allowed
to grow within the recipient under conditions that allow the tissue
to vascularize. Suitable conditions may include the use of pre or
post-operative procedures to prevent rejection of the implant as
well as the administration of factors (e.g., pleotrophin, FGF1,
GNDF, and the like) that stimulate tubulogenesis and/or
morphogenesis of the in vitro engineered kidney tissue.
Immunosuppression techniques (in the absence or combined with
genetically engineered techniques) such as cyclosporin A (CSA) to
prevent rejection of the donor tissue are known in the art.
EXAMPLES
[0111] Fibroblast growth factor-1 (FGF1) and glial cell-derived
neurotrophic factor (GDNF) were obtained from R&D Systems
(Minneapolis, Minn.). Mouse anti-E-cadherin antibodies were from BD
Biosciences Pharmingen.TM. (San Diego, Calif.) and goat anti-mouse
AlexaFluor.RTM. 594 was from Molecular Probes (Eugene, Oreg.).
FITC-conjugated D. biflorus (DB) lectin and rhodamine-conjugated
PNA were from Vector Laboratories (Burlingame, Calif.). Type I and
type IV collagens, and growth factor-reduced Matrigel.TM. were from
BD Biosciences (San Jose, Calif.). Antibiotics, DMEM:F12 1:1 (v:v)
and PBS were from GIBCO-BRL (Grand Island, N.Y.). Unless otherwise
noted, all other reagents are from Sigma (St. Louis, Mo.).
[0112] Embryos from timed pregnant Holtzman rats (Harlan,
Indianapolis, Ind.) at day 13 (E13) of gestation (day 0 being the
day of appearance of the vaginal plug) or timed pregnant HoxB7-GFP
mice embryos at E12 were dissected free of surrounding tissues. The
urogenital tract was isolated and WDs were dissected free of
surrounding tissue. The mesonephric tubules and intermediate
mesoderm was carefully stripped away leaving only the epithelial
tube of the WD. Metanphric kidneys were isolated and directly used
in the kidney culture as described below or further separated in to
the UB and MM tissues.
[0113] Unless otherwise stated, the incubations were performed at
37.degree. C. in an atmosphere of 5% CO.sub.2 and 100% humidity.
For the immunodetection of pleiotrophin either on western blots or
frozen sections of E1 3 mouse kidney, a goat anti-pleiotrophin
antibody (R&D systems) was used.
[0114] Conditioned medium secreted by metanephric
mesenchyme-derived cells is required for isolated UB, WD, or UB and
WD branching morphogenesis. To identity mesenchymal factors that
induce branching morphogenesis of the ureteric bud, Wolffian duct
bud, or ureteric and Wolffian duct bud (UB, WD, or UB and WD), a
metanephric mesenchyme (MM)-derived cell line (BSN cells) was
employed as a substitute for the embryonic MM. These cells were
derived from the embryonic day 11.5 (E11.5) MM from a SV40 large
T-expressing transgenic mouse and have been extensively
characterized. BSN cells are positive for vimentin and negative for
cytokeratin, E-cadherin, and ZO-1 by immunostaining, as well as
negative for Dolichos biflorus lectin-binding. By PCR the cells
express WT1 and are negative for c-ret. The cells also express mRNA
for growth factors such as HGF and TGF.beta. by northern blot. cDNA
array analysis has confirmed their non-epithelial character. Most
importantly, conditioned medium elaborated by BSN cells (BSN-CM)
acts similar to the MM by inducing branching morphogenesis of
cultured UBs, WDs, or UBs and WDs and the isolated UB, WD, or UB
and WD (in the presence of GDNF).
[0115] UBs, WDs, or UBs and WDs isolated from E13 rat embryos, when
suspended in an extracellular matrix gel and cultured in the
presence of BSN-CM (with GDNF), grew to form impressive multiply
branching tubular structures comparable to those seen in in vivo
kidney development (though the growth was non-directional) (FIG.
1B). In the absence of BSN-CM, however, the UBs, WDs, or UBs and
WDs failed to develop. Thus, BSN-CM contains an additional soluble
factor(s) necessary for epithelial cell branching morphogenesis.
Using this isolated UB, WD, or UB and WD culture model as an assay,
key morphogenetic factor present in the BSN-CM were identified.
Example 1
[0116] BSN cells were grown to confluency in DMEM/F12 supplemented
with 10% fetal calf serum (FCS). The growth media was removed and
the cells were then incubated in serum-free DMEM/F12 for 3-4 days
followed by collection of the conditioned medium. Swiss ST3 cells
(ATCC) were grown to confluence in DMEM with 10% FCS. Once the
cells were confluent, the growth media was replaced with DMEM
supplemented with 2% FCS and the cells were cultured for an
additional 3-4 days. The conditioned medium was collected and used
for the experiments. UBs, WDs, or UBs and WDs were cultured in DMEM
supplemented, with 10% FCS at 32.degree. C. in an atmosphere of 5%
CO.sub.2 and 100% humidity.
Example 2
[0117] Timed pregnant female Sprague-Dawley rats at day 13 of
gestation (day 0 coincided with appearance of the vaginal plug)
were sacrificed and the uteri were removed. The embryos were
dissected free of surrounding tissues and the kidneys were
isolated. For the culture of the whole kidney rudiment, 2-3 kidneys
were applied directly to the top of a polyester Transwell filter
(0.4 .mu.m pore size; Corning-Costar). The Transwells were then
placed within individual wells of a 24-well tissue culture dish
containing 400 .mu.l DMEM/F12 supplemented with 10% FCS with or
without purified pleiotrophin. Following 7 days of culture, the
kidneys were fixed in 2% paraformaldehyde and doublestained with
fluorescein-conjugated Dolichos biflorus, a lectin which binds
specifically to UB, WD, or UB and WD-derived structures, and
rhodamine conjugated peanut agglutinin, a lectin which binds to
structures derived from the MM. Fluorescent staining was detected
using a laser-scanning confocal microscope (Zeiss).
[0118] In the case of culture of the isolated UB, WD, or UB and WD,
the isolated kidneys were trypsinized for 15 min at 37.degree. C.
in L-15 media containing 2 .mu.g/ml trypsin (Sigma). Trypsin
digestion was arrested by the addition of 10% FCS and the kidneys
were removed to fresh L-15 where the UBs, WDs, or UBs and WDs were
isolated from surrounding MM by mechanical dissection. isolated
UBs, WDs, or UBs and WDs were suspended within an extracellular
matrix gel [1:1 mixture of growth factor reduced Matrigel.TM. (BD)
and Type 1 collagen (BD)] applied to the top of a polyester
Transwell filter (0.4 .mu.m pore size; Corning-Costar). The
Transwells were placed within individual wells of a 24-well tissue
culture dish containing 400 .mu.l of either whole BSN-CM, purified
tractions of BSN-CM, or D-12 which were supplemented with human
recombinant FGF1 (250 ng/ml; R&D Systems), rat recombinant GDNF
(125 ng/ml; R&D Systems) and 10% FCS and cultured.
Phase-contrast photomicrographs of the developing UB, WD, or UB and
WD were taken using a RT-Slider Spot Digital Camera (Diagnostic
Instruments Inc.) attached to a Nikon Eclipse TE300 Inverted
Microscope.
Example 3
[0119] Confluent monolayers of UBs, WDs, or UBs and WDs were
removed from tissue culture dishes by light trypsinization and the
cells. 20,000 cells/ml were suspended in an extracellular matrix
gel composed of 80% Type 1 collagen and 20% growth factor-reduced
Matrigel.TM.. 100 .mu.l of the UB, WD, or UB and WD cell-containing
gel was then aliquoted into individual wells of a 96-well tissue
culture plate. After gelation, 100 .mu.l of growth medium (DMEM/F12
with or without purified pleiotrophin) supplemented with 1% FCS was
applied to each well and the cultures were incubated at 32.degree.
C. in 5% CO.sub.2 and 100% humidity. Following 4 days of culture,
the percentage of cells/colonies with processes was counted as an
indicator of the tubulogenic activity. Phase-contrast
photomicrographs were taken as described herein.
Example 4
[0120] 1.5-2 L of BSN-CM collected as described herein was filtered
to remove extraneous cellular debris using a 0.22 .mu.m
polyethersulphone membrane filter (Corning). The BSN-CM was then
concentrated 40-fold using a Vivatlow.TM. 200 concentrator with a 5
kDa molecular weight cutoff (Sartorius). After adjusting the salt
concentration to 0.4 M NaCl, the concentrated BSN-CM was then
subjected to sequential liquid column chromatography using an AKTA
purifier (Amersham-Pharmacia). Initial fractionation was performed
using a heparin sepharose chromatography column (HiTrap.TM.
heparin, 5 ml; Amersham Pharmacia). The flow-through fraction was
collected and individual 5 ml fractions of the heparin-bound
proteins were eluted via increasing concentrations of NaCl (0.4
M-2.0M) buffered to pH 7.2 with 50 mM HEPES. Aliquots of each
fraction were subjected to buffer exchange by dia-filtration using
an Ultrafree.TM. 500 spin column (Millipore) according to the
manufacturer's instructions and then tested for morphogenetic
activity using the isolated UB, WD, OR UB AND WD culture
system.
[0121] An active fraction corresponding to the 1.2-1.4 M NaCl
eluate was identified based on its ability to induce branching
morphogenesis of the isolated UB, WD, or UB and WD. After adjusting
this fraction to 1.7 M ammonium sulfate (pH 7.2) it was subjected
to further fractionation using a Resource phenyl sepharose
hydrophobic interaction column (1 ml; Amersham-Pharmacia). The flow
through was collected and 1 ml fractions of bound proteins were
eluted with decreasing concentrations of ammonium sulfate (1.7 M-0
M). After buffer exchange, the individual fractions were again
tested for their ability to induce UB, WD, or UB and WD branching
morphogenesis.
[0122] The morphogenetically active fractions from the hydrophobic
interaction column were diluted 10-fold with 50 mM HEPES and
applied to a Resource S cation exchange column (1 ml;
Amersham-Pharmacia). The flow-through was collected and individual
1 ml fractions of bound proteins were eluted--using increasing NaCl
concentrations (0 M-2.0 M) and assayed for the ability to induce
branching morphogenesis.
[0123] The active fractions from the Resource S cation exchange
column were subjected to further fractionation using a Superdex.TM.
200 gel filtration column (Amersham-Pharmacia). Individual 1 ml
fractions were collected and assayed for morphogenetic activity. In
addition, the active fractions from the Resource S cation exchange
column were subjected to SDS-PAGE and the proteins were visualized
using coumassie blue (Colloidal Coumassie; Invitrogen) staining.
Individual protein bands were cut out of the gels and submitted for
microsequencing. Sequence analysis of the protein bands was
performed at the Harvard Microchemistry Facility by microcapillary
reverse phase HPLC nanoelectrospray tandem mass spectrometry
(pLC/MS/MS) on a Finnigan LCQ DECAT.TM. quadrupole ion trap mass
spectrometer.
[0124] SDS-PAGE and silver staining of BSN-CM revealed the presence
of many protein bands. As described above, liquid column
chromatography was used to fractionate BSN-CN and each fraction was
tested for its ability to induce branching morphogenesis of the
isolated UB, WD, or UB and WD. Of the multiple columns tested, a
heparin sepharose column was found to adsorb most of the
morphogenetic activity. Within this heparin-binding fraction, the
fraction, which eluted at a NaCl concentration of 1.2-1.4 M
possessed particularly strong morphogenetic activity. Silver stain
analysis of this fraction revealed the presence of prominent lower
molecular weight (40 kDa) protein bands. This active fraction was
then applied to a Resource phenyl sepharose hydrophobic interaction
column. A morphogenetic activity was eluted from this column at 1.4
1.2 M ammonium sulfate. Again, silver staining of this peak
fraction revealed prominent low molecular weight protein bands.
This active fraction was diluted 10-fold with 50 mM HEPES (pH 7.2)
buffer and applied to a Resource S cation exchange column. Each 1
ml fraction of the Resource S eluate was substituted for whole
BSN-CM in the isolated UB, WD, or UB and WD culture and compared
with BSN-CM itself. Of the 8 fractions eluted from the column,
Fraction 4, the peak protein fraction, induced significant UB, WD,
or UB and WD morphogenesis. SDS-PAGE analysis and silver staining
of this peak fraction revealed the presence of a single protein
band with an approximate molecular weight of 18 kDa. This protein
band was subjected to in-gel digestion followed by tandem mass
spectrometry and was identified as pleiotrophin. (This type of
experiment was performed at least 3 times during different
purifications, and pleiotrophin was always detected by mass
spectrometry).
[0125] The presence of pleiotrophin in the active fraction
(fraction 4) was confirmed by immunoblot analysis using
anti-pleiotrophin antibodies. The morphogenetic activity of
individual fractions corresponded to the presence of pleiotrophin
in that fraction. In a similar fashion, further purification of the
peak fraction from Resource S column was accomplished by applying
the active fraction to a Superdex.TM. 200 gel filtration column. A
single protein peak eluted at 15.93 ml, corresponding to a protein
with a molecular weight of approximately 18 kDa, and was positive
for pleiotrophin by immunoblot. This fraction induced isolated UB,
WD, or UB and WD branching morphogenesis. Taken together, these
results identify pleiotrophin as a morphogenetic factor present in
BSN-CM.
[0126] Previous studies have found that pleiotrophin can be
isolated to homogeneity from a conditioned medium elaborated by
Swiss 3T3 cells. Thus, using this alternative purification
procedure, a pure fraction of pleiotrophin was isolated from 3T3
conditioned medium (3T3-CM), as confirmed by silver stain,
immunoblot analysis and mass spectrometry. Like the pleiotrophin
that purified from BSN cells, this pure pleiotrophin was capable of
inducing impressive branching morphogenesis of the isolated UB, WD,
or UB and WD. Thus, pleiotrophin purified from two different cell
lines gave the same results.
[0127] Nevertheless, to provide further confirmation that
pleiotrophin is the factor inducing the morphogenetic changes
observed in the isolated UB, WD, or UB and WD culture the ability
of polyA-sepharose to adsorb pleiotrophin. Treatment of purified
pleiotrophin with polyA-sepharose beads results in the loss of
detectable pleiotrophin, either by silver staining or immunoblot
analysis. Importantly, this bead depleted fraction was no longer
capable of inducing UB, WD, or UB and WD branching morphogenesis.
Insect cell-derived recombinant human pleiotrophin is incapable of
inducing proliferation and experiments using recombinant human
pleiotrophin produced in the insect cell line (R&D systems) was
also unable to induce UB, WD, or UB and WD branching
morphogenesis.
Example 5
[0128] During the course of purification, differences in the
morphology of the branching UB, WD, or UB and WD, depending upon
the amount of pleiotrophin present in the fraction (detected by
immunoblotting) was observed. This was examined more carefully
using the purified protein in which the pleiotrophin concentration
was determined by immunoblotting using recombinant human
pleiotrophin as a standard. High concentration (.gtoreq.5 .mu.g/ml)
pleiotrophin resulted in robust proliferation with less elongation,
while lower concentrations of pleiotrophin (156 ng/ml-2.5 pg/ml)
induced dichotomous branching and elongation of the stalk, similar
to that seen with whole BSN-CM.
Example 6
[0129] In the course of purification, variation in the inductive
capacity of whole BSN-CM on UB, WD, or UB and WD branching was
encountered. It was found that the addition of fibroblast growth
factor1 (FGF1) could potentiate the activity of the BSN-CM,
although alone or in combination with GDNF it was not sufficient to
induce isolated UB, WD, or UB and WD branching morphogenesis. Based
on this finding, the growth media (either BSN-CM or individual
fractions) used in the culture of the isolated UB, WD, or UB and WD
was supplemented with 250 ng/ml of FGF1. However, it was found that
purified pleiotrophin supplemented with GDNF was capable of
inducing UB, WD, or UB and WD branching morphogenesis in the
absence of FGF1, although the UB, WD, or UB and WD grew faster when
FGF1 was added to the culture.
[0130] This result suggests that pleiotrophin and GDNF alone are
necessary and sufficient for the observed branching morphogenesis
of the isolated UB, WD, or UB and WD, though a FGF-like activity
could play a role in the process.
Example 7
[0131] Pleiotrophin also induces branching morphogenesis of UBs,
WDs, or UBs and WDs in three dimensional culture. As discussed
herein, E11 S mouse UB, WD, or UB and WD derived cells (UBs, WDs,
or UBs and WDs) develop into branching tubular structures with
lumens in the presence of BSN-CM. DNA array, PCR analysis, and
immunostaining have confirmed the epithelial and UB, WD, OR UB AND
WD-like characteristics of these cells. Using this model for UB,
WD, or UB and WD branching morphogenesis, pleiotrophin was also
capable of inducing the formation of branching structures of UBs,
WDs, or UBs and WDs. As in the isolated UB, WD, or UB and WD
culture model, the extent of UB, WD, or UB and WD branching
morphogenesis was found to be concentration-dependent, with higher
concentrations resulting in more extensive growth and branching.
Morphologically, the structures were comparable to those induced by
whole BSN-CM.
Example 8
[0132] Pleiotrophin is expressed in the embryonic kidney and
secreted from MM derived cells but not UB, WD, or UB and WD-derived
cells. By immunoblot, pleiotrophin was found in an extract of whole
embryonic day 13 rat kidney. To determine whether epithelial cells
or mesenchymal cells secrete pleiotrophin, conditioned medium
derived from the UB, WD, or UB and WD cell line and the BSN cell
line were compared. Only BSN-CM contained pleiotrophin. This is
consistent with a previous in situ hybridization study
(Vanderwinden et al., Anat. Embryol (Berl) 186:387-406, 1992),
which showed that the developing rat kidney mesenchyme (as early as
E13 of development) expresses pleiotrophin mRNA, but the ureteric
bud, Wolffian duct bud, or ureteric and Wolffian duct bud does not.
Another study had suggested the presence of pleiotrophin in the
basement membrane of epithelial tubules in the developing kidney of
E13 mouse embryos (Mitsiadis et al., Development 121:37-51, 1995).
When frozen sections of mouse E 13 kidneys stained with
anti-pleiotrophin antibodies were examined, a strong signal was
observed in the basement membrane of the UB, WD, or UB and WD with
weak staining in the surrounding MM. Since the MM expresses
pleiotrophin mRNA at the earliest stages of kidney development
(Vanderwinden et al., 1992), the data presented herein suggest that
pleiotrophin is secreted by the MM and binds to the basement
membrane of the UB, WD, or UB and WD where it can exert its
morphogenetic function.
Example 9
[0133] Exogenous pleiotrophin affects UB, WD, or UB and WD
morphology in embryonic kidney organ culture. While the
spatiotemporal expression pattern and in vitro data from the
isolated UB, WD, or UB and WD and the UB, WD, or UB and WD cell
culture model strongly support a direct role for pleiotrophin in
UB, WD, or UB and WD morphogenesis, it was also important to
determine its effect in a system that more closely approximates the
intact developing kidney. To study this pleiotrophin was applied to
whole embryonic kidney organ culture. Exogenously added
pleiotrophin disproportionately stimulated growth of the UB, WD, or
UB and WD. Pleiotrophin-treated kidneys exhibited an expanded UB,
WD, or UB and WD area in a concentration-dependent manner similar
to that seen in the isolated UB, WD, or UB and WD culture.
Furthermore, the central area of UB, WD, or UB and WD expansion
became more prominent at higher concentrations of pleiotrophin. The
whole kidney also appeared slightly larger following pleiotrophin
treatment. Nephron induction visualized with PNA lectin appeared to
be normal even in the presence of high concentrations of
pleiotrophin. Thus, not only isolated UB, WD, or UB and WD, but
also the UB, WD, or UB and WD in the context of the whole embryonic
kidney responded to pleiotrophin, supporting the notion that the
UB, WD, or UB and WD is the target for pleiotrophin action in the
developing kidney.
[0134] Based upon this data an essential role for direct contact
between the metanephric mesenchyme (MM) and the ureteric bud,
Wolffian duct bud, or ureteric and Wolffian duct bud (UB, WD, or UB
and WD) during metanephrogenesis was suggested. Induction of the
isolated MM was inhibited by the placement of a filter with c 0.1
.mu.m pore size between an inducer and the MM, suggesting an
absolute requirement for cell contact between the MM and an
inducer. However, a combination of soluble factors elaborated by an
immortalized UB, WD, or UB and WD D cell line supplemented with
either fibroblast growth factor (FGF)-2, or a combination of FGF2
and transforming growth factor are sufficient, in the absence of
direct contact between the UB, WD, OR UB AND WD and MM, to induce
the mesenchymal-epithelial transition and differentiation of the
proximal nephron in cultures of isolated MM. Likewise, soluble
factors produced by a MM cell line (BSN cells) supplemented with
glial cell-derived neurotrophic factor (GDNF) have been suggested
to be necessary and sufficient to induce extensive branching
morphogenesis of the UB, WD, or UB and WD. Thus, soluble factors
play a key role in both aspects of the mesenchymal-epithelial
interaction leading to the formation of a functionally mature
kidney. This constitutes an important revision in thinking relating
to kidney organogenesis.
[0135] The identification of specific soluble factors (e.g.,
MM-derived soluble factors) mediating UB, WD, or UB and WD
branching morphogenesis remains a central question in this field.
Hepatocyte growth factor (HGF) has been shown to induce the
formation of branching tubular structures with lumens in
three-dimensional cultures of epithelial cell lines derived from
adult kidneys (i.e., MDCK and mIMCD cells) (Barros et al., 1995;
Cantley et al., 1994; Montesano et al., 1991; Santos et al., 1993).
However, incubation of three-dimensional cultures of an embryonic
cell line derived from the UB, WD, or UB and WD (UBs, WDs, or UBs
and WDs) with HGF had a slight morphogenetic effect and the
formation of branching tubular structures with lumens was not
observed (Sakurai et al., 1997). Furthermore, HGF, alone or in the
presence of GDNF, does not induce branching morphogenesis of the
isolated UB, WD, or UB and WD (as seen with the MM cell conditioned
medium).
Example 10
[0136] When BSN-CM was treated with trypsin or exposure to
prolonged heat (100.degree. C.; 30 min), the morphogenetic activity
for the UB, WD, or UB and WD was completely abolished. Based on
this result, it is likely that the morphogenetic factor(s) in
BSN-CM is proteinaceous in nature.
[0137] Centrifugation filtration systems with different nominal
molecular weight cutoffs were used to concentrate BSN-CM.
Centricon.TM. filters with a 8 kDa molecular mass cutoff membrane
maintained biological activities in the retained fraction but not
in the flow-through, suggesting the morphogenetic activity is
larger than 8 kD.
Example 11
[0138] As discussed, the morphogenetic factor is heparin binding.
Thus, a heparin binding-column (Hitrap Heparin.TM.,
Amersham-Pharmacia) was employed. Each fraction was assayed in
isolated UB, WD, or UB and WD culture system in the presence of
GDNF and FGF-1. Strong proliferative/morphogenetic activity was
observed in the fractions eluted with 0.9-1.25 M NaCl. These
morphogenetically active fractions were adjusted to 1.7 M ammonium
sulfate and were applied to the Phenyl Sepharose column at pH 7.2.
Isolated UB, WD, or UB and WD culture showed that several different
activities were present in fractions eluted between 1.5-0.7 M
ammonium sulfate, The 1.5-1.35 M eluate fraction in FIG. 10 induced
UB, WD, or UB and WD proliferation but had little effect on
branching tubule formation or elongation. In contrast, the 0.9-0.7
M eluate exhibited branching morphogenesis and elongation, but less
robust proliferation. Interestingly, the activity found in
fractions 7-9 suggested a combination of both fraction 6 and 10.
This result suggests that although full-blown branching
morphogenesis (as seen in the UB, WD, or UB and WD culture in
fraction 9) may require a combination of multiple factors (e.g., a
proliferative factor present in fraction 6 plus a possible
elongation/branching factor present in fraction 10), individual
factors can be separated and purified. In fact, by SDS-PAGE and
silver staining, fraction 6, which appears to be mainly
proliferative, contains a few bands clustered between 18-31 kDa,
while fraction 10, which appears to promote elongation and
branching, contains one band visible at 31 kDa.
Example 12
[0139] Sequential use of a hydrophobic interaction column, a cation
exchange column, and a gel filtration column lead to the
purification of PTN from these heparin-bound active fractions.
However, as discussed above, BSN-CM is likely to contain more than
one morphogenetic factors. In fact while higher salt eluate
fractions (fraction 6) from phenyl sepharose column contained PTN
by western blotting, lower salt eluate from a phenyl sepharose
column (fraction 10) did not. In addition, when morphogenetically
active fractions eluted from a heparin column (adjusted to Tris HCl
buffer pH 8.0) were applied to an anion exchange (Q) column,
morphogenetic activity was eluted at 0.15-0.5 M NaCl fractions (4
and 5). This morphogenetic activity was preserved after applying
these fractions to a gel filtration column. This Q column-bound
activity is unlikely to be PTN because PTN (pI=9.3) should not bind
to the Q column at pH 8.0. By microsequencing analysis, a heparin
binding growth factor heregulin was present in these fractions.
This result was further confirmed by western blotting, which was
positive for heregulin alpha in these fractions. Recombinant human
heregulin alpha (250 .mu.g/ml) induced isolated UB, WD, or UB and
WD to grow to the similar morphology as fractions 4 and 5 in the
presence of GDNF and FGF1. Thus, it is very likely that heregulin
is one of the factors that induce UB, WD, or UB and WD growth.
Example 13
[0140] Heparin-bound fractions of BSN-CM are likely to contain many
morphogenetic growth-promoting factors other than PTN. Existence of
such factors are highly likely for the following reasons: (1) an
active fraction eluted from anion exchange (Q) column is not likely
to contain PTN; (2) a fraction elated from a phenyl sepharose
column at 0.7 M ammonium sulfate (fraction 10), which induced
elongation and branching of the UB, WD, or UB and WD tubules,
should not contain PTN. Considering the relatively low resolution
of hydrophobic interaction column, the existence of very low
concentrations of PTN cannot be excluded, however, a dose dependent
response suggests that it is unlikely that such a low concentration
of PTN can induce the UB, WD, or UB and WD morphogenesis observed;
and (3) a morphogenetically active fraction containing little, if
any, PTN by western blotting was obtained by sequential
chromatography over 3 columns including a heparin sepharose
column.
Example 14
[0141] Tissue culture media was obtained from Mediatech and bovine
fetal calf serum was obtained from Biowhittiker. Growth factor
reduced Matrigel.TM. and Type I collagen were obtained from Becton
Dickenson. FGF1 and GDNF were obtained from R&D systems.
FITC-conjugated DB were obtained from Vector Laboratories.
[0142] The Cellmax.TM. artificial capillary cell culture system was
inoculated with BSN cells, and conditioned media harvested as
described herein.
[0143] Isolated ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct buds were obtained from whole embryonic kidneys as
previously described. Briefly, the embryonic kidney was digested
with trypsin and the UB, WD, or UB and WD separated from the MM
using fine-tipped needles. The UBs, WDs, or UBs and WDs were
suspended within a matrix containing growth factor reduced
Matrigel.TM. and Type I collagen and buffered by HEPES,
NaHCO.sub.3, and DMEM to a pH of approximately 7.2. This mixture
containing the suspended UB, WD, or UB and WD was applied to the
top of the Transwell filter and BSN-conditioned media added to the
well. The BSN conditioned media is supplemented with GDNF (125
ng/ml) and FGF1 (31 ng/ml) and 10% FCS, and the isolated UBs, WDs,
or UBs and WDs cultured at 37.degree. C. and humidified 5% CO.sub.2
atmosphere. At specified time intervals, the cultured UB, WD, OR UB
AND WD is separated from the surrounding matrix by blunt
microdissection, sectioned into thirds, resuspended in new matrix
and cultured with fresh supplemented BSN conditioned media.
Example 15
[0144] Isolated metanephric mesenchyme were isolated as described
above and cultured on top of the Transwell filter. DMEM/F12 media
supplemented with FGF2 (100 ng/ml) and TGF.alpha. (10 ng/ml) was
added to the well to prevent MM apoptosis.
Example 16
[0145] Using blunt microdissection with fine tipped needles,
cultured or subcultured UBs, WDs, or UBs and WDs were cleanly
separated from surrounding matrix and placed on top of a Transwell
filter in close proximity to MM that was either freshly isolated or
cultured. BSN conditioned media supplemented with GDNF, FGF1 and
10% FCS was added to the well.
[0146] Cultured or subcultured embryonic kidneys, isolated buds,
and recombined kidneys were fixed in 4% paraformaldehyde and
processed for immunofluorescent staining with either
F1TC-conjugated DB or antibodies. Immunofluorescence was detected
with a Zeiss laser-scanning confocal microscope.
Example 17
[0147] Adult male rats (weighing 200-250 grams) were housed and fed
on standard rat chow, water ingestion and 12-hour cycles of light
and dark. All animals were maintained and experiments conducted in
accord with the National Institutes of Health (NM) Guide for the
care and Use of Lab Animals.
[0148] Rats were anesthetized with an intraperitoneal injection of
sodium pentobarbitol solution (50 mg/kg). The anesthetized animals
were placed on a warming blanket and a midline abdominal incision
made. Bilateral or unilateral occlusion of the renal pedicule were
maintained for 40 minutes to induce ischemia and the incision
temporarily closed until completion of vascular occlusion. If an
arterial catheter was required for the experiment one was placed in
the femoral artery and exteriorized in the dorsal scapular region.
If ureteral catheters were necessary, they were placed and
exteriorized. Upon completion of ischemic period, the arterial
occlusion are removed, the incisions were sutured or stapled closed
and the rats allowed to recover for designated reperfusion
time.
Example 18
[0149] Injury was induced with either mercuric chloride or the
antibiotic gentamicin. Mercuric chloride primarily induces injury
and subsequent cell proliferation in proximal straight tubules
(PST), whereas gentamicin predominantly injures proximal convoluted
tubules (PCT). Gentamicin nephrotoxicity were induced by LP
injections of 40 mg/ml in 0.9 percent saline, divided with three
daily injections over two days for a total of 400 mg/kg. Mercuric
chloride are administered at various doses (0.25, 0.5, 1.0 and 2.5
mg/kg). These doses have been reported to induce renal injury
ranging from minimal to marked.
Example 19
[0150] To mimic the usual clinical situation, some rats were
exposed to either gentamicin or mercuric chloride at the ischemic
injury. The renal injury was especially severe in these
animals.
[0151] To purify factors involved in embryonic nephrogenesis, BSN
cell conditioned media (BSN-CM) was collected after 2 to 4 days of
BSN cell confluency, spun at low speed to remove cell debris and
filtered (0.22 .mu.m filter). The media is then concentrated
(Vivaflow.TM. 200, 5 kDA cutoff) subjected to sequential liquid
column chromatography and ion techniques, and final purification
accomplished with HPLC and SDS-Page electrophoresis. The final
purified protein(s) was submitted for microsequencing to an out
side vender.
Example 20
[0152] Isolated ureteric bud, Wolffian duct bud, or ureteric and
Wolffian duct buds were obtained from whole embryonic kidneys as
described herein. Briefly, the embryonic kidney was lightly
digested with trypsin and the UB, WD, or UB and WD were separated
from the MM using fine-tipped needles. The UBs, WDs, or UBs and WDs
were suspended within a matrix containing growth factor reduced
Matrigel.TM. and Type I collagen and buffered by HEPES,
NaHCO.sub.3, and DMEM to a pH of approximately 7.2. This mixture
containing the suspended UB, WD, or UB and WD was applied to the
top of the Transwell filter and the purified factor is applied to
the well. The factor is supplemented with GDNF (125 ng/ml) and 10%
FCS, and the isolated UBs, WDs, or UBs and WDs are cultured at
37.degree. C. and humidified 5% CO.sub.2 atmosphere and branching
morphogenesis, was assayed.
Example 21
[0153] Plasma collections during the experiment were collected via
the rat tail vein under isoflurane anaesthesia. A large blood
volume was collected at the end of the experimental period by
sanguination under pentobarbitol (50 mg/kg) anacstiesia. Plasma
from these collections were analyzed for sodium, potassium, ionized
calcium, ionized magnesium (Nova 8 Electrolyte Analyzer), BUN and
crealinine by autoanalyzer (core facility). Urine collection during
and at the end of the experiment were done in metabolic cages. The
urine was analyzed colormetrically for creatinine, calcium,
magnesium, phosphate and chloride and protein. Sodium and potassium
are measured with a Nova 6 Electrolyte Analyzer.
Example 22
[0154] Cross sections of kidney from each rat were fixed on a
microscope slide and stained with hematoxylin and eosin. Slides
were read for the presence or absence of tubular epithelial
degeneration and/or necrosis.
Example 23
[0155] Tubular injury and cell proliferation were assessed on
PCNA/PAS sections. Staining was done on 5 .mu.m paraffin sections
from ethacam-fixed renal tissue. Proliferating cells were
immunostained with a rabbit anti-mouse monoclonal antibody (PC 10
from Dako) directed to proliferating cell nuclear antigen (PCNA).
After blocking (goat sera) and incubation with the primary
antibody, the sections were incubated with biotinylated goat-anti
rabbit antiserum in the presence of normal rat serum and stained by
the avidin-biotinylated horseradish peroxidase complex
(Vectastatin.TM., Vector Labs) using 3,3'-diaminobenzidine as the
chromogen. Sections were then counterstained with methyl green and
periodic acid-Schiff (PAS).
Example 24
[0156] Identification and determination of apoptosis was done using
the terminal deoxynucleotidyl transferase (TdT)-mediated UTP biotin
nick-end labeling (TUNEL) technique by using an Apoptag.TM. in situ
apoptosis detection kit (Oncor, Gaitheburg, Md.). Frozen sections
(Sum) were fixed in 10% neutral-buffered red formalin and post
fixed in ethanol: acetic acid at -20.degree. C. for comparison to
control tissue as described herein.
Example 25
[0157] Determination of the factor(s) was also performed in adult
rat kidney: After purification of unique factor(s) an antibody was
generated by immunizing rabbits with purified protein (Multiple
Peptide Systems, San Diego, Calif.). Kidney homogenates following
ischemic and/or nephrotoxin injury were fractionated on 4-15% SDS
polyacrylamide gels under reducing conditions and transferred to
PVDF membranes. After blocking with phosphate buffered saline
containing 5% nonfat milk, the blots were incubated Primary
antibody (rabbit anti-rat peptide) and visualized by enhanced
chemiluminescence system (Pierce). If peptide was present by
western blot analysis then the polyclonal was used for
immunohistochemical detection and localization.
Example 26
[0158] Tissue culture media were obtained from Mediatech and bovine
fetal calf serum (FCS) from Biowhittaker (East Rutherford, N.J.).
Transwell filters, pore size 0.4 .mu.m, were obtained from Costar
(Cambridge, Mass.). Growth factor reduced Matrigel.TM. was obtained
from Becton Dickenson (Franklin Lakes, N.J.). Glial-cell derived
neutrophic factor (GDNF), fibroblast growth factor-1 (FGF1) and
fibroblast growth factor-7 (FGF7) were obtained from R&D
Systems (Minneapolis, Minn.). FITC-conjugated Dolicus Biflorous and
rhodamine-conjugated peanut agglutinin (PNA) lectin were obtained
from Vector Laboratories (Burlingame, Calif.).
[0159] The entire urogenital tract was isolated from timed pregnant
Holtzman rats at embryonic day 13 (E13). Kidney rudiments were
dissected, and the Wolffian duct was transected and removed. In
some cases, the mesonephric tubules and most of the intermediate
mesoderm were mechanically separated from the WD leaving a thin
layer of the intermediate mesoderm that remained adherent to the
WD. In some cases, the intermediate mesoderm was carefully stripped
away in its entirety so that the epithelial tube of the WD remained
intact. The WD with mesonephros and the WD with intermediate
mesoderm were placed on 0.4 .mu.m pore size Transwell filters and
cultured at the air-media interface. The isolated WD devoid of all
intermediate mesoderm was suspended in a matrix containing growth
factor reduced Matrigel.TM. and DMEM/F12 (50:50 v/v) on a 0.4 .mu.m
pore sized Transwell filter. All cultures were carried out at
37.degree. C. in a fully humidified 5% CO.sub.2 atmosphere in the
presence of DMEM/F12 (50:50) media supplemented with 10% fetal calf
serum (FCS), GDNF (10 ng/mL for the WD plus mesonephros, 125 ng/ml
WD plus mesoderm and isolated WD) and, if necessary, either FGF1
(250 ng/ml) or FGF7 (50 ng/ml).
[0160] Budded Wolffian ducts are removed from the filter and were
lightly digested with trypsin, and the buds were separated from the
Wolffian duct and surrounding attached cells. The microdissected in
vitro-formed buds were suspended within a matrix containing growth
factor-reduced Matrigel.TM. and DMEM/F12 (50:50 v/v) on a 0.4 .mu.m
pore sized Transwell filter and BSN-conditioned media (prepared as
described previously (29)) added to the well. The BSN-CM was
supplemented with GDNF (125 ng/ml) and FGF1 (250 ng/ml). These in
vitro-formed UBs were then cultured at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere.
[0161] Optimization of Growth Factor and Matrix (Natural and
Artificial) Conditions for Isolated Ureteric Bud Branching
[0162] The entire urogenital tract was isolated from timed pregnant
Holtzman rats at embryonic day 13 (E13). Kidney rudiments were
dissected and lightly digested in trypsin for 15 minutes, after
which the ureteric buds were mechanically separated from the
metanephric mesenchyme. For the growth factor optimization, the
buds were suspended within a matrix containing growth
factor-reduced Matrigel.TM. and DMEM/F12 (50:50 v/v) on a 0.4 .mu.m
pore sized Transwell filter and BSN-conditioned media supplemented
with 10% FCS and 1% antibiotics was placed beneath the filter. GDNF
(125 ng/ml), FGF1 (250 ng/ml), FGF7 (50 ng/ml), HRG (500 ng/ml),
PTN (purified from BSN-CM) was added to the media in various
combinations to achieve a "minimal" and "most robust" growth factor
condition. For the matrix optimization, isolated buds were cultured
in a BSN-CM supplemented with 10% FCS, GDNF (125 ng/ml), FGF1 (250
ng/ml), and 1% antibiotics. Isolate buds were suspended in matrices
(natural and artificial) consisting of growth factor reduced
Matrigel.TM. diluted with DMEM/F12 (15%, 25%, 50%, 75% and 100%),
type I collagen (3.0 mg/ml), Puramatrix.TM., 1% alginate solution
(crosslinked with 100 mM CaCl.sub.2), and type IV collagen (0.75
mg/mL). In some cases, type I collagen, laminin 1, alginate or type
IV collagen were added to a 50% growth factor reduced Matrigel.TM.
solution. All matrix solutions were supplemented with DMEM and
buffered by HEPES and NaHCO.sub.3 to a pH of approximately 7.2. The
isolated buds were then cultured at 37.degree. C. in a humidified
5% CO.sub.2 atmosphere.
[0163] After 4-6 days the branched in vitro-formed UBs were
separated from the surrounding matrix by blunt dissection. Branched
buds with minimal matrix were placed on a 0.4 .mu.m pore sized
Transwell filter with DMEM/F12 (50:50) supplemented with 10% FCS in
lower well. Metanephric mesenchyme from kidneys at embryonic day 13
was placed next to and on the branched in vitro-formed UB. After
4-7 days of culture, 37.degree. C. and 5% humidified CO.sub.2, the
recombined kidney-like tissues were fixed in 4% parafomaldehyde,
extensively washed in PBS and processed for fluorescent
staining.
[0164] Tissues were fixed with 2% paraformaldehyde for 30 min at
4.degree. C., blocked with 50 mM NH4Cl overnight at 4.degree. C.,
and followed by incubation with 1% gelatin in 0.075% Saponin for 30
min at 37.degree. C. After two washes with Neuraminidase buffer
(150 mM NaCl/50 mM sodium acetate, pH 5.5), tissues were incubated
with Neuraminidase (1 unit/ml) for 4 hr at 37.degree. C. and then
with rhodamine-conjugated PNA (50 .mu.g/ml) and
fluorescent-conjugated DB (50 .mu.g/ml) for 60 min at 37.degree. C.
Tissues were postfixed with 2% paraformaldehyde and viewed with a
laser-scanning confocal microscope.
[0165] The E13 kidney tissues, E18 kidney tissues, adult kidney
tissues, and recombined kidney-like tissues were dissolved in lysis
buffer and the RNA was isolated using the RNAqueous.TM. Micro kit
from Ambion (Austin, Tex.). RNA was submitted to the UCSD Genechip
core facility for processing with the Affymetrix (Santa Clara,
Calif.) rat 230 2.0 whole genome chip. The in vivo time points were
analyzed in biological triplicates, while the recombined tissues
were analyzed in biological duplicates. All gene expression
analysis was performed using Agilent (Santa Clara, Calif.)
Genespring.TM. GX 7.3 software.
[0166] Recombined tissues on culture day 6 were gently detached
from Transwell filter, and suspended within cold L-15 medium. The
abdominal cavity of an adult male rat was opened under inhalant
anesthesia with isoflurane. A subcapsular tunnel was prepared on
the right kidney using the tip of microsurgery forceps. 2-6
recombined tissues were inserted into the subcapsular region
together with a small volume of L-15 medium (.about.40 .mu.l) by
micropipette. The abdominal cavity was closed by suturing muscle
and skin layers. After survival time of 14 days, the kidneys with
implants were excised and provided for histological analyses.
[0167] Host kidneys bearing implants were excised, fixed in 4%
paraformaldehyde/0.1 M phosphate buffered saline, pH 7.4 for 24 hr
at 4.degree. C., and incubated within a graded concentration of
sucrose solution (10%, 15%, and 20%). Samples were embedded in
Tissue Tek O.C.T. compound (Sakura Finetek, Torrance, Calif.), and
frozen rapidly. Cryosections were cut at a thickness of 4 .mu.m by
a Cryostat CM3050S (Leica, Heidelberg, Germany), pasted on slides
and air-dried. The specimens were stained with a mixture of
anti-rat PECAM-1 (CD31) antibody (1:100; clone TLD-3A12, mouse
IgG1k, Pharmingen) and anti-collagen type IV antibody (1:100;
rabbit polyclonal, Chemicon) at 4.degree. C. overnight. Then they
were reacted with a mixture of donkey anti-mouse IgG-Alexa Fluor
488, goat anti-rabbit IgG-Alexa Fluor 568, and
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) at room
temperature for 1 hr. Fluorescent labeled antibodies and DAPI were
purchased from Molecular Probes (Eugene, Oreg.). The specimens were
observed with a Nikon D-Eclipse C1 confocal laser scanning
microscope. Some cryosections were processed for routine histology
using hematoxylin and eosin (H&E) staining, and observed with a
Nikon Eclipse TE300 light microscope.
[0168] The first developmental event exclusive to metanephric
kidney development is the outgrowth of the UB from the Wolffian
duct. Using three different methods, this developmental event was
replicated in vitro (Table 1). First, the whole mesonephros was
isolated and cultured on a Transwell filter in the presence of a
media containing 10% serum and 10 ng/mL GDNF. After two days in
culture, numerous budding events occurred at multiple foci along
the WD (FIGS. 1A, B). Note that, assuming a single focus of budding
can yield a kidney (as occurs in vivo), if each of the multiple
buds can be cultured individually, then there is the possibility of
many kidneys arising from a single WD cultured in vitro. In the
second method, the mesonephric tubules, along with most of the
non-epithelial mesoderm, were removed from the WD prior to in vitro
culture. For impressive budding to occur, the GDNF concentration
had to be increased to .about.125 ng/mL and an additional growth
factor was required; either FGF1 at .about.250 ng/mL or FGF7 at
.about.50 ng/mL (FIGS. 1C, D). In the third method, WD was
completely cleared of all surrounding mesoderm prior to in vitro
culture, leaving essentially an epithelial tube. In this "minimal"
system, budding could not be achieved in 2D Transwell culture.
Rather, the WD had to be suspended in a 3D gel composed of diluted
Matrigel.TM. and then cultured in a medium containing 125 ng/mL
GDNF and either 250 ng/mL FGF1 or 50 ng/mL FGF7 (FIGS. 1E, F). With
all methods, although the overall surface area of the WD increases
due to budding at multiple foci, the WD does not appear to lengthen
significantly under any of the culture conditions.
TABLE-US-00001 TABLE 1 Wolffian duct budding conditions of Table 2.
Growth factor effects on ureteric bud survival, growth and shape
Dissected WD condition Factors Necessary for Bud Formation Whole
Mesonephros 2D filter culture, 10 ng/ml GDNF WD + intermediate 2D
filter culture, 125 ng/ml GDNF + 50 ng/ml FGF7 2D mesoderm filter
culture, 125 ng/ml GDNF + 250 ng/ml FGF1 Clean WD (no 3D-matrix
(Matrigel .TM.), 125 ng/ml GDNF + 50 ng/ml FGF7 intermediate
mesoderm) 3D-matrix (Matrigel .TM.), 125 ng/ml GDNF + 250 ng/ml
FGF1
[0169] The next step in kidney development after WD budding is UB
branching. It has previously been shown that rat isolated ureteric
buds suspended in extracellular matrix gels undergo robust
branching in the presence of GDNF and BSN-conditioned medium
(secreted by metanephric mesenchyme-derived cells). The invention
demonstrates in vitro-formed UB (created as described herein)
retained the ability to undergo branching similar to newly isolated
ureteric buds. FIG. 2 indicates how one bud was excised from the
budded Wolffian duct and then induced to branch in 3D culture using
culture conditions similar to those described for the "T-shaped" UB
dissected from E13 rats. Thus, the in vitro-formed UBs, while not
achieving the T-shaped structure assumed to be necessary for normal
development of the kidney based on knockout studies, can grow and
branch in vitro in a fashion similar to the excised T-shaped bud.
This indicates that the T-shaped bud stage can be bypassed in the
developmental strategy for tissue engineering. This also
demonstrates how one Wolffian duct can be used to generate multiple
ureteric buds, each capable of undergoing in vitro branching.
Assuming that the branching UB-like structures can somehow be
induced to form full nephrons, this potentially represents a point
at which multiple renal tissues can be generated from a single
WD.
[0170] Following the determination that the bud extracted from an
in vitro budded Wolffian duct retains branching ability, the
invention demonstrates the degree to which one could optimize
branching with soluble factors to more closely resemble in vivo
branching morphogenesis and to increase the extent of branching
since each tip represents another potential point of propagation to
create multiple renal tissues. Within the array of positive and
negative growth and sculpting factors that comprise BSN-CM,
pleiotrophin (PTN) has been identified as a significant
branch-promoting factor. Several fibroblast growth factors (FGFs)
can affect growth patterns of the isolated UB in the presence of
BSN-CM and GDNF. Furthermore, heregulin (HRG) has recently been
isolated from BSN-CM, which has been shown to induce non-branching,
GDNF-independent growth of the isolated UB.
[0171] The goal was to identify a growth factor (or a combination
of a few defined factors) that reproduced the most robust
branch-inducing conditions. Initially, the effect of a single
soluble factor on growth of the isolated UB was examined.
Consistent with previous reports, GDNF, PTN, HRG, or whole BSN-CM
did not support growth of the isolated UB by itself, while FGFs
supported survival and minimal growth (FIG. 3A and Table 2). Next,
additional growth factors were added to the culture medium
containing at least one fibroblast growth factor to enhance
survival. Addition of every factor tested (including GDNF, PTN,
HRG, and whole BSN-CM) to FGF1 or FGF7 stimulated growth of the
isolated UBs to a much greater extent than each FGF alone. In
addition, UB growth patterns were not altered significantly by
addition of the second growth factor to either FGF-1 or FGF-7 (FIG.
3B). Combinations of 2 factors were also tested that, in isolation,
were incapable of inducing UB growth. The invention demonstrates
that the combination of PTN and GDNF (in the absence of FGFs) could
induce branching growth of the isolated UB (FIG. 3C) and that whole
BSN-CM and GDNF could also induce branching growth, although less
reliably.
TABLE-US-00002 TABLE 2 Growth factor analysis on ureteric bud
survival, growth and shape UB survival Overall growth Shape BSN-CM
- NA NA GDNF - NA NA HRG - NA NA PTN - NA NA FGF1 + + tubular FGF7
+ ++ globular BSN + GDNF - (Rarely +) If survives ++ If survives,
branching HRG + GDNF - NA NA PTN + GDNF + +++ branching/ globular
FGF1 + GDNF + +-++ tubular FGF7 + GDNF + +++ globular BSN + FGF1 +
+ tubular HRG + FGF1 + ++ dilated tubular PTN + FGF1 + + tubular
BSN + FGF7 + +++ globular HRG + FGF7 + +++ globular PTN + FGF7 +
+++ globular BSN + FGF1 + GDNF + +++ branching tubular HRG + FGF1 +
GDNF + ++ dilated tubular PTN + FGF1 + GDNF + +++-++++ branching/
globular BSN + FGF7 + GDNF + ++++ globular HRG + FGF7 + GDNF + ++++
globular PTN + FGF7 + GDNF + ++++ globular
[0172] Added FGF-1, a branching facilitating FGF, to other
conditions that supported variable degrees of branching, including
PTN plus GDNF and BSN-CM plus GDNF. The combination of PTN, FGF1,
and GDNF sometimes induced excessive tip formation, and resulted in
stalk-less UBs (FIG. 3D), while the combination of whole BSN-CM,
FGF1, and GDNF induced the most robust branching growth of isolated
UBs in terms of both tip and stalk formation. It should be noted
that PTN had to be purified from mammalian sources (BSN-CM, 3T3-CM,
or PTN cDNA transfected mammalian cell conditioned medium) in order
to be biologically active in this assay.
[0173] Given that the extracellular matrix plays a significant role
in terms of scaffolding for isolated UB growth and branching as
well as modulation of growth factor effects, optimization of the
growth conditions for 3-dimensional ureteric bud branching was then
performed. Previous studies of isolated UB branching utilized a
matrix of "growth-factor reduced" Matrigel.TM. and Type I collagen
(50:50 v/v) supplemented with DMEM and buffered by HEPES and
NaHCO.sub.3 to a pH of approximately 7.2. First, it was determined
whether Matrigel.TM. or type I collagen alone could support
branching morphogenesis. FIG. 4 shows that type I collagen does not
support branching morphogenesis and is, in fact, inhibitory, while
Matrigel.TM. supports branching at a wide range of concentrations
with decreases in branching at the highest and lowest
concentrations (100% and 15%, respectively). In addition to the two
original components, a 1% alginate solution, crosslinked with 100
mM CaCl.sub.2, and Puramatrix', an inert self assembling peptide
matrix, were also tested with the isolated UB system; neither
supported UB branching.
[0174] Given that the mature kidney basement membrane is composed
of approximately 50% type IV collagen and the network forming
component of Matrigel.TM. is type IV collagen, other types of
collagen were tested to see if type IV collagen alone could support
branching morphogenesis or if the additional basement membrane
components, such as laminin I, were also necessary. Surprisingly,
type IV collagen alone did support branching morphogenesis, and
additional factors such as laminin I did not increase branching
capacity (FIG. 4I-K). However, ureteric buds in type IV collagen do
not grow as well as those in Matrigel.TM.; this may be due to the
fact that Matrigel.TM. contains a concentration of type IV collagen
(.about.3.3 mg/mL) more than triple that of the pure type IV
collagen commercially available (.about.1 mg/mL). These data
demonstrate that in vitro isolated UB branching morphogenesis is
dependent upon Collagen IV and does not require additional matrix
components. A summary of all matrix effects is provided in Table
3.
TABLE-US-00003 TABLE 3 Extracellular matrix effects on ureteric bud
development Network forming Matrix Molecules Branching Support
Matrigel .TM. (15-100%) Supports branching Type IV Collagen (0.75
mg/mL) Supports Branching Type I collagen (0.4-4.0 mg/mL) Does not
support branching Alginate (0.3-1.5%) Does not support branching
Puramatrix .TM. (50-100%) Does not support branching Matrigel .TM.
Matrix Additions Effect on Branching Laminin (0.2-0.75 mg/m) No
effect Alginate (0.1-0.9%) Diminished branching Type I Collagen
(0.4-3.0 mg/mL) Diminished branching Type IV Collagen (0.25-0.75
mg/mL) No effect
[0175] Taken together with the growth factor optimization studies,
a minimal set of conditions can be defined for UB growth and
branching as PTN plus GDNF in a type IV collagen matrix. The most
robust system in terms of both tip and stalk formation, however, is
BSN-CM supplemented with GDNF and FGF1 in a 50% Matrigel.TM.
solution.
[0176] Once a Wolffian duct has been budded in vitro and the
dissected in vitro-formed UB has branched, the next step is to
determine if 3D nephron formation can occur; the approach was to
recombine the branched structure with fresh MM. These experiments
differ from typical recombination studies with "T-shaped" UBs,
considered a key developmental stage (32-34), in that the branched
UB was derived from an in vitro budded Wolffian duct that does not
form a T-shaped structure, as already described. The recombined
tissue was cultured on a Transwell filter without any additional
growth factors (FIG. 5B). Several days after recombination,
branched structures had grown and elongated, and induction was
evident by phase contrast microscopy (FIG. 5C). This was confirmed
by confocal microscopy and lectin staining (FIG. 5D-F). Connections
between the collecting system and the more proximal portions of the
tubule derived from the MM were evident.
[0177] While the recombined tissues comprised of nephron structures
resembling the late stages of renal development, it was important
to verify the extent to which the MM and branched UB followed
developmental pathways by analyzing the global gene expression and
comparing it to the gene expression of early, late, post
developmental kidneys. Genes with a 3-fold difference between any
of the 4 conditions (E13, E18, Wk4, Recombined tissue) were
analyzed. 6,763 genes from the Affymetrix 230 2.0 Rat gene chip fit
that condition (genes flagged as "Absent" by the Affymetrix
algorithm and genes without annotation were excluded from this
analysis). The genes were organized into 10 groups based on their
developmental expression patterns as described in FIG. 6 with 1,080
genes not fitting into any group. Then, the expression of each
group of genes was analyzed in the recombined tissue to determine
how far the recombined tissue gene expression had progressed
relative to the three in vivo time points (See Table 4). Since the
recombined tissue had progressed in vitro for seven days, the
recombined tissue culture gene expression was hypothesized to reach
a mid-to-late kidney developmental level similar to that of E17-E18
based on morphology. In rats, development is known to continue in
the weeks following birth, so seven days of in vitro culture is not
expected to recapitulate the gene expression pattern of the 4 week
old kidneys. This comparison revealed that, while the in vitro
engineered kidney-like tissues are not identical to any of the in
vivo expression patterns, there is considerable resemblance to the
in vivo gene expression of the E18 kidney. More than 50% of the
genes that were up-regulated during the E18 time point were also
up-regulated in the recombined tissue. In addition, almost 75% of
the genes in Group VI properly down-regulated in the recombined
tissue to at least E18 levels. This indicates that a large number
of the developmental pathways are being properly regulated in the
recombined tissue of what was originally the microdissected in
vitro grown WD bud and fresh MM tissue. There also exists a group
of genes that are absent or present at the onset of kidney
development, followed by an up or down regulation during
development, and then a return to their initial state following
development (Groups VII and VIII respectively). These genes are
present or absent during development, and, therefore, it is not
possible to determine whether a particular gene is at the final or
initial state. It can only be determined whether a gene is in a
"developmental state" similar to E18. However, .about.53% of the
genes that were up-regulated at E18 were also up-regulated in the
recombined tissue, further suggesting that many of the natural
developmental pathways are being followed. Finally, there were
genes that were abnormally high or low in the recombined tissue
that did not change throughout development (these are genes in
which the recombined tissue is at least 3 fold higher or lower than
all three time points); these genes represented less than 5% of the
total number of genes in the analysis, suggesting that the in vitro
conditions cause few extraneous gene expression changes.
TABLE-US-00004 TABLE 4 Global microarray analysis of UB/MM
recombination Percent of Recombination Genes that Up-regulated
Total Genes Up-regulated Genes (Number of Genes) in Group Group I
13.19 (98) 743 Group II 50.00 (82) 164 Group III 49.76 (620) 1246
Percent of Recombination Genes that Down-regulated Down-regulated
Genes (Number of Genes) Group IV 15.03 (196) 1304 Group V 49.04
(51) 104 Group VI 74.34 (762) 1025 Percent of Recombination Genes
Developmentally that Up-regulated Up-regulated Genes (Number of
Genes) Group VII 53.65 (169) 315 Percent of Recombination Genes
Developmentally that Down-regulated Down-regulated Genes (Number of
Genes) Group VIII 27.94 (126) 451 Number of Genes Recombo
Abnormally High Group IX 241 Recombo Abnormally Low Group X 90
[0178] In order for a kidney tissue to be functional it must
contain a vasculature and glomeruli. While the microarray analysis
demonstrated that the recombined tissue recapitulates many of the
gene expression activities of renal development, few vascular genes
were up-regulated, and, in any case, this does not predict whether
the recombined tissue will be able to successfully integrate into a
host animal and be functional. Previous studies have demonstrated
that early avascular embryonic kidneys can be implanted into a host
animal to obtain a vasculature with functional glomeruli. The
recombined kidney-like tissue, which is derived from rat tissue,
were implanted under the renal capsule of a host rat. After 14
days, the host animal was sacrificed and the implanted tissue was
analyzed (FIG. 7). The implanted recombined tissue successfully
recruited a vasculature, formed multiple glomeruli, and expressed
the endothelial marker PECAM-1 in the cells of the glomerulus (FIG.
7D). Erythrocytes can be seen in the glomeruli of the recombined
tissue indicating blood flow to the implanted tissue (FIG. 7C,
arrows).
[0179] The disclosure provides a strategy for tissue engineering a
propagatable kidney-like tissue by following key kidney
developmental events in vitro in a stepwise fashion beginning with
a Wolffian duct, essentially a single epithelial tube. The strategy
results in tissues with spatially appropriate nephrons, glomeruli
and a vasculature in three dimensions. Furthermore, optimization of
each in vitro step was analyzed and provides details on a variety
of conditions that can be used at each step, potentially offering
multiple approaches within this strategy. FIG. 8 illustrates how
these systems can be put together to create in vitro engineered
renal structures from the initial components. This data also
suggests the possibility of creating multiple 3D kidney-like
tissues not only from a single Wolffian duct tissue, but,
potentially, from an epithelial tubule consisting of relatively
homogenous polarized epithelial cells in the presence of
mesenchymal cells and/or tissues.
[0180] The optimization studies revealed that the Wolffian duct,
the basic epithelial tubular structure from which the 3 dimensional
collecting duct system of the kidney arises, requires only the
presence of GDNF for budding if the whole mesonephros is cultured
in vitro, but requires additionally either FGF-1 or FGF-7 if the
mesonephric tubules and surrounding mesoderm are removed. Other
factors, including those present in BSN-CM, can be employed as
well. For example, activin inhibition has previously been
demonstrated to promote budding, in place of FGF-1 or FGF-7. As
shown here, a fully cleaned Wolffian duct, truly a polarized
epithelial tube, requires a suspended culture system using a
diluted Matrigel.TM. solution and the presence of growth factors in
order to undergo budding in vitro. These experiments suggest that
the surrounding mesoderm plays a role in budding by providing
growth factors and/or necessary matrix components, which for tissue
engineering purposes, are supplied exogenously here. Moreover, each
of the many buds can be excised, suspended in a 3D matrix, and
induced to undergo extensive branching morphogenesis. The branching
in vitro-formed UB appears highly similar, if not identical, to the
isolated UB in culture--both morphologically and in terms of
functional ability to induce MM.
[0181] From the perspective of tissue engineering, beginning from a
solitary epithelial tube, initial survival of the tubular cells is
an obvious issue if multiple steps are involved. In the process of
optimization of growth factors for the in vitro cultures, FGFs were
the only soluble factors tested that supported both isolated WD and
UB survival in vitro when applied as a single agent. However,
another set of conditions that increased isolated UB survival was
to simultaneous culture with PTN (or PTN-containing BSN-CM) and
GDNF. In this case, branching was also observed. FGFs also affected
the pattern of isolated UB growth. To achieve optimal branching
growth, FGF-1, a growth factor that supports branching and growth
of the isolated UB culture system, was combined with non-FGF
branch-supporting growth factor combinations (i.e. GDNF plus either
PTN or BSN-CM). In vitro, these growth factor conditions provided
the most robust (i.e. growth plus most in vivo-like patterning)
branch-stimulating conditions among the many soluble factor
combinations that were tested. The fact that a combination of
BSN-CM, FGF1, and GDNF gave more consistent branching growth than
PTN, FGF1 and GDNF, suggests one or more factors within BSN-CM
assist in culture. Conceivably these can include factors such as
TGF.beta.-superfamily members, including bone morphogenetic
proteins 2 and 4, which seem to modulate branching and are involved
in sculpting of the isolated UB culture system. Thus, it is
possible that a combination of PTN plus one additional
"UB-sculpting factor" could replace BSN-CM to attain an ideal
minimal set of conditions for a tissue engineering approach. The
combination of BSN-CM, FGF1, and GDNF supports the most robust
growth and branching of the UB in vitro.
[0182] Given the recent work on matrices and scaffolds for tissue
engineering of bone, cartilage and other organs, both artificial
and natural matrix conditions were analyzed for isolated 3D UB
branching. The UB appears to only branch in type IV collagen or
Matrigel.TM., which is type IV collagen based. Surprisingly, adding
laminin I, which enhances branching of cultured cells, to a type IV
collagen matrix did not further increase branching morphogenesis.
This suggests that the initial extracellular matrix scaffold does
not require all components of the final basement membrane and that
the UB itself can synthesize any necessary supplementary proteins
in an isolated system. In addition, two inert ECM molecules,
Alginate, which has been used extensively in cartilage tissue
engineering, and Puramatrix.TM., which was successfully used to
support neuronal migration and promote osteoblast differentiation,
were tested for branching support; the UB was unable to branch in
either of these two artificial matrices. This may be caused by the
inability of the UB to break down and remodel the artificial matrix
to allow room for new branches. Nevertheless, an "ideal minimal
system" for tissue engineering of the kidney according to the
scheme ought to continue consideration of other artificial matrices
as they become available.
[0183] The branched in vitro-formed UB, derived from the Wolffian
duct, when placed adjacent to freshly isolated MM, resulted in
mesenchymal-to-epithelial transformation and connections forming
nephron-containing kidney tissues. This indicates the ability of
the in vitro-formed UB, after being induced to branch in vitro, to
be further manipulated in vitro to form kidney-like tissues. To
determine the extent of similarity to the mid-to-late developing
kidney in vivo, the recombined tissue was analyzed in microarray
studies to reveal that more than 50% of the genes that up-regulated
during kidney development also up-regulated in the recombined
tissue. The comparison revealed that of the down-regulated genes,
more than 72% of genes properly turn off. This suggests that the
recombined tissue is following normal renal developmental pathways
and creating nephron structures that not only phenotypically appear
normal, but also resemble the transcriptome of the developing
kidney.
[0184] While fresh MM tissue was used for the recombination step,
this tissue is reported to contain pluripotent renal progenitor
cells. It may be most feasible, however, to begin with cells alone.
The BSN cell line is derived from the MM. The disclosure
demonstrates the ability of secreted products from this cell line
to induce optimal branching of the in vitro-formed UB (derived from
in vitro WD culture) but thus far have been unable to show that the
cells will recombine to form nephron-containing structures (data
not shown). This may require a matrix-based strategy to make cells
cohere or, alternatively, it is possible that these cells are too
differentiated (perhaps more like mesenchymal "cap" cells) to be
used for this application. Recently, it has been reported that
mouse ES cells can be induced to form MM-like cells, suggesting an
alternative approach.
[0185] Similar considerations apply to the creation of an
epithelial tubule like the WD from cells. Of note, it has been
shown that the UB cell line can form a tubule under conditions
somewhat similar to those shown as optimal for branching of the
isolated UB. It has also been shown that adult "progenitor-like"
cells from the injured mouse kidney can form tubular epithelial
structures in vitro and migrate to multiple compartments of the
developing kidney in organ culture.
[0186] These types of cells, or possibly others that have recently
been described that circumvent the use of and minimize concerns
about embryonic tissue, may be more acceptable. The disclosure
provides proof of concept for assembly of 3 dimensional renal
tissues with differentiated nephron-containing structures from an
epithelial tubule (the Wolffian duct), in whichever manner it is
ultimately constructed. The methods may be suitable for xenogenic
based approaches. Given the fact that they are tissue/organ
culture-based, and that there are at least two points for
propagation (at the level of the in vitro cultured WD and at the
level of the in vitro-formed UB), it may be possible to "humanize"
the tissue through transfection or similar strategies, or to induce
expression of immunomodulatory or other genes to diminish the
possibility of rejection and, potentially, improve functionality.
These techniques, not currently feasible in mammalian adult organs,
provide considerable flexibility for the goal of creating
immunocompatible tissues suitable for a particular genetic
profile.
[0187] Beginning with a single or limited in vitro propagatable
tissue may also help address the concern about animal viruses with
xeno-based approaches by creating a single or limited set of key
points in a tissue engineering strategy where intense quality
control or surveillance can be applied.
[0188] Vascularization was achieved by placing the recombined
tissue in the in vivo setting of the renal capsule. The recombined
tissue recruits a vasculature and forms glomeruli that
appropriately express a key endothelial marker when implanted
underneath the renal capsule of a host animal. Whether this
technique will be successful as a therapy for ESRD is unknown, but
again, it provides important "proof of concept" that an in vitro
engineered kidney-like tissue, designed in the manner described
herein, can survive and recruit a vasculature when placed in a host
animal despite the absence of a developed renal vasculature of its
own. Among the goals of kidney engineering are to design a kidney
or tissue that can replace damaged kidneys and/or alleviate the
problems associated with current allogenic transplants. The ability
to begin with an epithelial tube, the stripped Wolffian duct, and
take it in vitro to the point of vascularization represents an
important result in considering how developmental strategies can be
employed for the purpose of tissue engineering of the kidney.
Moreover, this method allows for the creation of multiple
recombined kidney-like tissues from a single Wolffian duct
progenitor tissue or, potentially, a pluripotent epithelial tubule
constructed from cells, whether adult, embryonic, amniotic or
other. Embryonic-derived tissues seem to elicit a reduced immune
response in rodents; therefore, in vitro manipulation of
xeno-tissues or primitive cells to create kidney tissue may result
in a less antigenic transplant than alternative options, such as
whole embryonic kidneys that have had a longer time to develop in
utero.
[0189] While many approaches are being taken towards engineering
kidney substitutes, that a kidney-like tissue formed by following
the natural developmental progression will be more likely to
recapitulate the 3 dimensional relationships necessary to maintain
vital renal functions as opposed to other cell-based kidney
engineering approaches. The disclosure provides guidelines for such
a strategy, at least in rodents, to stimulate renal progenitor
tissues to follow the natural developmental path resulting in the
in vitro engineered kidney-like tissue containing a branched
collecting duct system, nephrons, glomeruli and a vasculature. That
the strategy has strong potential for propagation of the engineered
kidney-like tissue, as well as modulation of functionality and
immunogenicity by transfection-type methods, adds to its
potential.
[0190] An approximately 100 .mu.m segment of WD was excised and
suspended within the isolated MM from one kidney in a 1 mg/mL type
I collagen solution (supplemented with DMEM and buffered by HEPES
and NaHCO.sub.3 to a pH of approximately 7.2). Before the gel was
completely solidified, the WD segment was placed in the crevice of
the MM left behind from the removal of the UB. The WD/MM tissue was
cultured in the presence of a DMEM:F12 medium supplemented with 10%
FBS (Hyclone.TM., Logan, Utah) and 1% antibiotics for 7 or 12 days.
All cultures were incubated at 37.degree. C. in a humidified 5%
CO.sub.2 and 100% humidity atmosphere.
[0191] Confluent monolayers of mouse SV40 large-T antigen
transfected UB cells (Barasch) or inner medullary collecting duct
(IMCD) cells (Rauchman) were trypsinized and suspended in DME/F12
(supplemented with 10% FBS and 1% antibiotics) at a concentration
of 1.times.10.sup.5 cells/mL. 20 .mu.L of the cell solution was
placed on the bottom of a Petri dish lid with 10 mL of PBS in the
Petri dish. The cells were incubated as a hanging drop for 2 days
at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. The cell
aggregates were removed from the hanging drops and placed on a 0.4
.mu.m Transwell filter surrounded by freshly isolated MM with 400
.mu.L DMEM:F12 media supplemented with 10% FBS and 1% antibiotics
placed below the filter and incubated for an additional 7 days.
[0192] The MM primary cell line was created by placing freshly
isolated MM cells directly on a cell-culture treated plate. The MM
cells were cultured on the plate for 5 days after which time the MM
cells were trypsinized and placed on new plates. To create
conditioned medium BSN, RIMM-18, MM primary, or 3T3 cells were
cultured on plates and allowed to reach confluence. After
confluence was reached, the medium was replaced with DMEM:F12 (no
antibiotics or FBS) and the cells were incubated for 3 days. After
3 days the conditioned medium was removed and concentrated 5 times
with a 5000 MW cutoff Millipore (Billerica, Mass.) filter.
[0193] Isolated ureteric buds were suspended in a growth
factor-reduced Matrigel.TM. solution (1:1 Matrigel.TM.:DMEM/F12),
and cultured with conditioned medium from the BSN, RIMM-18, MM
primary, or 3T3 cell lines supplemented with 10% FBS, 1%
antibiotics, 125 ng/mL FGF1, and 125 ng/mL GDNF. The suspended UBs
were then cultured for 7 days at which time tips were counted.
[0194] Embryonic metanephric kidneys were isolated and suspended in
extracellular matrix solutions of type I collagen, type IV
collagen, or growth factor-reduced Matrigel.TM. at the noted
concentrations. All matrix solutions were supplemented with DMEM
and buffered by HEPES and NaHCO.sub.3 to a pH of approximately 7.2.
Kidneys were cultured in the presence of 600 .mu.L DMEM:F12
supplemented with 10% FCS and 1% antibiotics for 7 days.
[0195] After the indicated number of days, kidney cultures were
fixed for 30 min with 4% paraformaldehyde in PBS. Fixed kidney
cultures were rinsed with TBS and extracted in absolute methanol at
-20.degree. C. for 20 min. Samples were then blocked for 1 hr in 3%
BSA in TBS-T at 4.degree. C. followed by incubation in
anti-E-cadherin, anti-cytokeratin, and/or anti-PAX-2 antibodies
(1:500 in blocking solution) for 24 hr at 4.degree. C. Samples were
then washed 3.times.8 hr in TBS-T, followed by incubation in
AlexaFluor594, AlexaFluor488 antibody (1:2000), and/or FITC-DB
(1:500) for 24 hr at 4.degree. C., and a final 3.times.8 hr washes
in TBS-T at 4.degree. C. Samples were then mounted on slides with
ProLong Gold.TM. antifade reagent (Invitrogen, Carlsbad, Calif.).
For PNA staining, following the blocking step the tissues were
washed twice with Neuraminidase buffer (150 mM NaCl, 50 mM sodium
acetate, pH 5.5), incubated with Neuraminidase (1 unit/ml) for 4 hr
at 37.degree. C. and then with rhodamine-conjugated PNA (50
.mu.g/ml) and FITC-DB (1:500) for 24 hr at 4.degree. C.
Fluorescently stained samples were imaged using either the Nikon
EZ-C1 confocal system.
[0196] Kidney cultures from HoxB7-GFP mice were fixed for 30 min
with 4% paraformaldehyde in PBS and rinsed 3.times.5 min in PBS.
Kidneys were then cleared with Focus Clear.TM. (Cedarlane
Laboratories, Burlington, N.C.) for 20 min and mounted on
depression slides with Mount Clear.TM. (Cedarlane Laboratories,
Burlington, N.C.). Samples were imaged on the FV300 Olympus
2-photon microscopy system. 3D Reconstructions, isosurfacing, and
3D measurements of fluorescent stacks were performed using Image
Pro Plus 3D Constructor 5.1 (Media Cybernetics, Bethesda, Md.).
Tissue thickness was determined as being the minimum distance
between two planes (or Feret minimum). Length to thickness ratio
was calculated as the Feret maximum to Feret minimum ratio. Kidney
volumes were estimated as the volume of an ellipsoid with the
dimensions of half the length, depth and width of a bounding box
around the branching structure. All samples were analyzed with
n.gtoreq.3 and with errors reported as the standard error of the
mean.
[0197] To investigate whether an epithelial tube with an apparently
homogenous cell population could function as a UB and retain the
capacities to undergo branching morphogenesis and induce MET, the
UB was removed from an E13 rat kidney and a segment of the WD from
an E13 rat kidney was put in its place. The E13 kidney with the UB
replaced by a segment of WD developed a branched collecting duct
system and induced MM to epithelialize in a manner similar to that
of traditional in vitro metanephric kidney culture. After 7 days of
in vitro culture, convoluted MM-derived epithelial structures
expressing E-cadherin were visible, suggesting the formation of
nascent nephrons. After 12 days of in vitro culture, in addition to
increased growth of the collecting duct system, a large number of
developing glomeruli were evident by PNA lectin staining. This
demonstrates that the epithelial tube of the WD can branch and form
a collecting duct structure that induces MM epithelialization and
glomerulus formation.
[0198] Since developmental approaches to tissue engineering begin
with a homogenous epithelial tube, the next step was to attempt to
construct an epithelial tube from a homogenous cell line possessing
the potential to act as a UB or WD.
[0199] Initially, the process of constructing an epithelial tissue
from cells was simulated using an immortalized mouse UB cell line.
This cell line is capable of undergoing tubulogenesis in
3-dimensional extracellular matrix gels in response to a
conditioned medium (CM) from a metanephric mesenchyme cell line
(BSN cell line). To test whether these cells are capable of
transforming into a UB tissue, the cells were induced to form cell
aggregates by growing the cells in hanging drop cultures. The
inductive and branching capacity of these UB cell aggregates was
then tested by establishing a culture system in which the
aggregates were combined with freshly isolated E13 rat MM. The UB
cell aggregates not only induced mesenchymal-to-epithelial
transition of the isolated MM cells, but connections between the UB
cell aggregate and isolated MM were also found, suggesting that
recombination of the MM with pre-formed aggregates of UB cells
results in the formation of a contiguous tissue segment,
reminiscent of the recombination of cultured isolated UB with MM
(REF). For example, the E-cadherin positive/DB negative MM-derived
tubule appears to be continuous with the D. biflorus
(DB)-positive/E-cadherin positive UB-cell-derived structure. While
the UB cell aggregate did not appear to branch (perhaps due to the
lack of a 3-D ECM), polarized epithelial multicellular structures
were observed.
[0200] A similar experiment was performed using the inner medullary
collecting duct (IMCD) cell line, a cell line derived from the
mouse adult collecting duct. This cell line is also capable of
undergoing branching tubulogenesis in 3D ECM cultures. In contrast
to UB cells, hanging drop aggregates of IMCD cells were capable of
self-organizing into tubules with lumens following recombination
with MM; additionally, although the IMCD cell aggregates did not
induce the MM to form multiple long tubules with lumens as is the
case with the UB cells, occasional formation of small comma-shaped
bodies could be found.
[0201] In addition to a UB-like epithelial tubule, construction of
a MM-like progenitor tissue derived from cultured cells would also
be advantageous for the bio-engineering of kidney or kidney-like
tissues. Therefore, it was investigated if any of the currently
available cultured MM cells have the potential to substitute for
the native progenitor tissue and whether the functions of the MM
can be recapitulated by a homogenous cell line. Initially, three
different MM-derived cell types (the well characterized BSN cell
line, a conditionally immortalized metanephric mesenchymal cell
line (RIMM-18), and primary rat E13 MM cells (which were found to
be vimentin positive and cytokeratin negative, similar to the BSN
cells and RIMM-18 cells (FIG. 12a-d)), as well as a 3T3 fibroblast
cell line (as a control) were tested for their ability to secrete
soluble factors capable of inducing isolated UB growth and
branching. Of the media tested only that elucidated by BSN cells or
by primary MM cells were capable of inducing isolated UB branching
morphogenesis (FIG. 12,g); however, the BSN-CM was substantially
more potent than the primary MM-CM (FIG. 12i). Although the RIMM-18
and 3T3 conditioned media did not induce UB branching, the 3T3-CM
(which contains the branch-promoting factor, pleiotrophin appeared
to induce slight globular growth of the isolated UB (FIG. 12h).
[0202] Experiments also shows that kidneys in the traditional
filter culture grew flat and along the filter, while kidneys
cultured in type I collagen or type IV collagen grew much thicker
and in a more 3D manner.
[0203] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *