U.S. patent application number 10/082261 was filed with the patent office on 2002-08-29 for methods and compositions for isolation and growth of kidney tubule stem cells, in vitro kidney tubulogenesis and ex vivo construction of renal tubules.
This patent application is currently assigned to The University of Michigan. Invention is credited to Humes, H. David.
Application Number | 20020119566 10/082261 |
Document ID | / |
Family ID | 46203791 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119566 |
Kind Code |
A1 |
Humes, H. David |
August 29, 2002 |
Methods and compositions for isolation and growth of kidney tubule
stem cells, in vitro kidney tubulogenesis and ex vivo construction
of renal tubules
Abstract
Methods, including culture media conditions, which provide for
isolation and purification of renal tubule stem cells and for in
vitro kidney tubulogenesis are disclosed. The methods rely on
culturing adult kidney cells in a culture media treated with
combinations of transforming growth factor-.beta..sub.1, epidermal
growth factor, and all-trans retinoic acid.
Inventors: |
Humes, H. David; (Ann Arbor,
MI) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
The University of Michigan
Ann Armbor
MI
|
Family ID: |
46203791 |
Appl. No.: |
10/082261 |
Filed: |
February 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10082261 |
Feb 26, 2002 |
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09494044 |
Jan 31, 2000 |
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6410320 |
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09494044 |
Jan 31, 2000 |
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08449912 |
May 25, 1995 |
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6060270 |
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08449912 |
May 25, 1995 |
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07844758 |
Mar 2, 1992 |
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5429938 |
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Current U.S.
Class: |
435/369 ;
435/385 |
Current CPC
Class: |
C12N 2510/00 20130101;
A61P 15/00 20180101; C12N 2533/54 20130101; C12N 5/0687 20130101;
A61K 48/00 20130101; C12N 2501/385 20130101; C12N 2502/28 20130101;
A61K 35/12 20130101; C12N 2501/148 20130101; C12N 2501/15 20130101;
A61P 13/02 20180101; C12N 2501/11 20130101 |
Class at
Publication: |
435/369 ;
435/385 |
International
Class: |
C12N 005/08 |
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. An ex vivo renal tubule tissue system prepared by a process
comprising culturing adult kidney cells in a culture medium
containing all-trans retinoic acid and either epidermal growth
factor or transforming growth factor in an amount effective for
achieving tubulogenesis.
2. The renal tubule tissue system of claim 1, wherein said cells
are further treated with transforming growth
factor-.beta..sub.1.
3. The renal tubule tissue system of claim 2, wherein said culture
medium contains a soluble factor selected from the group consisting
of fetal calf serum, prostaglandins, hydrocortisone,
triodothyronine, selenium, fibroblastic growth factor, hepatocyte
growth factor and combinations thereof.
4. The renal tubule tissue system of claim 2, wherein said culture
medium contains an insoluble factor selected from the group
consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
5. The renal tubule tissue system of claim 2, wherein said culture
medium contains a soluble factor selected from the group consisting
of fetal calf serum, prostaglandins, hydrocortisone,
triodothyronine, selenium, fibroblastic growth factor, hepatocyte
growth factor, and combinations thereof, and an insoluble factor
selected from the group consisting of Type I collagen, Type IV
collagen, laminin, proteoglycans, fibronectin, and combinations
thereof.
6. A transformed renal tubule cell produced by the process of
culturing renal tubule cells in a culture medium containing with
all-trans retinoic acid and either epidermal growth factor or
transforming growth factor-.alpha. in an amount effective to
achieve tubulogenesis, in the presence of a cell transformation
vector.
7. The transformed renal tubule cell of claim 6, wherein said cells
are further treated with transforming growth
factor-.beta..sub.1.
8. The transformed renal tubule cell of claim 7, wherein said
culture medium contains a soluble factor selected from the group
consisting of fetal calf serum, protaglandins, hydrocortisone,
triodothyronine, selenium, fibroblastic growth factor, hepatocyte
growth factor and combinations thereof.
9. The transformed renal tubule cell of claim 7, wherein said
culture medium contains an insoluble factor selected from the group
consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
10. The transformed renal tubule cell of claim 7, wherein said
culture medium contains a soluble factor selected from the group
consisting of fetal calf serum, prostaglandins, hydrocortisone,
triodothyronine, selenium, fibroblastic growth factor, hepatocyte
growth factor, and combinations thereof, and an insoluble factor
selected from the group consisting of Type I collagen, Type IV
collagen, laminin, proteoglycans, fibronectin, and combinations
thereof.
11. A method for growing kidney tubule stem cells ex vivo,
comprising culturing adult kidney cells in the presence of
all-trans retinoic acid, and either epidermal growth factor or
transforming growth factor-.alpha., in an amount effective for
growing kidney tubule stem cells.
12. The method of claim 11, wherein said cells are further treated
with transforming growth factor-.beta..sub.1.
13. The method of claim 12, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
14. The method of claim 12, wherein said culture medium contains an
insoluble factor selected from the group consisting of Type I
collagen, Type IV collagen, laminin, proteoglycans, fibronectin,
and combinations thereof.
15. The method of claim 12, wherein said culture medium contains a
soluble factor, selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor, and
combinations thereof, and an insoluble factor selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
16. A method for effecting tubulogenesis in a renal cell culture,
comprising culturing adult kidney cells in the presence of
all-trans retinoic acid and either epidermal growth factor or
transforming growth factor-.alpha. in an amount effective for
achieving tubulogenesis.
17. The method of claim 16, wherein said cells are further treated
with transforming growth factor-.beta..sub.1.
18. The method of claim 17, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
19. The method of claim 17, wherein said culture medium contains an
insoluble factor selected from the group consisting of Type I
collagen, Type IV collagen, laminin, proteoglycans, fibronectin,
and combinations thereof.
20. The method of claim 17, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor, and
combinations thereof, and an insoluble factor, selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
21. A method for constructing and maintaining an ex vivo renal
tubule tissue system, comprising culturing adult kidney cells in
the presence of all-trans retinoic acid and either epidermal growth
factor or transforming growth factor-.alpha. in an amount effective
for tubulogenesis.
22. The method of claim 21, wherein said cells are further treated
with transforming growth factor-.beta..sub.1.
23. The method of claim 22, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
24. The method of claim 22, wherein said culture medium contains an
insoluble factor selected from the group consisting of Type I
collagen, Type IV collagen, laminin, proteoglycans, fibronectin,
and combinations thereof.
25. The method of claim 22, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor, and
combinations thereof, and an insoluble factor selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
26. A method for effecting kidney repair, comprising administering
to a patient in need thereof all-trans retinoic acid and either
epidermal growth factor or transforming growth factor-.alpha. in an
amount effective for achieving kidney repair.
27. The method of claim 26, wherein said patient is further treated
with transforming growth factor-.beta..sub.1.
28. The method of claim 27, wherein said patient is treated with a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
29. The method of claim 27, wherein said patient is further treated
with an insoluble factor, selected from the group consisting of
Type I collagen, Type IV collagen, laminin, proteoglycans,
fibronectin, and combinations thereof.
30. The method of claim 27, wherein said patient is further treated
with a soluble factor, selected from the group consisting of fetal
calf serum, prostaglandins, hydrocortisone, triodothyronine,
selenium, fibroblastic growth factor, hepatocyte growth factor and
combinations thereof, and an insoluble factor selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
31. A method for effecting gene therapy upon renal tubule tissue,
comprising culturing adult kidney cells in a culture medium
containing all-trans retinoic acid and either epidermal growth
factor or transforming growth factor-.alpha. in an amount effective
to achieve tubulogenesis in the presence of a transforming
vector.
32. The method of claim 31, wherein said cells are further treated
with transforming growth factor-.beta..sub.1.
33. The method of claim 32, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
34. The method of claim 32, wherein said culture medium contains an
insoluble factor selected from the group consisting of Type I
collagen, Type IV collagen, laminin, proteoglycans, fibronectin,
and combinations thereof.
35. The method of claim 32, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor, and
combinations thereof, and an insoluble factor selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
36. A method for effecting kidney repair, comprising removing renal
cells from a patient in need of treatment, culturing said renal
cells in a culture medium containing all-trans retinoic acid and
either epidermal growth factor or transforming growth
factor-.alpha. in an amount effective for achieving a tubule tissue
system and returning said tubule tissue to said patient.
37. The method of claim 36, wherein said cells are further treated
with transforming growth factor-.beta..sub.1.
38. The method of claim 37, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof.
39. The method of claim 37, wherein said culture medium contains
insoluble factor selected from the group consisting of Type I
collagen, Type IV collagen, laminin, proteogiycans, fibronectin,
and combinations thereof.
40. The method of claim 37, wherein said culture medium contains a
soluble factor selected from the group consisting of fetal calf
serum, prostaglandins, hydrocortisone, triodothyronine, selenium,
fibroblastic growth factor, hepatocyte growth factor and
combinations thereof, and an insoluble factor selected from the
group consisting of Type I collagen, Type IV collagen, laminin,
proteoglycans, fibronectin, and combinations thereof.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to methods and compositions for
isolating, growing and transforming kidney tubule stem cells.
[0003] 2. Discussion of the Background
[0004] Renal failure is a common clinical syndrome, defined as a
decline in renal function, either acutely or chronically. The
clinical manifestations of this disorder arise from a decline in
glomerular filtration rate and an inability of the kidney to
excrete the toxic metabolic wastes produced by the body. The
complete treatment of this condition is dependent upon the
replacement of filtrative, reabsorptive, homeostatic and endocrine
functions of the kidney as an integrated organ structure.
[0005] In this regard, the function of a tissue is critically
dependent upon the spatial arrangement of its constituent cells.
The precise molecular determinants of such pattern formation both
in vitro and in vivo is complicated, but soluble factors, such as
growth factors, and insoluble factors, such as extracellular matrix
molecules, most likely play fundamental roles in this process.
Soluble molecules include both growth promoters (epidermal growth
factor) and growth inhibitors (transforming growth factor-.beta.).
Insoluble factors include complex extracellular matrices (collagen
gels, Matrigel) or extracellular matrix (ECM) molecules (laminin,
fibronectin, collagen types I and IV).
[0006] This critical interplay of structure and function is
demonstrated in the embryonic morphogenesis of the kidney, which is
dependent upon a finely orchestrated interaction between mesenchyme
and epithelium. The initial steps in differentiated nephrogenesis
are followed by the development of tubule epithelial cell polarity
and lumen formation. Coincident with the onset of cell polarity and
tubulogenesis, as defined by both morphologic and directional
transport properties, is the appearance of the A chain of laminin,
a cell attachment protein of the basement membrane, in the basal
regions of the mesenchymal cell aggregate. A sequential series of
growth and further differentiation processes then follows to result
eventually in a fully formed and functional kidney.
[0007] If this complex process of kidney organogenesis could be
mimicked in vitro, novel methods for the treatment of renal failure
could become available. Various tissue engineering products could
be constructed from both semi-synthetic and organic components for
complete replacement of renal function in patients with renal
failure. Such advances might also allow the development of
bioreactors comprising purely organic material for the substitution
of renal function in a patient whose kidneys are compromised. These
developments could also allow for kidney organogenesis, resulting
in the growth of an organic kidney in vitro, from the isolation of
renal tubule stem cells from a donor and subsequent growth and
differentiation. The in vitro kidney could later be transplanted to
the donor of the original renal cells, resulting in replacement of
renal function without any fear of transplant rejection or
immunosuppressive therapy. The availability of renal tubule stem
cells could also promote an efficient process for incorporation of
various genes into renal cells for gene therapy of various
diseases.
[0008] However, such developments are predicated upon the
development of a culture system which allows for isolation and
growth of kidney tubule stem cells and for in vitro
tubulogenesis.
[0009] Such a culture system has not been achieved in the prior
art, although in vivo kidney cells have demonstrated a potential
for differentiation and regeneration. As demonstrated by Humes et
al., J. Clin. Invest. 84:1757-61 (1989) and by Coimbra et al., Am.
J. Physiol. 259:F438-F443 (1990), complete recovery of renal
function can occur after severe nephrotoxic or ischemic acute renal
injury that was of a magnitude to produce complete renal failure.
Thus, some subset of renal proximal tubule cells apparently has the
ability in vivo to regenerate and form a fully functional,
differentiated epithelium. However, such results are doubtlessly
the result of the complex interaction of a large number of
biological factors responsible for growth, differentiation, pattern
formation and morphogenesis of the renal tubule.
[0010] Certain of these factors have been identified and employed
in renal cell cultures. TGF-.beta..sub.1 has been recently shown to
transform a monolayer of renal proximal tubule cells in primary
culture into a three-dimensional adhesive aggregate of cells, see
Humes et al, Lab. Invest. 64:538-545 (1991). EGF has been shown to
be a potent growth promoter for renal epithelial cells, see Norman
et al, Am. J. Physiol. 253:F299-F 309 (1987). Retinoic acid has
been reported to increase laminin synthesis in embryonic cell lines
by promoting laminin gene transcription, see Dziadek et al, Devel.
Biol. 111:372-382 (1985); Vasios et al, Proc. Natl. Acad. Sci.
(USA) 86:9099-9103 (1989); and Rogers et al, J. Cell. Biol.
110:1767-1777 (1990). However, these efforts of the prior art have
all failed to evoke tubulogenesis in renal cell cultures, such
tubulogenesis is the first step towards in vitro kidney
organogenesis.
SUMMARY OF THE INVENTION
[0011] Accordingly, it is an object of this invention to provide
novel methods, including culture media conditions, for in vitro
kidney tubulogenesis.
[0012] It is another object of the present invention to provide
novel methods for isolating and growing kidney tubule stem
cells.
[0013] It is another object of this invention to provide a novel,
functioning ex vivo kidney tubule tissue system.
[0014] It is another object of this invention to provide novel
methods, including culture media conditions, for culturing renal
tubule stem cells so as to effect gene therapy upon said cells.
[0015] The present invention provides methods and composition which
satisfy all of the above objects of this invention, and other
objects, as will be apparent from the description of the invention
given hereinbelow.
[0016] The present invention is based on the inventor's discovery
of novel methods, including culture media conditions, which provide
for isolation and growth of kidney tubule stem cells, for in vitro
kidney tubulogenesis and for ex vivo construction of renal tubules.
These methods rely on culturing renal proximal tubule cells in a
hormonally-defined culture medium which is treated with
combinations of transforming growth factor-.beta..sub.1
(TGF-.beta..sub.1), epidermal growth factor (EGF) and the retinoid,
all-trans retinoic acid (RA), while maintaining the culture under
physiologically acceptable conditions.
[0017] The inventor has discovered that the administration of
TGF-.beta..sub.1, EGF and all-trans retinoic acid transform a
confluent monolayer of renal proximal tubule cells into
three-dimensional cell aggregates containing lumens within the
interior of the cell clusters. The lumens were bordered by tubule
cells possessing a polarized epithelial cell phenotype with
extensive microvilli formation and tight junctional complexes along
the luminal border.
[0018] In one embodiment all three factors are used to isolate and
grow kidney tubule stem cells and to induce this phenotypic
transformation. In an alternate embodiment all-trans retinoic acid
and EGF are employed to isolate and grow kidney tubule stem cells
and to induce tubulogenesis. It is possible to substitute
transforming growth factor-.alpha. for EGF. These results
demonstrate that the growth factors, TGF-.beta..sub.1, and EGF, and
the retinoid, all-trans retinoic acid, promote tubulogenesis of
adult renal proximal tubule cells in kidney cell culture.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The inventor has found that the treatment of a
hormonally-defined renal cell culture with transforming growth
factor-.beta..sub.1, epidermal growth factor, and all-trans
retinoic acid transformed a confluent monolayer of renal proximal
tubule cells into epithelial cell aggregates containing lumens,
bordered by cells with a differentiated polarized epithelial cell
phenotype.
[0020] In a preferred embodiment of the present invention,
transforming growth factor-.beta..sub.1 is administered so as to
achieve a concentration of from 0.1 ng/ml-1 mg/ml, epidermal growth
factor in a concentration range of from 0.1 nM to 1 .mu.M, and
all-trans retinoic acid in a concentration range of from 0.01 .mu.M
to 100 .mu.M.
[0021] Another, optional but important, embodiment of the present
invention, resides in the addition of soluble factors to the renal
tubule stem cell culture. In a particularly preferred aspect of
this embodiment, these soluble factors include fetal calf serum,
prostagladins, hydrocortisone trioodothyronine, selenium,
fibroblastic growth factor, transforming growth factor-.alpha.,
hepatocyte growth factor, and combinations thereof.
[0022] These soluble factors are preferably added in the following
concentrations: fetal calf serum, 3-25% (volume/volume) of growth
media; prostaglandin E.sub.1, 1 to 100 .mu.g/ml; triiodothyronine,
0.1 nM to 1 .mu.M; selenium, 0.001 to 1.00 .mu.M; cholesterol, 1.0
nM to 0.10 .mu.M; transferrin, 1 to 50 Mg/ml; transforming growth
factor-.alpha., 0.1 nM to 1 .mu.M; insulin, 1-50 .mu.g/ml;
hydrocortisone, 1 nm to 1 .mu.M; and hepatocyte growth factor 0.1
ng/ml to 100 ng/ml.
[0023] Another, optional but important, embodiment of the present
invention, resides in the addition of insoluble factors to the
renal tubular stem cell culture. These insoluble factors include a
variety of extracellular matrix molecules. Included in these
extracellular matrix molecules are Type I collagen, Type IV
collagen, laminin, proteoglycans, fibronectin, and combinations
thereof.
[0024] These insoluble factors are preferably added in the
following concentrations: collagen, Type I, 1 to 5 mg/ml; collagen,
Type IV, 0.01 to 5 mg/ml; laminin, 10 to 1000 .mu.g/ml; heparin
sulfate, 10 to 1000 .mu.g/ml; and heparin, 10 to 1000 .mu.g/ml.
[0025] An additional preferred embodiment of the present invention
resides in the addition of both soluble factors and insoluble
factors to the renal tubule stem cell cultures.
[0026] In the present invention, the techniques used to obtain,
collect and grow the renal cells and the culture systems in which
the renal cells are grown are conventional ones and are described
in Taub et al, J. Biol. Chem., 254, 11440-11444; Taub et al, J.
Cell Physiol., 106 191-199; Taub et al, J. Supramol. Struct, 11,
207-216; Taub et al, J. Cell Physiol, 105, 369-378; Taub et al,
Proc. Nat. Accad. Sci. USA, 76, 3338-3342; Taub et al, Ann. New
York Acad Sci., 372, 406-421; Taub et al, J. Supramol. Struct., 15,
63-72; and Taub et al, J. Cell Physiol, 114, 153-161; which are
incorporated herein by reference.
[0027] In the present specification, the terms treatment and
treating refer to a process or means of exposing the cultured cells
to the substance being administered. The specific method of
treatment varies, depending on the properties of the substance. If
added in aliquots, the flow of the aliquot being added may be by
gravity, by pump, or by any other suitable means. The flow may be
in any direction or multiplicity of directions, depending upoh the
configuration and packing of the culture. Preferably, the substance
is added in a manner such that it contacts the cell mass. Most
preferably, it is added to the culture in a manner mimicking in
vivo perfusion, i.e., it is perfused through at least part of the
cell mass and up to the whole cell mass.
[0028] The term tubulogenesis denotes the de novo construction of
three-dimensional cell aggregates containing lumens within the
interior of the cell clusters. Such lumens are bordered by tubule
cells possessing a polarized epithelial cell phenotype with
extensive microvilli formation and tight junctional complexes along
the laminal border.
[0029] In one embodiment, the ex vivo renal tubule tissue system
may be implanted in a patient in need thereof. The renal cells
comprising such a tubule tissue system may be either transformed or
non-transformed cells. The implantation may be achieved by
conventional techniques, such as, by graft or insertion.
[0030] Moreover, the renal tubule stem cell cultures of the present
invention may serve as an important target in gene therapy. Gene
therapy is a rapidly growing field in medicine which is of
inestimable clinical potential. It comprises the insertion of genes
irro cells for the purpose of medicinal therapy. Research in gene
therapy has been on-going for several years and has been conducted
in several types of cells in vitro and in animal studies, and has
recently entered the first human clinical trial. Gene therapy has
many potential uses in treating disease and has been reviewed
extensively. See, e.g., Boggs, Int. J. Cell Cloning 8, 80 (1990),
Kohn et al, Cancer Invest. 7, 179 (1989), Lehn, Bone Marrow Transp.
5, 287 (1990), and Verma, Scientific Amer. 68 (1990). Suitable
genetic diseases and disorders that may be treated with the
transformed renal tubule-stem cells of the present invention are
described in Ellis, Inborn Errors of Metabolism, Croom Helm London,
1980, and Galjaard, Genetic Metabolic Diseases, Elsevier, N.Y.,
1980, which are incorporated herein by reference.
[0031] The renal tubule stem cell system is an ideal choice for
gene therapy. However, up to the present time renal tubule stem
cells have not been accessible. With the methods and compositions
of the present invention, renal tubule stem cells are now readily
available for gene therapy, can be maintained in culture for
unlimited periods of time, and upon reimplantation, may replace
kidney function.
[0032] The renal tubule stem cells can be transformed with one or
more genes providing for desired traits. Methods for transforming
mammalian cells are well known and there is an extensive literature
of which only a few references have been previously given. The
constructs may employ the naturally occurring transcriptional
initiation regulatory region, comprising the promoter and, as
appropriate the enhancer, or a different transcriptional initiation
region may be involved, which may be inducible or constitutive.
[0033] A large number of transcriptional initiation regions are
available which are inducible or constitutive, may be associated
with a naturally occurring enhancer, or an enhancer may be
provided, may be induced only in a particular cell type, or may be
functional in a plurality or all cell types. The transcriptional
initiation region may be derived from a virus, a naturally
occurring gene, may be synthesized, or combinations thereof.
[0034] Promoters which are available and have found use include the
chromosomal promoters, such as the mouse or human metallothionein-I
or II promoters, actin promoter, etc., or viral promoters, such as
SV40 early gene promoters, CMV promoter, adenovirus promoters,
promoters associated with LTRs of retroviruses, etc. These
promoters are available and may be readily inserted into
appropriate vectors which comprise polylinkers for insertion of the
transcriptional initiation region as well as the gene of interest.
In other instances, expression vectors are available which provide
for a polylinker between a transcriptional region, also providing
for the various signals associated with the processing of the
messenger for translation, i.e., the cap site and the
polyadenylation signal. The construction of the expression cassette
comprising the regulatory regions and the structural gene may
employ one or more of restriction enzymes, adapters, polylinkers,
in vitro mutagenesis, primer repair, resection, or the like.
[0035] The expression cassette will usually be part of a vector
which will include a marker and one or more replication systems.
The marker will allow for detection and/or selection of cells into
which the expression cassette and marker have been introduced.
Various markers may be employed, particularly markers which provide
for resistance to a toxin, particularly an antibiotic. Preferably,
gentamicin resistance is employed, which provides resistance to
G418 for a mammalian cell host. The replication systems may
comprise a prokaryotic replication system, which will allow for
cloning during the various stages of bringing together the
individual components of the expression cassette. The other
replication system may be used for maintenance of an episomal
element in the host cell, although for the most part the
replication system will be selected so as to allow for integration
of the expression cassette into a chromosome of the host.
[0036] The introduction of the expression cassette into the host
may employ any of the commonly employed techniques, including
transformation with calcium precipitated DNA, transfection,
infection, electroporation, ballistic particles, or the like. Once
the host cells have been transformed, they may be amplified in an
appropriate nutrient medium having a selective agent, to select for
those cells which comprise the marker. surviving cells may then be
amplified and used.
[0037] Host cells which may be employed include African green
monkey cell line CV1, mouse cells NIH-3T3, normal human bone marrow
fibroblasts, human spleen fibroblasts, normal mouse bone marrow
fibroblasts, and normal mouse spleen fibroblasts. It should be
noted that in some instances, depending upon the choice of vector
and cell line, the cells may become neoplastic.
[0038] Once the vector for expressing the appropriate trait has
been constructed, it may be used to transform the cells by any
convenient means. The cells will be allowed to grow for sufficient
time to ensure that the cells are viable and are capable of
producing the desired traits.
[0039] The term transforming vector or cell transformation vector
as used in the present invention refers to a DNA molecule, usually
a small plasmid or bacteriophage DNA capable of self-replication in
a host organism, and used to introduce a fragment of foreign DNA
into a host celll.
[0040] Other features of the present invention will become apparent
in the course of the following descriptions of exemplary
embodiments which are given for illustration of the invention and
are not intended to be limiting thereof.
[0041] Exemplary embodiments
[0042] Cell Culture
[0043] Rabbit renal proximal tubule cells were grown in primary
culture by techniques reported by Humes et al. in Lab. Invest.
64:538-45 (1991). The cells were grown in 35 mm Corning culture
dishes with serum-free, hormonally defined Dulbecco's Modified
Eagle Hams F-12 media (1:1, v/v) containing L-glutamine,
penicillin, strentomycin, 50 nM hydrocortisone, 5 .mu.g/ml of
insulin, and 5 .mu.g/ml of transferrin. The cultures were
maintained in a humidified 5% C0.sub.2/95% air incubator at
37.degree. C. Medium was changed every 3 to 5 days, depending on
nutrient requirements based upon cell number. Cultures became
confluent in 9 to 12 days. Once confluent, various agents as
described below were added in 20 .mu.l aliquots at various times to
promote changes in morphologic phenotype or pattern formation.
[0044] Morphology
[0045] Specimens for ultrastructural analysis were fixed with 2%
glutaraldehyde in Sorenson's buffer (pH 7.2, 310 mOsm).
Postfixation occurred in 1% OsO.sub.4 followed by dehydration in
ethanol. Specimens were transferred through propylene oxide into
monomer mixture (poly/Bed 812A, Araldite, DDSA and DMP-30) and
polymerized at 60.degree. C. Thin sections were stained with uranyl
acetate and lead citrate, and examined in a Zeiss 9-S2 transmission
electron microscope.
[0046] Materials
[0047] All reagents used were of the highest grade commercially
available. All organic reagents were obtained from Sigma Chemical
Company (St. Louis, Miss.) unless otherwise indicated. EGF
(recombinant human) was obtained from Amgen Biologicals (Thousand
Oaks, Calif., TGF .beta..sub.1 (porcine platelets) from R & D
Systems (Minneapolis, Minn.). TGF-.beta..sub.1 was dissolved in 4
mm HCl and 1 mg/ml bovine serum albumin; EGF in aqueous buffer, and
RA in 95% ethanol.
[0048] Results
[0049] Simultaneous treatment of density-arrested, confluent
monolayers of adult rabbit renal proximal tubule cells with
TGF-.beta..sub.1, EGF, and RA resulted in a dramatic phenotypic
transformation characterized by condensed aggregates of cells in
cord-like structures. Evaluation of these cellular aggregates by
light and transmission electron microscopy revealed the presence of
lumen formation within the interior of the cell aggregates. The
lumens were bordered by tubule cells possessing a polarized
epithelial phenotype with extensive microvilli formation and tight
junctional complexes along the luminal border. Of note, in each
aggregation of cells one or two lumen structures were usually
observed in random sectioning of tissue culture preparations. The
lumens and bordering polarized epithelial cells were surrounded by
nonpolarized, adherent cells which did not possess tight junctional
complexes.
[0050] All three factors are necessary for tubulogenesis, as
demonstrated by the following experiments.
EXAMPLE 1
[0051] A density-arrested, confluent monolayer of renal proximal
tubule cells in standard tissue culture was simultaneously treated
with TGF-.beta..sub.1 (10 ng/ml), EGF (1 nM), RA (0.1 .mu.M). After
144 hours of treatment, the cell culture had undergone a
transformation of the monolayer into adherent cell aggregates
containing an area with a well defined lumen, as seen by light
microscopy (magnification x400). Utilizing transmission microscopy,
the lumen could be seen to be bordered by tubule cells possessing a
polarized epithelial cell phenotype with well-developed epithelial
microvilli (magnification x6500). Further magnification (x19900)
with electron microscopy demonstrated that tight junctional
complexes existed between the cells bordering the lumen near the
apical surface.
COMPARATIVE EXAMPLE 2
[0052] Addition of TGF-.beta..sub.1 (10 ng/ml) to a
density-arrested, confluent monolayer of renal proximal tubule
cells resulted in a phenotypic transformation of the monolayer to
form solid aggregates of adherent cells as observed by light
microscopy (magnification x400). Cells lining the surface of the
cell aggregates possessed occasional broad based microvilli and
tight junctional complexes, but no lumen formation was
observed.
COMPARATIVE EXAMPLE 3
[0053] Simultaneous treatment of a confluent monolayer with
TGF-.beta..sub.1 (10 ng/ml) and EGF (1 nM) produced a similar
morphologic transformation into cell aggregates which were, in
general, larger than those seen with TGF-.beta..sub.1, due to
greater number of adherent cells within the condensed aggregate, as
observed by light microscopy (magnification x400). However, no
lumen formation or polarized epithelial phenotype was found.
COMPARATIVE EXAMPLE 4
[0054] The simultaneous exposure of the epithelial monolayer to
TGF-.beta..sub.1 (10 ng/ml) and RA (0.1 .mu.M) promoted
intracytoplasmic vacuolization as observed by light microscopy
(magnification x400). However, these areas never developed into
lumens with polarized epithelial phenotype, in contrast to the
experiment with TGF-.beta..sub.1, RA and EGF, administered in
combination.
COMPARATIVE EXAMPLE 5
[0055] Treatment of the monolayer with RA alone (0.1 .mu.m) or RA
(0.1 .mu.m) and EGF (1 nm) had no dramatic effect on the appearance
of the monolayer.
[0056] These results indicate that transforming growth
factor-.beta..sub.1, epidermal growth factor, and all-trans
retinoic acid play important roles in tubulogenesis.
[0057] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
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