 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
Departments of *Pediatrics, Pediatric Hemato-oncology and Functional Genomics Unit, and Nuclear Medicine, Sheba Medical Center, Tel Hashomer, Israel; Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and ||Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Correspondence to Dr. Benjamin Dekel, Department of Pediatrics, Sheba Medical Center, Tel Hashomer, Israel 52621. Phone: 972-3-530-2445; Fax: 972-8-9344141; E-mail: bmdekel{at}wicc.weizmann.ac.il
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several reports have documented development of murine metanephroi after transplantation (1–3). Rogers et al. (4) studied the feasibility of transplanting murine metanephroi in the omentum of rat hosts sufficiently near one of the ureters so as to render connection between the transplant and the host’s urine system possible. When the murine transplants were connected to the host’s urine system, they were shown to be functional, and clear inulin was infused into the host’s circulation. Such anastomosis was made possible after transplanted murine metanephroi had developed.
We have previously established human adult and fetal kidney grafts in immunodeficient rat hosts (5,6). The fetal transplants achieved rapid growth and long-term survival in vivo. Moreover, when studying the transplant immunology of the human kidney precursors, our small-animal model demonstrated a reduced alloimmune response of allogeneic human peripheral blood mononuclear cells to the metanephric transplants when compared with that of transplants of adult kidney tissue (5–7). Thus, the immunologic advantage of human metanephroi over the adult kidney in avoiding graft rejection was shown (8). Clearly, in vivo differentiation of human metanephric transplants into functional mature nephrons is critical if such were to be applicable as donor tissue in clinical practice. Moreover, a better understanding of the molecular signals that stimulate this process might lead to improvement in the differentiative potential after transplantation.
Therefore, in this report, with the use of cDNA arrays, we have examined the global mRNA expression of cell cycle and DNA replication regulators, transcription and growth factors, and signaling, transport, and adhesion and extracellular matrix (ECM) molecules in human fetal kidneys at various times during gestation. Moreover, we were able to monitor gene expression patterns in vivo in our model of transplanted human metanephroi and to determine to what extent they resemble those involved in the induction of the normal kidney or the transformation into an embryonic kidney malignancy (i.e., a Wilms tumor). In addition, we assessed the functional capacity of the newly formed nephrons in the developing human metanephric transplants.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human Fetal Kidneys
Normal human kidneys at 8, 12, 16, and 20 wk gestation were obtained after curettage of elective abortions. Studies with embryonic kidney tissue were approved by the Helsinki ethical committee. The kidneys were fixed in 10% paraffin, sectioned, and mounted on slides coated with poly-L-lysine, and sections were stained with hematoxylin and eosin for histologic evaluation.
Establishment of Human Kidney Grafts into NOD/SCID Mice
Human embryonic kidneys, 70 d gestation, were kept in sterile conditions at 4°C (for approximately 2 h) in either RPMI 1640 or Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (Biological Industries, Bet HaEmek, Israel). Transplantation of the human metanephroi was performed with the mice under general anesthesia (2.5% Avertin in phosphate-buffered saline, 10 ml/kg intraperitoneally). Both kidneys were exposed through a bilateral incision. A 1.5-mm incision was made at the caudal end of the kidney capsule, and a 1-mm3 fragment of human kidney was implanted under the left capsule. Mice that received transplants were treated postoperatively with ciprofloxacin in their drinking water for 7 d. Animals were killed at 2, 6, and 10 wk after transplant. Human renal tissue was initially assessed for engraftment by macroscopic examination (size, color) of the transplants at the subcapsular site. Kidneys and their capsules were then removed and fixed in 10% paraffin. Human grafts were sectioned and mounted on slides coated with poly-L-lysine, and sections were stained with hematoxylin and eosin for histologic evaluation.
RNA Preparation
Normal human embryonic kidney tissue and human embryonic kidney grafts dissected carefully from the subcapsular site of NOD/SCID mice were homogenized with a glass-Teflon tissue tearer in 1 ml of Tri-reagent (Molecular Research Center, Cincinnati, OH). RNA was isolated as described previously (9).
cDNA Array Hybridization
We used cDNA arrays of human cDNA spotted on a nylon membrane (Atlas filter arrays; Clontech, Palo Alto, CA) as previously described (9). We used the human 1.2 expression arrays (broad-coverage arrays), which contain 1176 cDNA. The filters also include housekeeping control cDNA. A complete list of the cDNA and controls and their accession numbers is available on the Internet (http://www.clontech.com/atlas/genelists/index.html).
Total RNA were treated with DNAse I according to the manufacturer’s instructions and used for cDNA synthesis. A 3-µl mix containing 5 µg of total RNA and 1 µl of 10x cDNA synthesis primer mix (specific for each filter array, provided by Clontech) was incubated at 70°C for 2 min, followed by incubation at 48°C for 2 min. To this mix, 8 µl of master mix containing 2 µl 5x reaction buffer, 1 µl 10x dNTP mix, 3.5 µl [32P]-dATP (3000 Ci/mmol, 10 mCi/ml; Amersham Pharmacia Biotech, Buckingham, UK), 0.5 µl 100 mM dithiothreitol, and 1 µl Moloney murine leukemia virus reverse transcriptase (50 U/µl) was added, mixed, and incubated for 25 min at 48°C. The reaction was terminated by adding 1 µl of 10x termination mix at room temperature.
The radioactively labeled cDNA mix was fractionated on a Chroma Spin-200 column (Clontech), and fractions that comprised the first peak of radioactivity were pooled for each cDNA synthesis reaction. In each set of hybridization, equal counts were taken for control and experimental labeled cDNA probes. The labeled cDNA probe was then mixed with 1/10 volume of 10x denaturing solution (1 M NaOH, 10 mM ethylenediaminetetraacetate) and incubated at 68°C for 20 min, followed by the addition of 5 µl (1 µg/µl) of cot-1 DNA and an equal volume of 2x neutralizing solution (1 M NaH2PO4, pH 7.0), and incubated at 68°C for 10 min. Denatured, labeled cDNA was then added to 5 ml of ExpressHyb solution (Clontech) with 1 mg of sheared salmon sperm DNA (Sigma Chemical Co., St. Louis, MO) and mixed. This hybridization solution was added to the Atlas cDNA Expression Array membrane, which was prehybridized in 10 ml of ExpressHyb hybridization solution at 68°C for 1 h. Hybridization proceeded overnight at 68°C in a roller bottle. Membranes were washed once with prewarmed 2x standard saline citrate/1% sodium dodecyl sulfate for 30 min and once or twice with 0.5x standard saline citrate/0.5% sodium dodecyl sulfate for 30 min at 68°C with constant agitation. The membranes were exposed to Fuji (Tokyo, Japan) x-ray films at 70°C with intensifying screens.
Analysis of Hybridization Signals
The cDNA microarray autoradiograms were scanned, and the images were analyzed by AtlasImage software, version 1.05 (Clontech). The background was calculated by default external background calculation, which takes into consideration the background signals at the blank space between the different panels of the arrays. Signal threshold was set as background-based signal threshold. The signal intensities were normalized globally by the sum method (AtlasImage; Clontech). A report of differentially expressed genes was generated on the basis of ratio and intensity differences.
Reverse Transcriptase-PCR Amplification
Total RNA was determined, and 1 µg of total RNA was reverse-transcribed into cDNA and amplified with the Access reverse transcriptase-PCR kit (Promega, Madison, WI) and with specific primers for human neu-differentiation factor (NDF), homeobox protein hLim1, mitogen-activated protein kinase p38, kidney glomeruli chloride channel, and our housekeeping gene, ß-actin. The primers used were as follows: 5'-ACC-ATC-AAG-CTC-TGC-GTG-ACT-G-3' (sense) and 5'-GCA-GGT-CAG-TTC AGT-TCC-AGG-TC-3' (antisense) for ß-actin (310 bp); 5'-CAA-AGA-AGG-CAG-AGG-CAA-AG-3' (sense) and 5'-ACC-ACT-TGA-ATC-TGA-GAG-AGG-3' (antisense) for human NDF (340 bp); 5'-CGT-CGT-CTT-CTT-CTC-3' (sense) and 5'-CAG-GTT-GCA-TTT-ACA-TTC-3' (antisense) for human Lim1 (506 bp); 5'-CCC-AGG-AGT-CCG-TAA-GTA-G-3' (sense) and 5'-ACT-GGA-GAC-AGG-TTC-TTG-3' (antisense) for human mitogen-activated protein (p38) kinase (200 bp); and 5'-TGA-TGT-GAT-ATG-GCT-GCA-AG-3' (sense) and 5'-TGT-AGG-CAA-AGG-CTC-CCT-C-3' (antisense) for human kidney chloride channel (700 bp). After amplification, the sample was separated on an agarose gel containing ethidium bromide, and bands were visualized and photographed with an ultraviolet transilluminator. The image densities were measured with Image software, version 1.62 (National Institutes of Health, Bethesda, MD), and semiquantitative results were expressed as a ratio of each reverse transcriptase-PCR product to ß-actin density (10). Three independent experiments were performed for each PCR determination.
Immunohistology
Immunohistochemistry for the stem cell leukemia (SCL) protein was performed via a monoclonal anti-SCL/TAL1 antibody (2TL242; provided by Dr. Karen Pulford, Oxford, England, UK). Briefly, 5-µm sections of were mounted on Super Frost/Plus glass (Menzel, Glazer, Braunschweig, Germany) and processed by the labeled streptavidin-biotin method with a Histostain Plus kit (Zymed, San Francisco, CA). Heat-induced antigen retrieval was performed by controlled microwave treatment with a model H2800 processor (Energy Bean Sciences, Agawan, MA) in 10 mM citrate buffer, pH 6.0, for 10 min at 97°C. The sections were treated with 3% H2O2 for 5 min. Consecutive sections were incubated for 1 h with the 2TL242 antibody. Negative control incubations were performed by substituting nonimmune serum for the primary antibody (the control sections were entirely negative).
Biotinylated second antibody was applied for 10 min, followed by incubation with horseradish peroxidase-conjugated streptavidin for 10 min. After each incubation, the slides were thoroughly washed with OptiMax wash buffer (Biogenex, San Ramon, CA). The immunoreaction was visualized by a horseradish peroxidase-based chromogen/substrate system, including diaminobenzidine (brown) chromogen (liquid diaminobenzidine substrate kit; Zymed). The sections were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted for microscopic examination.
Statistical Analyses
Comparisons between groups were evaluated by the unpaired t test. Data were expressed as mean ± SEM and were considered statistically significant at P 
0.05.
Dimercaptosuccinic Acid Radioisotope Scans after Intra-abdominal Transplantation of Human Metanephroi
In some experiments, metanephroi were implanted and sutured (5-0 suture) onto the testicular fat of NOD/SCID mice while they were under general anesthesia (2.5% Avertin in phosphate-buffered saline, 10 ml/kg intraperitoneally). During the same surgery, host mice had one kidney removed. Dimercaptosuccinic acid (DMSA) scans were performed at different times after transplantation. Briefly, each mouse was injected intravenous in a tail vein with 1 x 10-3 mCi technetium-99m DMSA. Animals were sedated 2 h after injection with 2.5% Avertin in phosphate-buffered saline, 10 ml/kg intraperitoneally. Whole-body images were acquired in the posterior view on a gamma camera equipped with a pinhole collimator (Elscint Sp4, Haifa, Israel). Images were acquired to a total of 100 K. After imaging, animals were killed, and the transplants were excised and imaged. The mice were further imaged in the same manner after the transplants had been removed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Functionality of Human Metanephric Transplants Established in NOD/SCID Mice
After establishment of human metanephric transplants in mice, we assessed their tubular function with 99technetium-DMSA renography. We performed intra-abdominal rather than renal subcapsular transplantation to discriminate between uptake of native kidney and transplanted metanephroi (see Materials and Methods). DMSA was administered at different time points after transplant, and animals were then killed. Table 1 summarizes the DMSA experiments. We could not detect DMSA in the younger transplants (3 to 4 wk after transplant). However, positive uptake was clearly demonstrated in the older metanephric grafts (8 to 10 wk after transplant) and declined upon their removal (Figure 2, A through C). Positive uptake of DMSA was found only when unilateral nephrectomy was performed and not when both native kidneys were left intact (Table 1). This might be related to decreased perfusion of the transplant (vascularized by peripheral vessels and not by end-to-end anastomosis) compared with the native kidneys (steal effect). Thus, maturation of the human metanephric transplants with time (3 to 10 wk) was associated with increased uptake of DMSA.
|
|
Figure 3 illustrates the results of hybridization to cDNA arrays when RNA used for probes was derived from the 12-, 16-, and 20-wk-old kidneys as well as from the t12-, t16-, and t20-wk transplants. A representative section (all of the E coordinates) from the atlas human 1.2 array (Clontech) representing 196 cDNA that profile transcription factors and DNA-binding proteins, ECM and adhesion proteins, cell surface antigens, and growth factors is shown. Expression levels of 1176 genes in 12-, 16-, 20-, t12-, t16-, and t20-wk kidneys were determined against baseline 8-wk-old kidneys (Figure 3G), as assessed by screening the differences in hybridization results with AtlasImage software. Only when expression levels deviated from that in the 8-wk-old kidneys by a factor >2.0 or <0.5 in at least 2 of the samples from the 12-, 16-, and 20-wk old kidneys or from the t12-, t16-, and t20-wk transplants were they considered substantial.
|
|
|
|
|
In contrast, c2 contained genes that were relatively upregulated after transplantation of metanephroi, most of which were related to cell and oxidative stress (9 of 19; glutathione-S-transferases 1, 3, and 12, heat shock proteins 90, 27, 40, and 60 kD, and glutathione and thioredoxin reductase) or angiogenesis (5 of 19; vascular endothelial growth factor [VEGF], VEGF receptors 1 and 2, angiopoietin 1 receptor, and endothelial monocyte-activating polypeptide 2). Thus, our approach could not only determine similar global patterns of gene expression in both normal and transplanted developing kidneys, but also could pinpoint sets of genes that are increased in the metanephric transplants and fall under specific functional categories.
Although cluster analysis provides for profiles of gene expression and the behavior of groups of genes, it does not identify differences at the level of a single gene. We therefore set out to determine differences between average mRNA levels of specific genes in the normal and transplanted kidneys. An example of such an analysis, here applied to specific genes that function in transport and growth and whose expression changed substantially in the induction of the normal and transplanted kidney, is illustrated in Figure 6. Analysis of genes that encode transport proteins (potassium [kv12] and chloride channel [ICLN] proteins, glucose [GLUT1], zinc, and Golgi transporters) demonstrated that although they increased steadily throughout development in both normal and transplanted metanephroi, thus showing similar temporal expression profiles (see cluster c3), mRNA levels were significantly decreased in the developed transplant (t20) compared with levels in the corresponding kidney at 20 wk gestation (Figure 6).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We therefore took a fresh look at the temporal program associated with the induction of the human kidney by use of cDNA arrays representing approximately 1200 known human genes. A coordinated induction of groups of genes was demonstrated during normal nephron formation, including DNA-binding and DNA transcription factors, proto-oncongenes, signaling molecules, growth factors or hormones and their receptors, cell adhesion and cytoskeletal molecules, and ECM glycoproteins and receptors, and genes associated with transport and metabolism. Genes induced in this program encode products that can (1) participate in the molecular regulation of growth and branching of the ureteric duct (e.g., PTN, c-ret, EGF-R, FGF-R, BMP-2, BMP-4, TGF-ß, activin, follistatin, MMP-2) (17,21–23); (2) participate in the molecular regulation of mesenchymal-to-epithelial conversion, formation of basal lamina, cell polarization, and postinductive tubulogenesis (e.g., hLim1, c-ret, c-ros-1, collagen type IV, integrins 
6, 3, ß1, ZO-1, junction plakoglobin, BDNF/NT3) (24–27); and (3) modulate angiogenesis, neovascularization, and glomerulogenesis (e.g., VEGF, VEGF-R, EMAP-2, angiopoietin-1 receptor, ephrin receptors, PDGFR, PDGFRß, integrin 3) (25,28–30).
In addition, we observed genes that have no recognized role in any aspect of kidney development. The NDF gene, for instance, encodes a protein that acts either as a differentiation factor for neuronal development or as a mitogen for mammary tumor cells (31). A recent in vitro study revealed that mesenchymally derived HGF, kerotinacyte growth factor could activate autocrine NDF signaling in their epithelial targets (32). Thus, the NDF protein might act in concert with these growth factors to direct epithelial proliferation, morphogenesis, and differentiation. Another example is the SCL gene. SCL, an established regulator of hematopoiesis, has been shown to play a pivotal role in hemangioblast formation and endothelial development (33). Thus, its presence in the developing metanephroi might suggest a role in vasculogenesis.
Although many genes were induced during the developmental process, the biologic relevance of our data became apparent only after assigning the genes into functional categories and clustering them into groups on the basis of the similarity of their temporal expression profiles. We could demonstrate the induction of nonspecific regulatory replicative machinery (genes that function in cell cycle, replication and DNA repair, transcription, and growth) that characterizes early development, and thereafter the predominance of groups of genes committed to a more specialized task and terminal differentiation (adhesion and ECM molecules, transporters, and genes related to metabolism and protein turnover). The general outline of these results is similar to that found by Stuart et al. (34) when describing the transcriptional program associated with rat kidney maturation. Given the complex nature of the molecular program we obtained for several stages of normal kidney growth, it is likely that redundancies among growth factors and other molecules may exist in term of the role they play in kidney development in vivo.
Strikingly, many of the details of the molecular program required to generate and build a nephron after transplantation of human kidney precursors into living animals are similar, in a global sense, to normal kidney induction. Comparison of temporal expression profiles demonstrated that the time course for development of normal human kidneys is applicable to that for development of transplanted human metanephroi. Comparison of the expression profiles of developing metanephric transplants to a Wilms tumor specimen revealed no similarity and reassured us that the threat of malignant transformation after transplantation of human embryonic kidney precursors does not exist. A functional test devised by intravenous administration of DMSA, a radioisotope known to be extracted and secreted by mature, functional renal tubules, revealed that this function is maintained in the transplanted metanephroi that had developed. Thus, after transplantation, human metanephroi integrate into host tissue microenvironment (including host blood vessels), survive, and function with available growth and regulatory signals.
Nevertheless, our analysis has revealed several limitations after transplantation of human metanephroi. First, early after grafting, we found elevated levels of genes that function in cell stress and angiogenesis, a response that might be related to both ischemia and neovascularization of the embryonic kidney graft (35). Relative ischemia and consequent injury might account for the mostly low expression levels of human genes in the early time points after transplantation compared with normal kidneys. Minimizing the time to grafting or developing a preservation system for the human metanephroi (36) could reduce the magnitude of ischemia. Second, the failure of genes encoding for various transporters to sufficiently increase their levels after transplantation, similar to normal kidney development, could possibly limit transplant function with time. Kim et al. (37) have demonstrated diminished renal expression of aquaporin water channels in rats with experimental ureteral obstruction. We have not yet been able to establish adequate anastomosis of the developing ureter of the human metanephric transplants, and thus gene expression of at least some of the channels in the transplants might have been similarly altered.
Finally, a comparison of a subset of genes encoding growth factor proteins, which have a recognized and important role in kidney development (21,24,25), detected mostly decreased transcript levels in the transplants compared with normal kidneys. There is a possibility that the xenogeneic microenvironment can compensate and support the differentiation of the transplanted human metanephroi by secreting diffusible proteins, or alternatively via growth factors present in the peripheral circulation of host mice. Nevertheless, these growth factors can be easily administered alone or in combination at a specific time after transplantation to further stimulate donor human metanephroi (Figure 6). On the basis of our results, an attractive option would be for instance to administer insulin growth factor early after transplantation and administer HGF and fibroblast growth factor later on.
The concept of gene dosage and nephron development/endowment has emerged from recent studies (thoroughly reviewed by Clark and Bertram [24]). These data demonstrate an enormous range in nephron number in human kidneys and suggest that low nephron numbers might be associated with acquired kidney disease (38,39). Several processes, which are regulated by specific genes, ultimately govern the number of nephrons during the development of human metanephroi—hence molecular regulation of nephron endowment. Furthermore, the dosage of specific genes also appears critical to the establishment of adequate nephron endowment. Thus, if transplanted human metanephroi maintain a temporal profile of gene expression similar to normal kidney development and the dosage of specific genes is reduced, nephrogenesis evidently pursues, but nephron endowment is limited. Future studies aimed at increasing gene dosage in the transplanted human metanephroi could determine whether nephron endowment improves.
We suggest that our approach of screening global gene expression in transplanted differentiating embryonic kidney cells versus normally developing cells might be relevant to other systems that follow the fate of embryonic cells after transplantation and elucidating factors that might drive them toward a specific lineage and organ. Furthermore, as far as directing embryonic cells solely toward human kidneys in vivo, our results indicate that it might be worthwhile to first use human embryonic precursors that have already differentiated into the nephric lineage rather than more pluripotent cells (40).
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Metsuyanim, N. Pode-Shakked, K. M. Schmidt-Ott, G. Keshet, G. Rechavi, D. Blumental, and B. Dekel Accumulation of Malignant Renal Stem Cells Is Associated with Epigenetic Changes in Normal Renal Progenitor Genes Stem Cells, July 1, 2008; 26(7): 1808 - 1817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Marshall, M. R. Dilworth, M. Clancy, C. A. Bravery, and N. Ashton Renal: Increasing renal mass improves survival in anephric rats following metanephros transplantation Exp Physiol, January 1, 2007; 92(1): 263 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Little Regrow or Repair: Potential Regenerative Therapies for the Kidney J. Am. Soc. Nephrol., September 1, 2006; 17(9): 2390 - 2401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Dekel, S. Metsuyanim, K. M. Schmidt-Ott, E. Fridman, J. Jacob-Hirsch, A. Simon, J. Pinthus, Y. Mor, J. Barasch, N. Amariglio, et al. Multiple Imprinted and Stemness Genes Provide a Link between Normal and Tumor Progenitor Cells of the Developing Human Kidney. Cancer Res., June 15, 2006; 66(12): 6040 - 6049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Dekel, E. Shezen, S. Even-Tov-Friedman, H. Katchman, R. Margalit, A. Nagler, and Y. Reisner Transplantation of Human Hematopoietic Stem Cells into Ischemic and Growing Kidneys Suggests a Role in Vasculogenesis but Not Tubulogenesis Stem Cells, May 1, 2006; 24(5): 1185 - 1193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Brodie and H. D. Humes Stem Cell Approaches for the Treatment of Renal Failure Pharmacol. Rev., September 1, 2005; 57(3): 299 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Prindull and D. Zipori Environmental guidance of normal and tumor cell plasticity: epithelial mesenchymal transitions as a paradigm Blood, April 15, 2004; 103(8): 2892 - 2899. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Steer and S. K. Nigam Developmental approaches to kidney tissue engineering Am J Physiol Renal Physiol, January 1, 2004; 286(1): F1 - F7. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Hammerman Xenotransplantation of developing kidneys Am J Physiol Renal Physiol, October 1, 2002; 283(4): F601 - F606. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||
