Human embryonic stem cells differentiate into functional renal proximal tubular–like cells
2013; Elsevier BV; Volume: 83; Issue: 4 Linguagem: Inglês
10.1038/ki.2012.442
ISSN1523-1755
AutoresKarthikeyan Narayanan, Karl Maria Schumacher, Farah Tasnim, Karthikeyan Kandasamy, Annegret Schumacher, Ming Ni, Shujun Gao, Began Gopalan, Daniele Zink, Jackie Y. Ying,
Tópico(s)Organ Donation and Transplantation
ResumoRenal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular–like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells. Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular–like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells. Renal proximal tubular cells (PTCs) are required for applications in disease models,1.Huang L. Haylor J.L. 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Zhang K. et al.Effects of quantum dots on different renal proximal tubule cell models and on gel-free renal tubules generated in vitro.Nanotoxicology. 2012; 6: 121-133Crossref PubMed Scopus (14) Google Scholar and dedifferentiate under in vitro conditions.5.Pfaller W. Gstraunthaler G. Nephrotoxicity testing in vitro—what we know and what we need to know.Environ Health Perspect. 1998; 106: 559-569Crossref PubMed Scopus (127) Google Scholar,11.Elberg G. Guruswamy S. Logan C.J. et al.Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures.Cell Tissue Res. 2008; 331: 495-508Crossref PubMed Scopus (28) Google Scholar Because of these problems, it would be desirable to develop protocols for the differentiation of stem cells into PTCs and other renal cell types. Amniotic fluid stem cells,12.Perin L. Giuliani S. 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Grange C. et al.Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 25.Ronconi E. Sagrinati C. Angelotti M.L. et al.Regeneration of glomerular podocytes by human renal progenitors.J Am Soc Nephrol. 2009; 20: 322-332Crossref PubMed Scopus (429) Google Scholar, 26.Sagrinati C. Netti G.S. Mazzinghi B. et al.Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys.J Am Soc Nephrol. 2006; 17: 2443-2456Crossref PubMed Scopus (570) Google Scholar or renal precursor–like cells derived from these stem cell types have been injected into renal ex vivo or in vivo models. The results of these studies are valuable for the development of cell-based renal therapies. For other approaches, including kidney tissue engineering and in vitro nephrotoxicology, the development of in vitro differentiation protocols would be required, which should yield relatively mature differentiated renal cell types. In vitro differentiation protocols have been applied to mouse ESCs (mESCs), and, according to the results, renal progenitor–like cells were obtained.18.Vigneau C. Polgar K. Striker G. et al.Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo.J Am Soc Nephrol. 2007; 18: 1709-1720Crossref PubMed Scopus (134) Google Scholar,19.Kim D. Dressler G.R. Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia.J Am Soc Nephrol. 2005; 16: 3527-3534Crossref PubMed Scopus (228) Google Scholar In other studies with mESCs, differentiated cells were obtained that expressed some PTC markers and/or markers of other renal cell types.20.Fuente Mora C. Ranghini E. Bruno S. et al.Differentiation of podocyte and proximal tubule-like cells from a mouse kidney-derived stem cell line.Stem Cells Dev. 2012; 21: 296-307Crossref PubMed Scopus (33) Google Scholar, 21.Bruce S.J. Rea R.W. Steptoe A.L. et al.In vitro differentiation of murine embryonic stem cells toward a renal lineage.Differentiation. 2007; 75: 337-349Crossref PubMed Scopus (104) Google Scholar, 22.Kramer J. Steinhoff J. Klinger M. et al.Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.Differentiation. 2006; 74: 91-104Crossref PubMed Scopus (38) Google Scholar A study performed with human ESCs (hESCs) demonstrated upregulation of renal markers during in vitro differentiation.23.Batchelder C.A. Lee C.C. Matsell D.G. et al.Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors.Differentiation. 2009; 78: 45-56Crossref PubMed Scopus (65) Google Scholar The most interesting results were obtained with presumable human kidney stem cells. A study performed with renal-derived CD133+ cells demonstrated that these cells could be differentiated in vitro into epithelial cells, which displayed characteristics of PTCs and distal tubular cells.24.Bussolati B. Bruno S. Grange C. et al.Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar However, CD133+ cells represented 30%, Figure 2b) were obtained in the presence of 10ng/ml of BMP2 and 2.5ng/ml of BMP7. Further, we tested the effects of retinoic acid (RA) and activin-A, which had been used in previous studies to generate renal epithelial or proximal tubular precursor cells from murine ESCs.18.Vigneau C. Polgar K. Striker G. et al.Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo.J Am Soc Nephrol. 2007; 18: 1709-1720Crossref PubMed Scopus (134) Google Scholar,19.Kim D. Dressler G.R. Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia.J Am Soc Nephrol. 2005; 16: 3527-3534Crossref PubMed Scopus (228) Google Scholar The same concentrations of RA (0.1μmol/l) and activin-A (10ng/ml) as in these previous studies were applied here, and in all of the experiments BMP2 and BMP7 were used at concentrations of 10 and 2.5ng/ml, respectively. Supplementation with activin-A and RA alone or in combination had no effects (data not shown). When activin-A was applied in combination with BMP2 and BMP7, the yield of AQP1-expressing cells was not further increased (Figure 2b and c). Combinations of activin-A and RA with either BMP2 or BMP7 yielded relatively high numbers of AQP1-expressing cells, but the numbers of these cells were not higher than those obtained with a combination of BMP2 and BMP7 alone (Figure 2b and c). However, when all four growth factors were applied in combination, the yield of AQP1-expressing cells was in the range of ∼38%, and higher than that obtained with all other combinations tested (Figure 2c). As high yields of AQP1-expressing cells were also obtained with a combination of BMP2 and BMP7, all subsequent experiments were performed with complete serum-containing REGM supplemented with these growth factors. To further characterize the differentiated hESCs, we analyzed 21 markers by RT-PCR. One of these markers was kidney-specific cadherin, which appears to be exclusively transcribed in the kidney, where its expression is largely confined to the renal epithelium.40.Shen S.S. Krishna B. Chirala R. et al.Kidney-specific cadherin, a specific marker for the distal portion of the nephron and related renal neoplasms.Mod Pathol. 2005; 18: 933-940Crossref PubMed Scopus (103) Google Scholar In addition, AQP1 and GGT were assessed, as well as the following markers expressed in the proximal tubule: aminopeptidase N (CD13), 25-hydroxyvitamin D3 1α-hydroxylase (Vit D3 Hydr), megalin (MEG), Na+/K+ ATPase, glucose transporter 5 (GLUT5), sodium-dependent glucose co-transporter 2 (SGLT2), Na+HCO3- co-transporter 1 (NBC1), organic anion transporter 1 (OAT1), OAT3, organic cation transporter 1 (OCT1), organic cation/carnitine transporter (OCTN2), p-glycoprotein (MDR1), proton-coupled peptide transporter 1 (PEPT1), and PEPT2. The following markers were specific for other parts of the nephron: thiazide-sensitive sodium-chloride co-transporter (NCCT, distal tubule),41.Simon D.B. Nelson-Williams C. Bia M.J. et al.Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.Nat Genet. 1996; 12: 24-30Crossref PubMed Scopus (1044) Google Scholar Na+/K+/2Cl- co-transporter (NKCC2, thick ascending limp of Henle's loop),42.Carota I. Theilig F. Oppermann M. et al.Localization and functional characterization of the human NKCC2 isoforms.Acta Physiol. 2010; 199: 327-338PubMed Google Scholar podocalyxin-like (PODXL, glomerulus),29.Chabardes-Garonne D. Mejean A. Aude J.C. et al.A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci USA. 2003; 100: 13710-13715Crossref PubMed Scopus (147) Google Scholar and AQP3 (collecting duct).29.Chabardes-Garonne D. Mejean A. Aude J.C. et al.A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci USA. 2003; 100: 13710-13715Crossref PubMed Scopus (147) Google Scholar Marker expression was analyzed in the undifferentiated and differentiated hESCs, as well as in HPTCs. The results (Figure 3) revealed remarkably similar gene expression profiles for differentiated hESCs and HPTCs. In most of the cases, the same gene was either up- or downregulated in both cell types (compared with undifferentiated hESCs). In some cases, differences were observed. This applied, for instance, to AQP1, which was expressed at higher levels in differentiated hESCs, and to SGLT2 and GLUT5, which were expressed at significantly lower levels in differentiated hESCs (compared with HPTCs and undifferentiated hESCs). Comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels revealed that GLUT5 was expressed at relatively high levels in undifferentiated hESCs and HPTCs (∼6% of GAPDH expression) and was still expressed above basal levels in differentiated hESCs (∼3% of GAPDH expression). MEG, OAT3, OCT1, and PEPT2 were significantly downregulated in differentiated hESCs. In all of these cases, expression levels were also very low in HPTCs, which was likely because of partial dedifferentiation. The collecting duct–specific marker AQP3 and the distal tubule–specific marker NCCT were expressed at significantly higher levels in differentiated hESCs and HPTCs in comparison with undifferentiated hESCs. However, the expression levels of these genes were still very low (∼0.1% of GAPDH expression for HPTCs and even lower for the differentiated hESCs). Also, the expression levels of NKCC2 and PODXL, which were not upregulated during differentiation, remained very low in differentiated hESCs and HPTCs. Altogether, the gene expression patterns revealed that hESCs had differentiated into renal epithelial cells (kidney-specific cadherin was strongly upregulated and expressed at levels of ∼4% of GAPDH expression; note that the cells also expressed CK18 (Figure 1)), which expressed a variety of markers specific for the proximal tubules, whereas markers specific for other parts of the nephrons or collecting ducts were not expressed or were expressed at very low levels. The gene expression pattern of differentiated hESCs was overall similar to that of commercial HPTCs. Immunostaining experiments confirmed the expression of AQP1, CK18, and PAX2 at the protein level (Supplementary Figure S4 online). These analyses also demonstrated the expression of the epithelial marker E-cadherin and of Wilm's tumor 1 (Supplementary Data online, Supplementary Figure S4 online), which is expressed during kidney development.43.Kreidberg J.A. WT1 and kidney progenitor cells.Organogenesis. 2010; 6: 61-70Crossref PubMed Scopus (51) Google Scholar Together, the immunostaining results confirmed that differentiated hESCs expressed markers characteristic of renal epithelial cells and their precursors. Interestingly, Wilm's tumor 1 and CK18 were expressed by almost all of the cells (Supplementary Figures S
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