- Email: [email protected]
Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro
Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro Ryuji Morizane, Toshiaki Monkawa *, Hiroshi Itoh Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 8 October 2009 Available online 31 October 2009 Keywords: Tubular cell Activin BMP7 GDNF iPS ES
a b s t r a c t Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The prevalence of end-stage renal disease (ESRD) has been progressively growing, and is a global healthcare problem. The potency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to undergo unlimited self-renewal and extensive differentiation makes them an attractive source for kidney regeneration therapies. iPS cells can also contribute to the elucidation of human kidney generation and the mechanism of genetic disorder such as polycystic kidney. Mouse ES cells isolated from blastocysts as well as mouse iPS cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF) [1]. Withdrawal of LIF, ES and iPS cells give rise to embryoid bodies (EB) that can be differentiated to several different cells types of mesodermal, endodermal, and ectodermal lineages. Although the in vivo cell organization and patterning is not achieved, sequential gene expressions during EB differentiation are highly reproducible and similar to in vivo kidney generation observed in post-implantation mouse embryos [2].
* Corresponding author. Address: Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: +81 3 5363 3512. E-mail address: [email protected] (T. Monkawa). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.148
However, kidney is a complex organ and consists of many types of cells, thus to generate kidney in vitro needs a complicated strategy. There are some studies in which differentiation of mouse ES cells to renal tubular cells in vitro [3–5] was tried, but there is little evidence suggesting that these cells contribute to the structural development of glomeruli. There is no report that shows the differentiation of mouse iPS cells to renal lineage cells. We studied whether not only mouse ES but also iPS cells could differentiate to cells of renal lineage in vitro, and tested a variety of inducing factors known to be involved in renal generation in vivo to know whether they could enhance the differentiation to renal lineage cells.
Materials and methods Culture of undifferentiated murine ES and iPS cells. The murine ES cell, EB3 is transfected with Oct3/4–Blasticidin resistant gene, so that differentiated cells are eliminated in the maintenance medium with Blasticidin. Thus, the pluripotency could be maintained without feeder cells, such as mouse embryonic fibroblasts (MEFs). The transfected EB3 cells were maintained in 10% fetal calf serum (FCS) and the Glasgow modification of Eagle’s minimal essential medium (GMEM, Sigma) with 1000 U/ml LIF (Chemicon), 0.1 mM 2-mercapto-ethanol (Sigma), and Blasticidin (Funakoshi) on
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
gelatin-coated dish. EB3 was kindly provided from Niwa (Riken Center for Developmental Biology, Kobe, Japan). The murine iPS cells, iPS-MEF-Nanog-38C2 established by Yamanaka [6], have been generated from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4. The iPS cells were cultured on mitotically inactivated MEFs in the culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM, GIBCO), 15% FCS, 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol, 1000 U/ml LIF according to the protocol previously reported [7]. Renal differentiation of murine ES and iPS cells. To initiate the differentiation, we formed embryoid bodies (EB) in ‘‘Hanging drops” [8] composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. On day 3 of differentiation, we transferred the EBs onto gelatin-coated culture dishes with 10 EBs per 1 ml medium. From day 4 to 18, the differentiation medium was replaced every 2 days. The differentiation medium was composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. Several inducing factors were added separately or in combination, i.e. Activin (10 ng/ml, R&D systems), GDNF (15 ng/ml, 150 ng/ml R&D systems), BMP7 (0.5 ng/ml, 15 ng/ml, 100 ng/ml, R&D systems), LIF (1 U/ml), Gremlin (5 lg/ml, R&D systems), and Gdf11 (25 ng/ml, R&D systems). RNA extraction, cDNA synthesis, PCR and real-time PCR data analysis. We isolated total RNA with RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Potentially contaminating genomic DNA was digested by DNAse (QIAGEN) for 15 min at room temperature. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). We performed real-time PCR with the use of TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and Step One Plus Real-Time PCR systems (Applied Biosystems). The cycling conditions were as follows: Predenaturation 95.0 °C 20 s then 40 cycles of 1-s denaturation at 95.0 °C and 20 s at 60.0 °C. The standard serial 10-fold dilutions of template cDNA and two reactions of negative control were repeated on every plate. Expression levels were normalized against GAPDH. Statistical analysis. Results are given as mean ± SD. Statistical analysis of the data was performed with t-test or ANOVA followed by Tukey’s post-hoc test. P < 0.05 was considered significant.
Results Differentiation of ES and iPS cells toward renal lineage The ES and iPS cells were differentiated through EB formation in the differentiation medium composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. Embryoid bodies (EB) were formed in ‘‘Hanging drops” composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. In a preliminary experiment, more cells were needed for iPS cells than ES cells to make EBs. 100 EBs were transferred to gelatin-coated 10 cm culture dish on day 3, and medium was changed every 2 days from day 4. Genetic markers of ES cell pluripotency, mesoderm induction and renal lineage cells were examined using real-time PCR over 18 days of EB differentiation (Fig. 1). Oct3/4, a genetic marker of pluripotency [9] showed significant reduction in expression after the initiation of differentiation in both ES and iPS cells, though the expression of Oct3/4 in iPS cells sustained later than in ES cells. The mesoderm induction was highlighted by progressive increase in Brachyury [10] expression that showed transient increase after the initiation of differentiation and followed by gradual decrease. After the transient expression of mesoderm marker, metanephric mesenchyme markers, Six2 [11], WT-1 [12] and Pax2 [13] showed progressive increase. Podocyte markers, WT-1 and Nephrin [14] were expressed on day 18, although high expression of Nephrin was also shown on
1335
day 0. And expression of tubular specific marker, KSP [15] showed progressive increase to day 18. It was suggested that matured renal lineage cells, podocyte and tubular cells can be differentiated from both murine ES and iPS cells, although iPS cells showed a tendency to remain undifferentiated state. Transfected four factors seemed to suppress the differentiation. Activin enhances the differentiation to mesoderm Activin (10 ng/ml), which was reported to enhance ES cells to differentiate to mesoderm [16], was added from day 0 to 4, during EB formation. The expression of Brachyury, a mesoderm marker was examined on day 4 by real-time PCR (Fig. 2A). The expression of Brachyury was enhanced by Activin in ES. This result shows that Activin enhances the differentiation of ES cells to mesoderm during EB formation. Screening for renal inducing factors in ES cells After ‘‘Hanging drop”, 15 EBs were transferred to a 12-well gelatin-coated dish on day 3, and medium was changed every 2 days from day 4 to 14. Several inducing factors known to be involved in renal genesis were added to the differentiation medium from day 4 to 14 in addition to Activin from day 0 to 4, i.e. Activin (10 ng/ml), GDNF [17] (150 ng/ml), BMP7 [18] (15 ng/ml), LIF [19] (1 U/ml), Gremlin [20] (5 lg/ml), Gdf11 [21] (25 ng/ml) and Wnt4 [22] conditioned medium obtained from NIH3T3 transfected with CMVWnt4. The expressions of renal lineage markers were analyzed on day 14 by real-time PCR. Pax2 and WT-1 are considered as markers of metanephric mesenchyme, KSP as tubular cells, Nephrin as podocytes and Ngn2 [23] as neuronal lineage cells (Fig. 2B). GDNF and BMP7 enhanced the expression of Pax2 and WT-1 while suppressing Ngn2 expression. In contrast, Gremlin enhanced the expression of Ngn2 as well as those of Pax2 and WT-1. This result shows that GDNF and BMP7 enhance the differentiation of ES cells to metanephric mesenchyme, but Gremlin enhanced the differentiation to neuronal lineage cells. Activin enhanced the expression of Pax2 and KSP while suppressing Ngn2 expression. These results indicate that Activin enhances the differentiation of ES cells not only to mesoderm but also to tubular cells. No inducer was found to enhance the expression of Nephrin, and the result indicates that it is difficult to promote the differentiation of ES cells to podocytes. Concentrations of GDNF and BMP7 We studied how GDNF and BMP7 could affect the differentiation of ES cells at concentrations of 15 ng/ml, 150 ng/ml and 0.5 ng/ml, 15 ng/ml, 100 ng/ml, respectively. EBs were generated by ‘‘Hanging drop” in differentiation medium with 10 ng/ml Activin, and 15 EBs were transferred to a 12-well culture dish with gelatin-coat on day 3. Activin was removed from day 4, and these inducing factors were added to medium from day 4 to 14. We did Real-time PCR to evaluate the expressions of Pax2, WT-1 and KSP (Fig. 2C and D). GDNF induced slightly higher expression of KSP at 150 ng/ml than at 15 ng/ml. This dose-responsiveness is compatible with a report [24] which showed that nephron formation was enhanced by GDNF at a concentration of 150 ng/ml, but not at 15 ng/ml. BMP7 induced the highest expression of KSP at 15 ng/ml compared to 0.5 ng/ml and 100 ng/ml. Enhancement of the differentiation of ES and iPS cells to tubular cells We extended the culture period till day 18 to enhance the differentiation to tubular cells and examined how Activin affected
1336
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
Fig. 1. Real-time PCR shows the expression of mesoderm and renal lineage genes in ES and iPS cells. The ES and iPS cells were differentiated through EB formation in the differentiation medium composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. EBs were formed in ‘‘Hanging drops” composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. 100 EBs were transferred to gelatin-coated 10 cm culture dish on day 3, and medium was changed every 2 days from day 4. Genetic markers of ES cell pluripotency, mesoderm induction and renal lineage cells were examined using triplicate real-time PCR over 18 days of EB differentiation. Expression levels were normalized against GAPDH.
the differentiation of ES and iPS cells. The cells were cultured with Activin from day 0 to 18. Differentiated cells were harvested on day 18, and real-time PCR was performed (Fig. 3). In ES cells, Activin showed an effect of differentiating cells to tubular cells, which was indicated by the enhanced KSP expression
of about 3.7-fold (P < 0.05). Compared with ES cells, iPS showed a tendency to remain undifferentiated state, but Activin also enhanced the expression of KSP in iPS cells about 1.2-fold (P < 0.05). It is suggested that Activin enhance the differentiation of both ES and iPS cells to tubular cells.
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
1337
Fig. 2. (A) Real-time PCR shows the enhancement of the differentiation to mesoderm by Activin. EBs were formed in ‘‘Hangind drops” in 30 ll of differentiation medium with Activin 10 ng/ml for 3 days. One hundred EBs were transferred to 10 cm culture dish with gelatin-coat, and cultured in differentiation medium with Activin 10 ng/ml. On day 4, cells were harvested and triplicate real-time PCR was performed. Expression levels were normalized against GAPDH. (B) The effects of several inducing factors were evaluated by real-time PCR. Several inducing factors involving in renal genesis were added to the differentiation medium from day 4 to 14 in addition to Activin from day 0 to 4, i.e. Activin (10 ng/ml), GDNF (150 ng/ml), BMP7 (15 ng/ml,), LIF (1 U/ml), Gremlin (5 lg/ml), Gdf11 (25 ng/ml) and Wnt4. On day 14, cells were harvested and duplicate real-time PCR was performed. Expression levels were normalized against GAPDH. (C, D) Real-time PCR shows the optimal concentration of GDNF and BMP7 for the differentiation to renal lineage. Activin was added from day 0 to 4, and from day 4 to 14 GDNF and BMP7 were added to differentiation medium at concentrations of 15 ng/ml, 150 ng/ml and 0.5 ng/ml, 15 ng/ml, 100 ng/ml, respectively. We did duplicate real-time PCR to evaluate the expressions of Pax2, WT-1 and KSP. Expression levels were normalized against GAPDH.
Fig. 3. Real-time PCR shows the enhancement of KSP expression in both (A) ES and (B) iPS cells. EBs were generated by ‘‘Hanging drop” in differentiation medium, and 100 EBs were transferred to a 10 cm culture dish with gelatin-coat on day 3. The cells were cultured with Activin from day 0 to 18. Differentiated cells were harvested on day 18, and triplicate real-time PCR was performed. Expression levels were normalized against GAPDH.
Discussion ES cells and iPS cells have an extensive differentiation potency, and can be an attractive source for kidney regeneration therapies. However, there are few reports about the differentiation of ES cells to renal lineage in vitro and no reports about iPS cells to renal lineage. Bruce [25] reported that mouse ES cells have a potency to dif-
ferentiate to renal lineage cells. We examined the expressions of renal lineage genes in both ES and iPS cells. Our data show that iPS cells seem reluctant to external stimuli of inducing differentiation and have a tendency to remain undifferentiated state, but both ES and iPS cells have a potency to differentiate into matured renal lineage cells. Vigneau [16] reported that Activin enhances the differentiation of mouse ES cells to mesoderm, and Brachyury-positive cells have a potency to differentiate to tubular cells. Our data also show that the expression of Brachyury is enhanced by Activin in ES cells. Interestingly, Activin also had an effect of tubular cell induction. This effect was shown by the enhancement of KSP expression in ES cells cultured with Activin. This result is consistent with a report from Maeshima [26], who showed that Activin induced the expressions of epithelial differentiation markers in cultured metanephric mesenchymal cells. It is suggested that Activin has an effect of mesenchymal-to-epithelial transformation (MET) induction as in mouse ES and iPS cells as metanephric mesenchyme. There are some reports showed that GDNF have different effects depending on the concentrations. At as a low concentration as 10 ng/ml, GDNF promotes neuronal differentiation [27], whereas at as a high concentration as 150 ng/ml it induces nephron formations. Reciprocal interactions between the ureteric bud and the metanephric mesenchyme are needed to promote growth and differentiation of the embryonic kidney. GDNF is secreted by metanephric mesenchyme, and promotes ureteric bud branching. Ureteric bud secretes Wnt11, which enhances the secretion of GDNF from metanephric mesenchyme. Wnt11 and GDNF cooper-
1338
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
ate in a positive autoregulatory feedback loop to coordinate ureteric branching and GDNF-expressing mesenchyme, and to ensure continued metanephric development [28]. GDNF at 150 ng/ml may have an effect of metanephric mesenchyme and ureteric bud induction. Our data showed that GDNF 150 ng/ml enhances Pax2 and WT-1 expression on day 14 in ES cells. Thus, GDNF may promote the differentiation of ES cells to metanephric mesenchyme and ureteric bud. BMP7 has an essential role involving with nephrogenesis, and its loss of function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. Although BMP7 can prevent apoptosis in explants of metanephric mesenchyme, BMP7 is not capable of responding to signals promoting tubulogenesis [18]. Our result is consistent with this report, which showed BMP7 promoted the expression of Pax2 and WT-1 in ES cells, but not KSP. Thus, BMP7 may have an effect of the induction to metanephric mesenchyme, but not to tubular cells. Activin, GDNF and BMP7 showed some effects to the differentiation of ES cells. Kim [3] reported that the combination of Activin, BMP7 and retinoic acid promotes the differentiation of mouse ES cells to intermediate mesoderm, from which the kidneys arise. We examined how the combination of Activin, GDNF and BMP7 affect the differentiation of ES cells, but KSP was not enhanced by the combination compared with Activin alone on day 18 (data not shown). We also examined the combination of Activin and retinoic acid (data not shown). They enhanced the expression of metanephric mesenchyme markers, but failed to enhance the differentiation to tubular cells. Thus, we concluded that only Activin promotes the differentiation of ES cells to tubular cells in vitro. We compared iPS to ES cells about the differentiation to renal lineage. Our results indicate that matured renal cells, tubular cells and podocytes can be differentiated from both ES and iPS cells. However, iPS cells had a tendency to remain undifferentiated state, and showed lower expression of KSP than ES cells. Similar findings were also reported by Mauritz, which showed the population of beating cells like cardiomyocytes was lower in iPS cells than in ES cells [29]. Our data showed an enhancement of KSP expression by Activin in iPS cells, but the enhancement was lower than in ES cells. Our results suggest that transfected four factors strongly suppress the differentiation. Further researches are needed to know whether iPS cells generated from three factors [30] or two factors [31] are capable of being differentiated to renal cell lineage. Since the establishment of human iPS cells by Yamanaka [32], a new gate for regenerative medicine and basic research have been open. iPS cells can be an attractive source for the cell replacement therapy of kidney disease, and in vitro induction of tubular cells and podocytes leads to drug discovery. In addition, iPS cells can contribute to the elucidation of human kidney generation and the mechanism of genetic disorder such as polycystic kidney. Our data show that both mouse ES and iPS cells have a potency to differentiate to renal lineage cells, and that Activin enhances the differentiation of ES and iPS cells to tubular cells. GDNF and BMP7 may enhance the differentiation of ES cells to metanephric mesenchyme. Further research is needed to establish the culture method for ES and iPS cells which differentiate them to renal lineage cells.
Acknowledgments We thank Shizuka Fujii for technical assistance. This work was supported in part by G-COE program and Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.10.148. References [1] M.J. Evans, M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos, Nature 292 (1981) 154–156. [2] G. Keller, M. Kennedy, T. Papayannopoulou, M.V. Wiles, Hematopoietic commitment during embryonic stem cell differentiation in culture, Mol. Cell. Biol. 13 (1) (1993) 473–486. [3] D. Kim, G.R. Dressler, Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia, J. Am. Soc. Nephrol. 16 (12) (2005) 3527–3534 (Epub 2005 November 2). [4] T. Kobayashi, H. Tanaka, H. Kuwana, S. Inoshita, H. Teraoka, S. Sasaki, Y. Terada, Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells, Biochem. Biophys. Res. Commun. 336 (2) (2005) 585–595. [5] B.M. Steenhard, K.S. Isom, P. Cazcarro, J.H. Dunmore, A.R. Godwin, P.L. St. John, D.R. Abrahamson, Integration of embryonic stem cells in metanephric kidney organ culture, J. Am. Soc. Nephrol. 16 (6) (2005) 1623–1631 (Epub 2005 May 4). [6] K. Okita, T. Ichisaka, S. Yamanaka, Generation of germline-competent induced pluripotent stem cells, Nature 448 (7151) (2007) 313–317 (Epub 2007 June 6). [7] K. Takahashi, K. Okita, M. Nakagawa, S. Yamanaka, Induction of pluripotent stem cells from fibroblast cultures, Nat. Protoc. 2 (12) (2007) 3081–3089. [8] V.A. Maltsev, A.M. Wobus, J. Rohwedel, M. Bader, J. Hescheler, Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents, Circ. Res. 75 (2) (1994) 233–244. [9] J. Nichols, B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Schöler, A. Smith, Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor, Cell 95 (3) (1998) 379–391. [10] P.N. Goodfellow, Developmental genetics: a long story and a short tail, Nature 343 (6259) (1990) 597–598. [11] M. Self, O.V. Lagutin, B. Bowling, J. Hendrix, Y. Cai, G.R. Dressler, G. Oliver, Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney, EMBO J. 25 (21) (2006) 5214–5228. [12] J.F. Armstrong, K. Pritchard-Jones, W.A. Bickmore, N.D. Hastie, J.B. Bard, The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo, Mech. Dev. 40 (1–2) (1993) 85–97. [13] M. Dehbi, M. Ghahremani, M. Lechner, G. Dressler, J. Pelletier, The paired-box transcription factor, PAX2, positively modulates expression of the Wilms’ tumor suppressor gene (WT1), Oncogene 13 (3) (1996) 447–453. [14] L.B. Holzman, P.L. St. John, I.A. Kovari, R. Verma, H. Holthofer, D.R. Abrahamson, Nephrin localizes to the slit pore of the glomerular epithelial cell, Kidney Int. 56 (4) (1999) 1481–1491. [15] X. Shao, J.E. Johnson, J.A. Richardson, T. Hiesberger, P. Igarashi, A minimal Kspcadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract, J. Am. Soc. Nephrol. 13 (7) (2002) 1824–1836. [16] C. Vigneau, K. Polgar, G. Striker, J. Elliott, D. Hyink, O. Weber, H.J. Fehling, G. Keller, C. Burrow, P. Wilson, Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo, J. Am. Soc. Nephrol. 18 (6) (2007) 1709–1720 (Epub 2007 May 2). [17] F. Costantini, R. Shakya, GDNF/Ret signaling and the development of the kidney, Bioessays 28 (2) (2006) 117–127. [18] A.T. Dudley, R.E. Godin, E.J. Robertson, Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme, Genes Dev. 13 (12) (1999) 1601–1613. [19] J. Barasch, J. Yang, C.B. Ware, T. Taga, K. Yoshida, H. Erdjument-Bromage, P. Tempst, E. Parravicini, S. Malach, T. Aranoff, J.A. Oliver, Mesenchymal to epithelial conversion in rat metanephros is induced by LIF, Cell 99 (4) (1999) 377–386. [20] O. Michos, A. Gonçalves, J. Lopez-Rios, E. Tiecke, F. Naillat, K. Beier, A. Galli, S. Vainio, R. Zeller, Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis, Development 134 (13) (2007) 2397–2405 (Epub 2007 May 23). [21] A.F. Esquela, S.J. Lee, Regulation of metanephric kidney development by growth/differentiation factor 11, Dev. Biol. 257 (2) (2003) 356–370. [22] K. Stark, S. Vainio, G. Vassileva, A.P. McMahon, Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4, Nature 372 (6507) (1994) 679–683. [23] R. Mizuguchi, M. Sugimori, H. Takebayashi, H. Kosako, M. Nagao, S. Yoshida, Y. Nabeshima, K. Shimamura, M. Nakafuku, Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtypespecific properties of motoneurons, Neuron 31 (2001) 757–771. [24] E. Moreau, J. Vilar, M. Lelièvre-Pégorier, C. Merlet-Bénichou, T. Gilbert, Regulation of c-ret expression by retinoic acid in rat metanephros: implication in nephron mass control, Am. J. Physiol. 275 (6 Pt. 2) (1998) F938–F945.
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339 [25] S.J. Bruce, R.W. Rea, A.L. Steptoe, M. Busslinger, J.F. Bertram, A.C. Perkins, In vitro differentiation of murine embryonic stem cells toward a renal lineage, Differentiation 75 (5) (2007) 337–349 (Epub 2007 February 5). [26] A. Maeshima, S. Yamashita, K. Maeshima, I. Kojima, Y. Nojima, Activin A produced by ureteric bud is a differentiation factor for metanephric mesenchyme, J. Am. Soc. Nephrol. 14 (6) (2003) 1523–1534. [27] E. Roussa, K. Krieglstein, GDNF promotes neuronal differentiation and dopaminergic development of mouse mesencephalic neurospheres, Neurosci. Lett. 361 (1–3) (2004) 52–55. [28] A. Majumdar, S. Vainio, A. Kispert, J. McMahon, A.P. McMahon, Wnt11 and Ret/ Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development, Development 130 (14) (2003) 3175–3185. [29] C. Mauritz, K. Schwanke, M. Reppel, S. Neef, K. Katsirntaki, L.S. Maier, F. Nguemo, S. Menke, M. Haustein, J. Hescheler, G. Hasenfuss, U. Martin,
1339
Generation of functional murine cardiac myocytes from induced pluripotent stem cells, Circulation 118 (5) (2008) 507–517 (Epub 2008 July 14). [30] M. Nakagawa, M. Koyanagi, K. Tanabe, K. Takahashi, T. Ichisaka, T. Aoi, K. Okita, Y. Mochiduki, N. Takizawa, S. Yamanaka, Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts, Nat. Biotechnol. 26 (1) (2008) 101–106 (Epub 2007 November 30). [31] J.B. Kim, H. Zaehres, G. Wu, L. Gentile, K. Ko, V. Sebastiano, M.J. Araúzo-Bravo, D. Ruau, D.W. Han, M. Zenke, H.R. Schöler, Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors, Nature 454 (7204) (2008) 646–650 (Epub 2008 June 29). [32] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131 (5) (2007) 861–872.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro Ryuji Morizane, Toshiaki Monkawa *, Hiroshi Itoh Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 8 October 2009 Available online 31 October 2009 Keywords: Tubular cell Activin BMP7 GDNF iPS ES
a b s t r a c t Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The prevalence of end-stage renal disease (ESRD) has been progressively growing, and is a global healthcare problem. The potency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to undergo unlimited self-renewal and extensive differentiation makes them an attractive source for kidney regeneration therapies. iPS cells can also contribute to the elucidation of human kidney generation and the mechanism of genetic disorder such as polycystic kidney. Mouse ES cells isolated from blastocysts as well as mouse iPS cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF) [1]. Withdrawal of LIF, ES and iPS cells give rise to embryoid bodies (EB) that can be differentiated to several different cells types of mesodermal, endodermal, and ectodermal lineages. Although the in vivo cell organization and patterning is not achieved, sequential gene expressions during EB differentiation are highly reproducible and similar to in vivo kidney generation observed in post-implantation mouse embryos [2].
* Corresponding author. Address: Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: +81 3 5363 3512. E-mail address: [email protected] (T. Monkawa). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.148
However, kidney is a complex organ and consists of many types of cells, thus to generate kidney in vitro needs a complicated strategy. There are some studies in which differentiation of mouse ES cells to renal tubular cells in vitro [3–5] was tried, but there is little evidence suggesting that these cells contribute to the structural development of glomeruli. There is no report that shows the differentiation of mouse iPS cells to renal lineage cells. We studied whether not only mouse ES but also iPS cells could differentiate to cells of renal lineage in vitro, and tested a variety of inducing factors known to be involved in renal generation in vivo to know whether they could enhance the differentiation to renal lineage cells.
Materials and methods Culture of undifferentiated murine ES and iPS cells. The murine ES cell, EB3 is transfected with Oct3/4–Blasticidin resistant gene, so that differentiated cells are eliminated in the maintenance medium with Blasticidin. Thus, the pluripotency could be maintained without feeder cells, such as mouse embryonic fibroblasts (MEFs). The transfected EB3 cells were maintained in 10% fetal calf serum (FCS) and the Glasgow modification of Eagle’s minimal essential medium (GMEM, Sigma) with 1000 U/ml LIF (Chemicon), 0.1 mM 2-mercapto-ethanol (Sigma), and Blasticidin (Funakoshi) on
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
gelatin-coated dish. EB3 was kindly provided from Niwa (Riken Center for Developmental Biology, Kobe, Japan). The murine iPS cells, iPS-MEF-Nanog-38C2 established by Yamanaka [6], have been generated from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4. The iPS cells were cultured on mitotically inactivated MEFs in the culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM, GIBCO), 15% FCS, 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol, 1000 U/ml LIF according to the protocol previously reported [7]. Renal differentiation of murine ES and iPS cells. To initiate the differentiation, we formed embryoid bodies (EB) in ‘‘Hanging drops” [8] composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. On day 3 of differentiation, we transferred the EBs onto gelatin-coated culture dishes with 10 EBs per 1 ml medium. From day 4 to 18, the differentiation medium was replaced every 2 days. The differentiation medium was composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. Several inducing factors were added separately or in combination, i.e. Activin (10 ng/ml, R&D systems), GDNF (15 ng/ml, 150 ng/ml R&D systems), BMP7 (0.5 ng/ml, 15 ng/ml, 100 ng/ml, R&D systems), LIF (1 U/ml), Gremlin (5 lg/ml, R&D systems), and Gdf11 (25 ng/ml, R&D systems). RNA extraction, cDNA synthesis, PCR and real-time PCR data analysis. We isolated total RNA with RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Potentially contaminating genomic DNA was digested by DNAse (QIAGEN) for 15 min at room temperature. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). We performed real-time PCR with the use of TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and Step One Plus Real-Time PCR systems (Applied Biosystems). The cycling conditions were as follows: Predenaturation 95.0 °C 20 s then 40 cycles of 1-s denaturation at 95.0 °C and 20 s at 60.0 °C. The standard serial 10-fold dilutions of template cDNA and two reactions of negative control were repeated on every plate. Expression levels were normalized against GAPDH. Statistical analysis. Results are given as mean ± SD. Statistical analysis of the data was performed with t-test or ANOVA followed by Tukey’s post-hoc test. P < 0.05 was considered significant.
Results Differentiation of ES and iPS cells toward renal lineage The ES and iPS cells were differentiated through EB formation in the differentiation medium composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. Embryoid bodies (EB) were formed in ‘‘Hanging drops” composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. In a preliminary experiment, more cells were needed for iPS cells than ES cells to make EBs. 100 EBs were transferred to gelatin-coated 10 cm culture dish on day 3, and medium was changed every 2 days from day 4. Genetic markers of ES cell pluripotency, mesoderm induction and renal lineage cells were examined using real-time PCR over 18 days of EB differentiation (Fig. 1). Oct3/4, a genetic marker of pluripotency [9] showed significant reduction in expression after the initiation of differentiation in both ES and iPS cells, though the expression of Oct3/4 in iPS cells sustained later than in ES cells. The mesoderm induction was highlighted by progressive increase in Brachyury [10] expression that showed transient increase after the initiation of differentiation and followed by gradual decrease. After the transient expression of mesoderm marker, metanephric mesenchyme markers, Six2 [11], WT-1 [12] and Pax2 [13] showed progressive increase. Podocyte markers, WT-1 and Nephrin [14] were expressed on day 18, although high expression of Nephrin was also shown on
1335
day 0. And expression of tubular specific marker, KSP [15] showed progressive increase to day 18. It was suggested that matured renal lineage cells, podocyte and tubular cells can be differentiated from both murine ES and iPS cells, although iPS cells showed a tendency to remain undifferentiated state. Transfected four factors seemed to suppress the differentiation. Activin enhances the differentiation to mesoderm Activin (10 ng/ml), which was reported to enhance ES cells to differentiate to mesoderm [16], was added from day 0 to 4, during EB formation. The expression of Brachyury, a mesoderm marker was examined on day 4 by real-time PCR (Fig. 2A). The expression of Brachyury was enhanced by Activin in ES. This result shows that Activin enhances the differentiation of ES cells to mesoderm during EB formation. Screening for renal inducing factors in ES cells After ‘‘Hanging drop”, 15 EBs were transferred to a 12-well gelatin-coated dish on day 3, and medium was changed every 2 days from day 4 to 14. Several inducing factors known to be involved in renal genesis were added to the differentiation medium from day 4 to 14 in addition to Activin from day 0 to 4, i.e. Activin (10 ng/ml), GDNF [17] (150 ng/ml), BMP7 [18] (15 ng/ml), LIF [19] (1 U/ml), Gremlin [20] (5 lg/ml), Gdf11 [21] (25 ng/ml) and Wnt4 [22] conditioned medium obtained from NIH3T3 transfected with CMVWnt4. The expressions of renal lineage markers were analyzed on day 14 by real-time PCR. Pax2 and WT-1 are considered as markers of metanephric mesenchyme, KSP as tubular cells, Nephrin as podocytes and Ngn2 [23] as neuronal lineage cells (Fig. 2B). GDNF and BMP7 enhanced the expression of Pax2 and WT-1 while suppressing Ngn2 expression. In contrast, Gremlin enhanced the expression of Ngn2 as well as those of Pax2 and WT-1. This result shows that GDNF and BMP7 enhance the differentiation of ES cells to metanephric mesenchyme, but Gremlin enhanced the differentiation to neuronal lineage cells. Activin enhanced the expression of Pax2 and KSP while suppressing Ngn2 expression. These results indicate that Activin enhances the differentiation of ES cells not only to mesoderm but also to tubular cells. No inducer was found to enhance the expression of Nephrin, and the result indicates that it is difficult to promote the differentiation of ES cells to podocytes. Concentrations of GDNF and BMP7 We studied how GDNF and BMP7 could affect the differentiation of ES cells at concentrations of 15 ng/ml, 150 ng/ml and 0.5 ng/ml, 15 ng/ml, 100 ng/ml, respectively. EBs were generated by ‘‘Hanging drop” in differentiation medium with 10 ng/ml Activin, and 15 EBs were transferred to a 12-well culture dish with gelatin-coat on day 3. Activin was removed from day 4, and these inducing factors were added to medium from day 4 to 14. We did Real-time PCR to evaluate the expressions of Pax2, WT-1 and KSP (Fig. 2C and D). GDNF induced slightly higher expression of KSP at 150 ng/ml than at 15 ng/ml. This dose-responsiveness is compatible with a report [24] which showed that nephron formation was enhanced by GDNF at a concentration of 150 ng/ml, but not at 15 ng/ml. BMP7 induced the highest expression of KSP at 15 ng/ml compared to 0.5 ng/ml and 100 ng/ml. Enhancement of the differentiation of ES and iPS cells to tubular cells We extended the culture period till day 18 to enhance the differentiation to tubular cells and examined how Activin affected
1336
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
Fig. 1. Real-time PCR shows the expression of mesoderm and renal lineage genes in ES and iPS cells. The ES and iPS cells were differentiated through EB formation in the differentiation medium composed of DMEM, 10% FCS and 0.1 mM 2-mercaptoethanol. EBs were formed in ‘‘Hanging drops” composed of 500 ES cells or 700 iPS cells in 30 ll of differentiation medium for 3 days. 100 EBs were transferred to gelatin-coated 10 cm culture dish on day 3, and medium was changed every 2 days from day 4. Genetic markers of ES cell pluripotency, mesoderm induction and renal lineage cells were examined using triplicate real-time PCR over 18 days of EB differentiation. Expression levels were normalized against GAPDH.
the differentiation of ES and iPS cells. The cells were cultured with Activin from day 0 to 18. Differentiated cells were harvested on day 18, and real-time PCR was performed (Fig. 3). In ES cells, Activin showed an effect of differentiating cells to tubular cells, which was indicated by the enhanced KSP expression
of about 3.7-fold (P < 0.05). Compared with ES cells, iPS showed a tendency to remain undifferentiated state, but Activin also enhanced the expression of KSP in iPS cells about 1.2-fold (P < 0.05). It is suggested that Activin enhance the differentiation of both ES and iPS cells to tubular cells.
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
1337
Fig. 2. (A) Real-time PCR shows the enhancement of the differentiation to mesoderm by Activin. EBs were formed in ‘‘Hangind drops” in 30 ll of differentiation medium with Activin 10 ng/ml for 3 days. One hundred EBs were transferred to 10 cm culture dish with gelatin-coat, and cultured in differentiation medium with Activin 10 ng/ml. On day 4, cells were harvested and triplicate real-time PCR was performed. Expression levels were normalized against GAPDH. (B) The effects of several inducing factors were evaluated by real-time PCR. Several inducing factors involving in renal genesis were added to the differentiation medium from day 4 to 14 in addition to Activin from day 0 to 4, i.e. Activin (10 ng/ml), GDNF (150 ng/ml), BMP7 (15 ng/ml,), LIF (1 U/ml), Gremlin (5 lg/ml), Gdf11 (25 ng/ml) and Wnt4. On day 14, cells were harvested and duplicate real-time PCR was performed. Expression levels were normalized against GAPDH. (C, D) Real-time PCR shows the optimal concentration of GDNF and BMP7 for the differentiation to renal lineage. Activin was added from day 0 to 4, and from day 4 to 14 GDNF and BMP7 were added to differentiation medium at concentrations of 15 ng/ml, 150 ng/ml and 0.5 ng/ml, 15 ng/ml, 100 ng/ml, respectively. We did duplicate real-time PCR to evaluate the expressions of Pax2, WT-1 and KSP. Expression levels were normalized against GAPDH.
Fig. 3. Real-time PCR shows the enhancement of KSP expression in both (A) ES and (B) iPS cells. EBs were generated by ‘‘Hanging drop” in differentiation medium, and 100 EBs were transferred to a 10 cm culture dish with gelatin-coat on day 3. The cells were cultured with Activin from day 0 to 18. Differentiated cells were harvested on day 18, and triplicate real-time PCR was performed. Expression levels were normalized against GAPDH.
Discussion ES cells and iPS cells have an extensive differentiation potency, and can be an attractive source for kidney regeneration therapies. However, there are few reports about the differentiation of ES cells to renal lineage in vitro and no reports about iPS cells to renal lineage. Bruce [25] reported that mouse ES cells have a potency to dif-
ferentiate to renal lineage cells. We examined the expressions of renal lineage genes in both ES and iPS cells. Our data show that iPS cells seem reluctant to external stimuli of inducing differentiation and have a tendency to remain undifferentiated state, but both ES and iPS cells have a potency to differentiate into matured renal lineage cells. Vigneau [16] reported that Activin enhances the differentiation of mouse ES cells to mesoderm, and Brachyury-positive cells have a potency to differentiate to tubular cells. Our data also show that the expression of Brachyury is enhanced by Activin in ES cells. Interestingly, Activin also had an effect of tubular cell induction. This effect was shown by the enhancement of KSP expression in ES cells cultured with Activin. This result is consistent with a report from Maeshima [26], who showed that Activin induced the expressions of epithelial differentiation markers in cultured metanephric mesenchymal cells. It is suggested that Activin has an effect of mesenchymal-to-epithelial transformation (MET) induction as in mouse ES and iPS cells as metanephric mesenchyme. There are some reports showed that GDNF have different effects depending on the concentrations. At as a low concentration as 10 ng/ml, GDNF promotes neuronal differentiation [27], whereas at as a high concentration as 150 ng/ml it induces nephron formations. Reciprocal interactions between the ureteric bud and the metanephric mesenchyme are needed to promote growth and differentiation of the embryonic kidney. GDNF is secreted by metanephric mesenchyme, and promotes ureteric bud branching. Ureteric bud secretes Wnt11, which enhances the secretion of GDNF from metanephric mesenchyme. Wnt11 and GDNF cooper-
1338
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339
ate in a positive autoregulatory feedback loop to coordinate ureteric branching and GDNF-expressing mesenchyme, and to ensure continued metanephric development [28]. GDNF at 150 ng/ml may have an effect of metanephric mesenchyme and ureteric bud induction. Our data showed that GDNF 150 ng/ml enhances Pax2 and WT-1 expression on day 14 in ES cells. Thus, GDNF may promote the differentiation of ES cells to metanephric mesenchyme and ureteric bud. BMP7 has an essential role involving with nephrogenesis, and its loss of function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. Although BMP7 can prevent apoptosis in explants of metanephric mesenchyme, BMP7 is not capable of responding to signals promoting tubulogenesis [18]. Our result is consistent with this report, which showed BMP7 promoted the expression of Pax2 and WT-1 in ES cells, but not KSP. Thus, BMP7 may have an effect of the induction to metanephric mesenchyme, but not to tubular cells. Activin, GDNF and BMP7 showed some effects to the differentiation of ES cells. Kim [3] reported that the combination of Activin, BMP7 and retinoic acid promotes the differentiation of mouse ES cells to intermediate mesoderm, from which the kidneys arise. We examined how the combination of Activin, GDNF and BMP7 affect the differentiation of ES cells, but KSP was not enhanced by the combination compared with Activin alone on day 18 (data not shown). We also examined the combination of Activin and retinoic acid (data not shown). They enhanced the expression of metanephric mesenchyme markers, but failed to enhance the differentiation to tubular cells. Thus, we concluded that only Activin promotes the differentiation of ES cells to tubular cells in vitro. We compared iPS to ES cells about the differentiation to renal lineage. Our results indicate that matured renal cells, tubular cells and podocytes can be differentiated from both ES and iPS cells. However, iPS cells had a tendency to remain undifferentiated state, and showed lower expression of KSP than ES cells. Similar findings were also reported by Mauritz, which showed the population of beating cells like cardiomyocytes was lower in iPS cells than in ES cells [29]. Our data showed an enhancement of KSP expression by Activin in iPS cells, but the enhancement was lower than in ES cells. Our results suggest that transfected four factors strongly suppress the differentiation. Further researches are needed to know whether iPS cells generated from three factors [30] or two factors [31] are capable of being differentiated to renal cell lineage. Since the establishment of human iPS cells by Yamanaka [32], a new gate for regenerative medicine and basic research have been open. iPS cells can be an attractive source for the cell replacement therapy of kidney disease, and in vitro induction of tubular cells and podocytes leads to drug discovery. In addition, iPS cells can contribute to the elucidation of human kidney generation and the mechanism of genetic disorder such as polycystic kidney. Our data show that both mouse ES and iPS cells have a potency to differentiate to renal lineage cells, and that Activin enhances the differentiation of ES and iPS cells to tubular cells. GDNF and BMP7 may enhance the differentiation of ES cells to metanephric mesenchyme. Further research is needed to establish the culture method for ES and iPS cells which differentiate them to renal lineage cells.
Acknowledgments We thank Shizuka Fujii for technical assistance. This work was supported in part by G-COE program and Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.10.148. References [1] M.J. Evans, M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos, Nature 292 (1981) 154–156. [2] G. Keller, M. Kennedy, T. Papayannopoulou, M.V. Wiles, Hematopoietic commitment during embryonic stem cell differentiation in culture, Mol. Cell. Biol. 13 (1) (1993) 473–486. [3] D. Kim, G.R. Dressler, Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia, J. Am. Soc. Nephrol. 16 (12) (2005) 3527–3534 (Epub 2005 November 2). [4] T. Kobayashi, H. Tanaka, H. Kuwana, S. Inoshita, H. Teraoka, S. Sasaki, Y. Terada, Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells, Biochem. Biophys. Res. Commun. 336 (2) (2005) 585–595. [5] B.M. Steenhard, K.S. Isom, P. Cazcarro, J.H. Dunmore, A.R. Godwin, P.L. St. John, D.R. Abrahamson, Integration of embryonic stem cells in metanephric kidney organ culture, J. Am. Soc. Nephrol. 16 (6) (2005) 1623–1631 (Epub 2005 May 4). [6] K. Okita, T. Ichisaka, S. Yamanaka, Generation of germline-competent induced pluripotent stem cells, Nature 448 (7151) (2007) 313–317 (Epub 2007 June 6). [7] K. Takahashi, K. Okita, M. Nakagawa, S. Yamanaka, Induction of pluripotent stem cells from fibroblast cultures, Nat. Protoc. 2 (12) (2007) 3081–3089. [8] V.A. Maltsev, A.M. Wobus, J. Rohwedel, M. Bader, J. Hescheler, Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents, Circ. Res. 75 (2) (1994) 233–244. [9] J. Nichols, B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Schöler, A. Smith, Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor, Cell 95 (3) (1998) 379–391. [10] P.N. Goodfellow, Developmental genetics: a long story and a short tail, Nature 343 (6259) (1990) 597–598. [11] M. Self, O.V. Lagutin, B. Bowling, J. Hendrix, Y. Cai, G.R. Dressler, G. Oliver, Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney, EMBO J. 25 (21) (2006) 5214–5228. [12] J.F. Armstrong, K. Pritchard-Jones, W.A. Bickmore, N.D. Hastie, J.B. Bard, The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo, Mech. Dev. 40 (1–2) (1993) 85–97. [13] M. Dehbi, M. Ghahremani, M. Lechner, G. Dressler, J. Pelletier, The paired-box transcription factor, PAX2, positively modulates expression of the Wilms’ tumor suppressor gene (WT1), Oncogene 13 (3) (1996) 447–453. [14] L.B. Holzman, P.L. St. John, I.A. Kovari, R. Verma, H. Holthofer, D.R. Abrahamson, Nephrin localizes to the slit pore of the glomerular epithelial cell, Kidney Int. 56 (4) (1999) 1481–1491. [15] X. Shao, J.E. Johnson, J.A. Richardson, T. Hiesberger, P. Igarashi, A minimal Kspcadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract, J. Am. Soc. Nephrol. 13 (7) (2002) 1824–1836. [16] C. Vigneau, K. Polgar, G. Striker, J. Elliott, D. Hyink, O. Weber, H.J. Fehling, G. Keller, C. Burrow, P. Wilson, Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo, J. Am. Soc. Nephrol. 18 (6) (2007) 1709–1720 (Epub 2007 May 2). [17] F. Costantini, R. Shakya, GDNF/Ret signaling and the development of the kidney, Bioessays 28 (2) (2006) 117–127. [18] A.T. Dudley, R.E. Godin, E.J. Robertson, Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme, Genes Dev. 13 (12) (1999) 1601–1613. [19] J. Barasch, J. Yang, C.B. Ware, T. Taga, K. Yoshida, H. Erdjument-Bromage, P. Tempst, E. Parravicini, S. Malach, T. Aranoff, J.A. Oliver, Mesenchymal to epithelial conversion in rat metanephros is induced by LIF, Cell 99 (4) (1999) 377–386. [20] O. Michos, A. Gonçalves, J. Lopez-Rios, E. Tiecke, F. Naillat, K. Beier, A. Galli, S. Vainio, R. Zeller, Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis, Development 134 (13) (2007) 2397–2405 (Epub 2007 May 23). [21] A.F. Esquela, S.J. Lee, Regulation of metanephric kidney development by growth/differentiation factor 11, Dev. Biol. 257 (2) (2003) 356–370. [22] K. Stark, S. Vainio, G. Vassileva, A.P. McMahon, Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4, Nature 372 (6507) (1994) 679–683. [23] R. Mizuguchi, M. Sugimori, H. Takebayashi, H. Kosako, M. Nagao, S. Yoshida, Y. Nabeshima, K. Shimamura, M. Nakafuku, Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtypespecific properties of motoneurons, Neuron 31 (2001) 757–771. [24] E. Moreau, J. Vilar, M. Lelièvre-Pégorier, C. Merlet-Bénichou, T. Gilbert, Regulation of c-ret expression by retinoic acid in rat metanephros: implication in nephron mass control, Am. J. Physiol. 275 (6 Pt. 2) (1998) F938–F945.
R. Morizane et al. / Biochemical and Biophysical Research Communications 390 (2009) 1334–1339 [25] S.J. Bruce, R.W. Rea, A.L. Steptoe, M. Busslinger, J.F. Bertram, A.C. Perkins, In vitro differentiation of murine embryonic stem cells toward a renal lineage, Differentiation 75 (5) (2007) 337–349 (Epub 2007 February 5). [26] A. Maeshima, S. Yamashita, K. Maeshima, I. Kojima, Y. Nojima, Activin A produced by ureteric bud is a differentiation factor for metanephric mesenchyme, J. Am. Soc. Nephrol. 14 (6) (2003) 1523–1534. [27] E. Roussa, K. Krieglstein, GDNF promotes neuronal differentiation and dopaminergic development of mouse mesencephalic neurospheres, Neurosci. Lett. 361 (1–3) (2004) 52–55. [28] A. Majumdar, S. Vainio, A. Kispert, J. McMahon, A.P. McMahon, Wnt11 and Ret/ Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development, Development 130 (14) (2003) 3175–3185. [29] C. Mauritz, K. Schwanke, M. Reppel, S. Neef, K. Katsirntaki, L.S. Maier, F. Nguemo, S. Menke, M. Haustein, J. Hescheler, G. Hasenfuss, U. Martin,
1339
Generation of functional murine cardiac myocytes from induced pluripotent stem cells, Circulation 118 (5) (2008) 507–517 (Epub 2008 July 14). [30] M. Nakagawa, M. Koyanagi, K. Tanabe, K. Takahashi, T. Ichisaka, T. Aoi, K. Okita, Y. Mochiduki, N. Takizawa, S. Yamanaka, Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts, Nat. Biotechnol. 26 (1) (2008) 101–106 (Epub 2007 November 30). [31] J.B. Kim, H. Zaehres, G. Wu, L. Gentile, K. Ko, V. Sebastiano, M.J. Araúzo-Bravo, D. Ruau, D.W. Han, M. Zenke, H.R. Schöler, Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors, Nature 454 (7204) (2008) 646–650 (Epub 2008 June 29). [32] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131 (5) (2007) 861–872.