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Pdx-1-independent differentiation of mouse embryonic stem cells into insulin-expressing cells
diabetes research and clinical practice 79 (2008) e8–e10
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/diabres
Brief report
Pdx-1-independent differentiation of mouse embryonic stem cells into insulin-expressing cells I. Takayama, S. Miyazaki, F. Tashiro, J. Fujikura, J. Miyazaki, E. Yamato * Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Japan
article info
abstract
Article history:
To investigate whether insulin-producing cells obtained from ES cells via the nestin-positive
Received 17 July 2007
cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell
Accepted 21 August 2007
line and analyzed its differentiation into insulin-producing cells. As a result, pdx-1 knockout
Published on line 27 September 2007
ES cell expressed insulin 2 gene at the final differentiated cells. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated
Keywords:
from this population of nestin-expression enriched ES cells, and suggested that the insulin-
ES cells
producing cells derived from ES cells may be different from the pancreatic beta cells in terms
Pdx-1
of their lineage. # 2007 Published by Elsevier Ireland Ltd.
Insulin
1.
Introduction
The differentiation of ES cells into lineage-restricted cells is thought to be a promising technique for future therapeutic use, including diabetes treatment. Lumelsky et al. developed a five-stage method for directing the differentiation of mouse ES cells into insulin-secreting cells that included a selection step to enrich the culture for nestin-positive cells [1]. Although several investigators, including us, have reported the successful differentiation of mouse ES cells into insulin-producing cells using Lumelsky et al.’s method with some modifications, the insulin-producing cells obtained by this method were not mature enough to secrete high levels of insulin in response to glucose [2–5]. Pdx-1 is known to transactivate insulin gene expression through conserved enhancer elements, and it is an essential regulator of pancreatic endocrine development and adult islet beta cell function. In mice lacking pdx-1, the development of the pancreas is blocked at a very early stage. Recent studies
have also suggested that pdx-1 is one of the earliest markers for pancreatic endocrine and exocrine precursor cells. Thus, pdx-1 is crucial for the proper differentiation of insulin-producing cells in pancreatic islets [6]. To investigate whether insulinproducing cells obtained from ES cells via the nestin-positive cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell line and analyzed its differentiation into insulin-producing cells.
2.
Materials and methods
A 6.0-kb XhoI-XbaI fragment including exon 1 of the mouse pdx-1 gene was isolated from a 129SvJ mouse genome lambda phage library. The targeting vector was constructed by inserting the IRES beta-geo gene connected to the mouse pgk-promoter-driven puromycin-resistance gene into the ScaI site of exon 1 of the mouse pdx-1 gene (Fig. 1a). ES cells (EB3 cells originated from E14tg2a ES cells [2,3]) were selected with
* Corresponding author at: Division of Stem Cell Regulation Research, G6, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 3824; fax: +81 6 6879 3829. E-mail address: [email protected] (E. Yamato). 0168-8227/$ – see front matter # 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.diabres.2007.08.013
diabetes research and clinical practice 79 (2008) e8–e10
e9
Fig. 1 – (a) Targeted disruption of the pdx-1 gene. A 6.0-kb XhoI-XbaI fragment including exon 1 of the mouse pdx-1 gene was isolated from a 129SvJ mouse genomic library in lambda phage vectors. The targeting vector was constructed by inserting the IRES b-geo gene connected to a mouse pgk promoter-driven puromycin-resistance gene into the ScaI site of exon 1 of the mouse pdx-1 gene. An alignment of the endogenous pdx-1 locus and the mutant allele after homologous recombination is shown. DT-A: MC1 promoter with diphtheria toxin A-fragment gene. (b) Gene expression in the pdx-1 K/O ES cells at each stage of in vitro differentiation. RT-PCR analysis was performed with RNAs from the differentiated cells. (+/ +): wild-type ES cells; (+/S) hetero K/O ES cells; (S/S) homo K/O ES cells; (C): RNA from MIN6 insulinoma cells as the positive control.
puromycin to establish ES clones harboring the targeted allele. Southern blot analysis with XbaI revealed that ES clones were heterozygous for the targeted allele (hetero K/O ES clone, data not shown). To obtain clones with homozygous pdx-1 knockout alleles, heterozygous ES clones were cultured with a high concentration of puromycin. The selected clones were verified to be homozygotes for the targeted allele by southern blot analysis and PCR (homo K/O ES clone, data not shown). The pdx-1 null ES cells showed similar growth to heterozygote and wild-type ES cells in undifferentiated culture condition and during differentiation. The differentiation protocol followed our method [2,3], which is based on Lumelsky et al.’s and proceeds through five differentiation stages: ES cells (stage 1), embryoid body-stage (stage 2), selection stage under serum-free-condition (stage 3),
proliferation stage (stage 4), and differentiation stage (stage 5). The gene expression pattern of the ES cells during differentiation was analyzed by RT-PCR (Fig. 1b). Total RNA was prepared from the undifferentiated and differentiating parental ES cells and the heterozygous and homozygous pdx-1 knockout cell lines at stages 1–5. The primer sequences and PCR conditions used for RT-PCR were previously described [2].
3.
Results and discussion
pdx-1 gene expression was observed at stage 3 in wild-type ES and hetero K/O ES cells, but was absent from pdx-1 homo K/O ES cells during differentiation. The cell morphology during ES differentiation at each stage was the same in the three groups (data not shown). In addition, there was no notable difference
e10
diabetes research and clinical practice 79 (2008) e8–e10
in the gene expression pattern during differentiation in the three groups, including insulin 2 gene expression (Fig. 1b). The gene expression level of insulin 2 gene was also confirmed by realtime PCR analysis (data not shown). Moreover, our study showed that the insulin 1 gene, which is specifically expressed in pancreatic B cells, was not expressed during differentiation at any stage in any of the groups. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated from this population of nestin-expression enriched ES cells, and suggested that the insulin-producing cells derived from ES cells may be different from the pancreatic beta cells in terms of their lineage. Indeed, we previously reported the differentiation of insulin-producing cells by this method from a feeder-free ES cell line harboring the beta-geo gene under the control of the mouse insulin 2 promoter. Our results suggested that insulinexpressing cells had already appeared in the visceral endoderm by the time of embryoid body formation. These data proved that the insulin-expressing cells derived from ES cells were different from pancreatic beta cells in their origin. In agreement with our result, Houard et al. found that the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6 and that the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of beta cells [7]. Our results also showed that the isl1, Beta2, and Pax6 genes were expressed in the pdx-1 homo K/O ES cells. The expression of these transcription factors may be responsible for the expression of the insulin 2 and glucagon genes. Interestingly, these transcription factors are also expressed in cells of the neuronal lineage. Likewise, the somatostatin gene, which was expressed in the pdx-1 homo K/O ES cells, is also expressed in cells of the neuronal lineage. Thus, as suggested by Sipione et al., insulin-positive cells that differentiate in vitro from ES cells are not of pancreatic lineage, but of neuronal lineage [8]. In our experiment, undifferentiated ES cells express neurogenin3 (ngn3) and the expression level was decreased in stage 2–4. However, in stage 5, the high expression of ngn3 was observed, while insulin2 expression level was high in this stage. The ngn3 was expressed in the progenitor of endocrine cells of pancreas, but not expressed in the mature beta cells. Considering that the expression of ngn3 is known to be expressed in the neuronal cell, the differentiated cells in stage 5 was not, at least, mature pancreatic beta cells, rather differentiated cells of neural cell lineage. We have also analyzed the expression of other pancreasrelated gene, nkx2.2, nkx6.1, pax4, Glut2, Kir6.2, prohormone convertase (PC2), GK (glucokinase), p48, amylase, and carboxypeptidase A (CXPA). No expressions were observed in Nkx2.2,
Pax4, Glut2 and amylase genes in any stage of three groups. Nkx6.1, Pax6, Kir6.2, PC2, GK, p48, CXPA genes were detected at stage 4 and stage 5, but there were no difference in the expression of these genes by RT-PCR in any stages of differentiation of three groups of ES cells (data not shown). We previously reported that the pdx-1 gene expression in the insulin-expressing cells was quite low [2] and that pdx-1 expression clearly enhanced the expression of the insulin 2 gene and of insulin production in differentiated cells from a newly established ES cell line in which the exogenous pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus [3]. Thus, the low expression of the pdx-1 gene in the insulin-producing cells is one of the causes of the immature phenotype of these cells. In conclusion, pdx-1 was not essential for insulin gene expression in cells differentiated from ES cells via enrichment of the nestin-positive population, suggesting that these differentiated cells may not be of pancreatic lineage. Further investigation into how to direct the differentiation of ES cells into functional insulin-producing cells is needed to develop an ES cell-based regeneration therapy for diabetes mellitus.
references
[1] N. Lumelsky, O. Blondel, P. Laeng, I. Velasco, R. Ravin, R. McKay, Differentiation of embryonic stem cells to insulinsecreting structures similar to pancreatic islets, Science 292 (2001) 1389–1394. [2] Y. Moritoh, E. Yamato, Y. Yasui, S. Miyazaki, J. Miyazaki, Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells, Diabetes 52 (2003) 1163–1168. [3] S. Miyazaki, E. Yamato, J. Miyazaki, Regulated expression of pdx-1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells, Diabetes 53 (2004) 1030– 1037. [4] G. Keller, Embryonic stem cell differentiation: emergence of a new era in biology and medicine, Genes Dev. 19 (2005) 1129–1155. [5] J. Rajagopal, W.J. Anderson, S. Kume, O.I. Martinez, D.A. Melton, Insulin staining of ES cell progeny from insulin uptake, Science 299 (2003) 363. [6] J.F. Habener, D.N. Kemp, M.K. Thomas, Transcriptional regulation in pancreatic development, Endocrinology 146 (2005) 1025–1034. [7] N. Houard, G.G. Rousseau, F.P. Lemaigre, HNF-6independent differentiation of mouse embryonic stem cells into insulin-producing cells, Diabetologia 46 (2003) 378–385. [8] S. Sipione, A. Eshpeter, J.G. Lyon, G.S. Korbutt, R.C. Bleackley, Insulin expressing cells from differentiated embryonic stem cells are not beta cells, Diabetologia 47 (2004) 499–508.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/diabres
Brief report
Pdx-1-independent differentiation of mouse embryonic stem cells into insulin-expressing cells I. Takayama, S. Miyazaki, F. Tashiro, J. Fujikura, J. Miyazaki, E. Yamato * Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Japan
article info
abstract
Article history:
To investigate whether insulin-producing cells obtained from ES cells via the nestin-positive
Received 17 July 2007
cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell
Accepted 21 August 2007
line and analyzed its differentiation into insulin-producing cells. As a result, pdx-1 knockout
Published on line 27 September 2007
ES cell expressed insulin 2 gene at the final differentiated cells. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated
Keywords:
from this population of nestin-expression enriched ES cells, and suggested that the insulin-
ES cells
producing cells derived from ES cells may be different from the pancreatic beta cells in terms
Pdx-1
of their lineage. # 2007 Published by Elsevier Ireland Ltd.
Insulin
1.
Introduction
The differentiation of ES cells into lineage-restricted cells is thought to be a promising technique for future therapeutic use, including diabetes treatment. Lumelsky et al. developed a five-stage method for directing the differentiation of mouse ES cells into insulin-secreting cells that included a selection step to enrich the culture for nestin-positive cells [1]. Although several investigators, including us, have reported the successful differentiation of mouse ES cells into insulin-producing cells using Lumelsky et al.’s method with some modifications, the insulin-producing cells obtained by this method were not mature enough to secrete high levels of insulin in response to glucose [2–5]. Pdx-1 is known to transactivate insulin gene expression through conserved enhancer elements, and it is an essential regulator of pancreatic endocrine development and adult islet beta cell function. In mice lacking pdx-1, the development of the pancreas is blocked at a very early stage. Recent studies
have also suggested that pdx-1 is one of the earliest markers for pancreatic endocrine and exocrine precursor cells. Thus, pdx-1 is crucial for the proper differentiation of insulin-producing cells in pancreatic islets [6]. To investigate whether insulinproducing cells obtained from ES cells via the nestin-positive cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell line and analyzed its differentiation into insulin-producing cells.
2.
Materials and methods
A 6.0-kb XhoI-XbaI fragment including exon 1 of the mouse pdx-1 gene was isolated from a 129SvJ mouse genome lambda phage library. The targeting vector was constructed by inserting the IRES beta-geo gene connected to the mouse pgk-promoter-driven puromycin-resistance gene into the ScaI site of exon 1 of the mouse pdx-1 gene (Fig. 1a). ES cells (EB3 cells originated from E14tg2a ES cells [2,3]) were selected with
* Corresponding author at: Division of Stem Cell Regulation Research, G6, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 3824; fax: +81 6 6879 3829. E-mail address: [email protected] (E. Yamato). 0168-8227/$ – see front matter # 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.diabres.2007.08.013
diabetes research and clinical practice 79 (2008) e8–e10
e9
Fig. 1 – (a) Targeted disruption of the pdx-1 gene. A 6.0-kb XhoI-XbaI fragment including exon 1 of the mouse pdx-1 gene was isolated from a 129SvJ mouse genomic library in lambda phage vectors. The targeting vector was constructed by inserting the IRES b-geo gene connected to a mouse pgk promoter-driven puromycin-resistance gene into the ScaI site of exon 1 of the mouse pdx-1 gene. An alignment of the endogenous pdx-1 locus and the mutant allele after homologous recombination is shown. DT-A: MC1 promoter with diphtheria toxin A-fragment gene. (b) Gene expression in the pdx-1 K/O ES cells at each stage of in vitro differentiation. RT-PCR analysis was performed with RNAs from the differentiated cells. (+/ +): wild-type ES cells; (+/S) hetero K/O ES cells; (S/S) homo K/O ES cells; (C): RNA from MIN6 insulinoma cells as the positive control.
puromycin to establish ES clones harboring the targeted allele. Southern blot analysis with XbaI revealed that ES clones were heterozygous for the targeted allele (hetero K/O ES clone, data not shown). To obtain clones with homozygous pdx-1 knockout alleles, heterozygous ES clones were cultured with a high concentration of puromycin. The selected clones were verified to be homozygotes for the targeted allele by southern blot analysis and PCR (homo K/O ES clone, data not shown). The pdx-1 null ES cells showed similar growth to heterozygote and wild-type ES cells in undifferentiated culture condition and during differentiation. The differentiation protocol followed our method [2,3], which is based on Lumelsky et al.’s and proceeds through five differentiation stages: ES cells (stage 1), embryoid body-stage (stage 2), selection stage under serum-free-condition (stage 3),
proliferation stage (stage 4), and differentiation stage (stage 5). The gene expression pattern of the ES cells during differentiation was analyzed by RT-PCR (Fig. 1b). Total RNA was prepared from the undifferentiated and differentiating parental ES cells and the heterozygous and homozygous pdx-1 knockout cell lines at stages 1–5. The primer sequences and PCR conditions used for RT-PCR were previously described [2].
3.
Results and discussion
pdx-1 gene expression was observed at stage 3 in wild-type ES and hetero K/O ES cells, but was absent from pdx-1 homo K/O ES cells during differentiation. The cell morphology during ES differentiation at each stage was the same in the three groups (data not shown). In addition, there was no notable difference
e10
diabetes research and clinical practice 79 (2008) e8–e10
in the gene expression pattern during differentiation in the three groups, including insulin 2 gene expression (Fig. 1b). The gene expression level of insulin 2 gene was also confirmed by realtime PCR analysis (data not shown). Moreover, our study showed that the insulin 1 gene, which is specifically expressed in pancreatic B cells, was not expressed during differentiation at any stage in any of the groups. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated from this population of nestin-expression enriched ES cells, and suggested that the insulin-producing cells derived from ES cells may be different from the pancreatic beta cells in terms of their lineage. Indeed, we previously reported the differentiation of insulin-producing cells by this method from a feeder-free ES cell line harboring the beta-geo gene under the control of the mouse insulin 2 promoter. Our results suggested that insulinexpressing cells had already appeared in the visceral endoderm by the time of embryoid body formation. These data proved that the insulin-expressing cells derived from ES cells were different from pancreatic beta cells in their origin. In agreement with our result, Houard et al. found that the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6 and that the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of beta cells [7]. Our results also showed that the isl1, Beta2, and Pax6 genes were expressed in the pdx-1 homo K/O ES cells. The expression of these transcription factors may be responsible for the expression of the insulin 2 and glucagon genes. Interestingly, these transcription factors are also expressed in cells of the neuronal lineage. Likewise, the somatostatin gene, which was expressed in the pdx-1 homo K/O ES cells, is also expressed in cells of the neuronal lineage. Thus, as suggested by Sipione et al., insulin-positive cells that differentiate in vitro from ES cells are not of pancreatic lineage, but of neuronal lineage [8]. In our experiment, undifferentiated ES cells express neurogenin3 (ngn3) and the expression level was decreased in stage 2–4. However, in stage 5, the high expression of ngn3 was observed, while insulin2 expression level was high in this stage. The ngn3 was expressed in the progenitor of endocrine cells of pancreas, but not expressed in the mature beta cells. Considering that the expression of ngn3 is known to be expressed in the neuronal cell, the differentiated cells in stage 5 was not, at least, mature pancreatic beta cells, rather differentiated cells of neural cell lineage. We have also analyzed the expression of other pancreasrelated gene, nkx2.2, nkx6.1, pax4, Glut2, Kir6.2, prohormone convertase (PC2), GK (glucokinase), p48, amylase, and carboxypeptidase A (CXPA). No expressions were observed in Nkx2.2,
Pax4, Glut2 and amylase genes in any stage of three groups. Nkx6.1, Pax6, Kir6.2, PC2, GK, p48, CXPA genes were detected at stage 4 and stage 5, but there were no difference in the expression of these genes by RT-PCR in any stages of differentiation of three groups of ES cells (data not shown). We previously reported that the pdx-1 gene expression in the insulin-expressing cells was quite low [2] and that pdx-1 expression clearly enhanced the expression of the insulin 2 gene and of insulin production in differentiated cells from a newly established ES cell line in which the exogenous pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus [3]. Thus, the low expression of the pdx-1 gene in the insulin-producing cells is one of the causes of the immature phenotype of these cells. In conclusion, pdx-1 was not essential for insulin gene expression in cells differentiated from ES cells via enrichment of the nestin-positive population, suggesting that these differentiated cells may not be of pancreatic lineage. Further investigation into how to direct the differentiation of ES cells into functional insulin-producing cells is needed to develop an ES cell-based regeneration therapy for diabetes mellitus.
references
[1] N. Lumelsky, O. Blondel, P. Laeng, I. Velasco, R. Ravin, R. McKay, Differentiation of embryonic stem cells to insulinsecreting structures similar to pancreatic islets, Science 292 (2001) 1389–1394. [2] Y. Moritoh, E. Yamato, Y. Yasui, S. Miyazaki, J. Miyazaki, Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells, Diabetes 52 (2003) 1163–1168. [3] S. Miyazaki, E. Yamato, J. Miyazaki, Regulated expression of pdx-1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells, Diabetes 53 (2004) 1030– 1037. [4] G. Keller, Embryonic stem cell differentiation: emergence of a new era in biology and medicine, Genes Dev. 19 (2005) 1129–1155. [5] J. Rajagopal, W.J. Anderson, S. Kume, O.I. Martinez, D.A. Melton, Insulin staining of ES cell progeny from insulin uptake, Science 299 (2003) 363. [6] J.F. Habener, D.N. Kemp, M.K. Thomas, Transcriptional regulation in pancreatic development, Endocrinology 146 (2005) 1025–1034. [7] N. Houard, G.G. Rousseau, F.P. Lemaigre, HNF-6independent differentiation of mouse embryonic stem cells into insulin-producing cells, Diabetologia 46 (2003) 378–385. [8] S. Sipione, A. Eshpeter, J.G. Lyon, G.S. Korbutt, R.C. Bleackley, Insulin expressing cells from differentiated embryonic stem cells are not beta cells, Diabetologia 47 (2004) 499–508.