Development of definitive endoderm from embryonic stem cells in culture

Kubo, Atsushi; Shinozaki, Kazuo; Shannon, John M.; Kouskoff, Valérie; Kennedy, Marion; Woo, Savio L. C.; Fehling, Hans Jöerg; Keller, Gordon

doi:10.1242/dev.01044

Cited by 773 publications

(696 citation statements)

References 81 publications

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“…In these embryological contexts, our data showed that differentiation of endoderm or mesendoderm can be actively directed by exposing hEBs to serum and activin. Consistent with our study, a recent study using mouse ESCs [24] reported the positive effect of serum and activin on the differentiation of definitive endoderm. A more recent study using hESCs [35] also demonstrated that, under two-dimensional differentiation conditions, a population enriched with definitive endoderm can be generated by treatment with activin A at a low serum concentration (0.5%).…”

Section: Discussionsupporting

confidence: 93%

“…1a, when hEBs were treated with serum for 4 days and further differentiated in serum-free conditions for 6 days (S4/SF6), the expression of the genes encoding forkhead box protein A2 (FOXA2) and SRY-box containing gene 17 (SOX17) was significantly increased compared with control cultures (SF10). We also observed that the serum treatment markedly upregulated the expression of the mix1 homeobox-like 1 gene (MIXL1), a primitive streak marker, and T [human homologue of the mouse T (brachyury)], which is known to be expressed in mesendoderm [24] (Fig. 1a).…”

Section: Differentiation Of Hescs Into Endodermmentioning

confidence: 82%

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Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

et al. 2007

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Aims/hypothesis The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation. Materials and methods The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation. Results Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice. Conclusions/interpretation Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.

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Section: Discussionsupporting

confidence: 93%

Section: Differentiation Of Hescs Into Endodermmentioning

confidence: 82%

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

et al. 2007

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“…We observed mRNA within 2-3 days for nestin (an early neural ectoderm marker) and brachyury (Brachy, an early mesoderm marker), whereas β-H1 globin (β-globin, a mesoderm marker) and α-fetoprotein (α-FP, an endoderm marker) mRNAs accumulated later (days 5-6). These patterns of expression are as previously reported for multilineage differentiation following LIF withdrawal [8,9,32,[38][39][40][41].…”

Section: Progesterone Receptor Gene Is Expressed Early During Differesupporting

confidence: 87%

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

Sauter

McDermid

Weinberg

et al. 2005

Experimental Cell Research

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The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERα, ERβ), are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen dependent manner by endogenous ERs or by transiently overexpressed ERα. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation

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“…The recent report of a technique for efficient generation of definitive endoderm from ES cells by culture in lower concentrations of serum and Activin A [7][8][9] has been an important advance for subsequent production of hepatocyte-like cells. We have found that induction of definitive endoderm from ES cells was a necessary step for subsequent hepatic differentiation in vitro.…”

Section: Protocols Have Been Developed To Differentiate and Enrich Vamentioning

confidence: 99%

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

et al. 2007

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This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

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Development of definitive endoderm from embryonic stem cells in culture

Cited by 773 publications

References 81 publications

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

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