In vitro derivation of functional insulin-producing cells from human embryonic stem cells

Abstract

The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

In vitro organogenesis from undifferentiated cells in Xenopus

“…Recently, using an approach similar to that used for the induction of pancreas from the animal caps of Xenopus embryos, a few groups have succeeded in using RA and activin to differentiate from murine ES cells a pancreaslike tissue that contains endocrine cells, exocrine cells, and duct-like structures (Shi et al, 2005;Jiang et al, 2007;Nakanishi et al, 2007). These advances show that the experimental system using animal caps of Xenopus is a useful tool to screen for novel substances that promote the differentiation of vertebrate stem cells into endodermal tissues and organs.…”

CD24: A Novel Surface Marker for PDX1-Positive Pancreatic Progenitors Derived from Human Embryonic Stem Cells

“…Significant progresses have been made in directing human ESCs to differentiate into pancreatic progenitors and insulin-producing beta cells by stepwise induction [2][3][4][5][6][7][8]. However, although pancreatic progenitors can be efficiently generated from human ESCs, efficient in vitro differentiation into mature functional beta cells that are able to reverse diabetes in animal models has not been achieved.…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…In the final, fourth stage cells were transferred into maturation medium containing nicotinamide and IGF2 and cultured for 5 days and without IGF2 for another 2 days to generate IPCs. Flow cytometric analysis indicated that only about 4 % of cells were positive for C-peptide (Jiang et al 2007a).…”

“…The question is in what way the immature endocrine cells implanted into heterotopic environment, such as the epididymal fat pads, subcutaneous tissue or under kidney capsule differentiate into pancreatic cells. Jiang et al (2007a) differentiated ESCs into IPCs using four-stage protocol. In the first stage, ESCs were differentiated into definitive endoderm via treatment with sodium butyrate and activin-A for 7 days.…”

“…Despite significant improvements in novel approaches for generation of IPCs from ESCs, the final yield of IPCs is still very low (0.8-7.3 % of the final heterogenous cell population) (D' Amour et al 2006;Jiang et al 2007a). In addition, the amount of insulin secreted by IPCs is significantly lower as compared to native b cells.…”