Directed Differentiation of Human Embryonic Stem Cells into the Pancreatic Endocrine Lineage

Abstract

Human embryonic stem (hES) cells represent a potentially unlimited source of transplantable beta-cells for the treatment of diabetes. Here we describe a differentiation strategy that reproducibly directs HES3, an National Institutes of Health (NIH)-registered hES cell line, into cells of the pancreatic endocrine lineage. HES3 cells are removed from their feeder layer and cultured as embryoid bodies in a three-dimensional matrix in the presence of Activin A and Bmp4 to induce definitive endoderm. Next, growth factors known to promote the proliferation and differentiation of pancreatic ductal epithelial cells to glucose-sensing, insulin-secreting beta-cells are added. Pdx1 expression, which identifies pancreatic progenitors, is detected as early as day 12 of differentiation. By day 34, Pdx1+ cells comprise between 5% and 20% of the total cell population and Insulin gene expression is up-regulated, with release of C-peptide into the culture medium. Unlike another recent report of the induction of insulin+ cells in differentiated hES cell populations, we are unable to detect the expression of other pancreatic hormones in insulin+ cells. When transplanted into severe combined immunodeficiency (SCID) mice, differentiated cell populations retain their endocrine identity and synthesize insulin.

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

“…These protocols induce definitive endoderm differentiation at the first stage, then pancreatic specialization and maturation at the following stages. A stepwise combination of different growth factors and small molecules was utilized to direct human ES cells to differentiate into insulin-producing cells [6][7][8][9]. In these reported protocols, insulin-positive cells were that exhibited some characteristics of pancreatic islet cells were generated.…”

“…In these reported protocols, insulin-positive cells were that exhibited some characteristics of pancreatic islet cells were generated. Most of these cells released insulin and C-peptide, expressed certain islet transcription factors and contained secretory granules [6][7][8][9]. However, these insulin-positive cells mostly exhibited immature islet characteristics such as the co-expression of insulin/ C-peptide and glucagon [6,7] and low insulin/C-peptide content in the differentiated cells [8,9].…”

“…Most of these cells released insulin and C-peptide, expressed certain islet transcription factors and contained secretory granules [6][7][8][9]. However, these insulin-positive cells mostly exhibited immature islet characteristics such as the co-expression of insulin/ C-peptide and glucagon [6,7] and low insulin/C-peptide content in the differentiated cells [8,9]. In general, the differentiation efficiency of the insulin-producing cells derived from human ES cells was quite low and the insulin/C-peptide-secreting level of these cells in vitro was much lower than that of adult human islet cells.…”

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.…”

BMPS and Liver: More Questions than Answers

“…Furthermore, BMP4 has been reported to be necessary to induce definitive endoderm from mouse and human embryonic stem cells in vitro [80]. BMP2 appears to have a similar effect to that of BMP4 [81,82], whereas BMP7 has been shown to potentiate mesoderm differentiation and inhibit definitive endoderm differentiation [79,83]. Oval cells are adult liver stem cells that resemble embryonic hepatoblasts as they express both hepatocyte and bile duct markers.…”