Generation of Homogeneous PDX1+ Pancreatic Progenitors from Human ES Cell-derived Endoderm Cells

Abstract

One key step in producing insulin-secreting cells from human embryonic stem (hES) cells is the generation of pancreatic and duodenal homeobox gene 1 (PDX1)-expressing pancreatic progenitor cells. All-trans retinoic acid (RA) has important roles in pancreas development and is widely used to induce pancreatic differentiation of ES cells. When RA was added directly to the activin A-induced hES cells, <20% cells were positive for the pancreatic marker PDX1, whereas the other cells were mainly hepatic cells. We found that when the activin A-induced hES cells were replated and seeded at low cell densities, the addition of RA induced significant pancreatic differentiation and over 70% of cells in culture expressed PDX1. When the endodermal cells were isolated with the surface marker CXCR4 from the activin A-induced culture and further differentiated with RA, a homogeneous PDX1(+) cell population (over 95% pure) was generated. The PDX1(+) cells could further differentiate into cells that expressed pancreatic transcription factors and pancreatic endocrine or exocrine markers. We also found that RA inhibited the hepatic differentiation of endodermal cells that were seeded at low cell densities, and this inhibition may have been through the inhibition of Smad1/5/8 activity. Thus, we present a highly efficient and reproducible protocol for generating PDX1(+) pancreatic progenitor cells from hES cells.

Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

“…This triplicates the relative number of PDX1-expressing cells [90]. It suggests that fewer cell-to-cell contacts are beneficial for the induction of PDX1.…”

“…Many groups have now developed such protocols [22,[90][91][92][93][94][95] for a variety of starting stem cell populations that include human ES cells [22,90], human iPS cells [95], and human parthenogenetic stem cells (generation of DE only) [96]. In these protocols, the first step is to differentiate the stem cells into DE.…”

“…For example, the DE cell surface marker CXCR4 has been used to enrich DE to near homogeneity by FACS from ES cell-derived DE cultures [90,116] prior to re-plating and differentiating these cultures further with improved efficiency [90]. Enrichment of PE cells, a later stage in the development of beta-cells, can be obtained by FACS purifying for CD142 (the cell surface glycoprotein coagu-lation factor III) [67] or discoidin domain receptor tyrosine kinase 1 [117].…”

Differentiation of nestin-positive cells derived from bone marrow into pancreatic endocrine and ductal cells in vitro

“…1C, induction). The concentration of RA adopted in our study was previously optimized in embryonic stem cell differentiation model (Cai et al 2009). Recently, the role of RA in pancreatic development and early endocrine lineage determination has been shown (Ostrom et al 2008).…”

Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells

“…Several treatments, forskolin (an adenylate cyclase activator) and dexamethasone (a synthetic adrenocortical steroid) [28] , hepatocyte growth factor, insulin growth factor 1 and glucagon-like peptide 1 [9] , have been used to enhance pancreatic β cell maturation in vitro. Several studies have previously reported the successful production of insulin-secreting cells in vitro from either hESCs or hiPSCs [15,16,18,19,24,25,29,30,36,[58][59][60] . However these differentiated β cells showed little ability to respond to glucose, which is considered one of the well-known and important parameters that recognize functional and mature β cells.…”