Induction of Differentiation of Embryonic Stem Cells into Insulin-Secreting Cells by Fetal Soluble Factors

Abstract

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/␤geo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bromodeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering ␤ cells from stem cells. STEM CELLS 2006;24:258 -265

A Role for the Host in the Roadmap to Diabetes Stem Cell Therapy

“…It was also shown that differentiated cells do not form teratomas (4,5) and they mature 30 days after transplantation (4). Subsequently, in vitro differentiation protocols for human embryonic stem cells (hESCs) or induced pluripotent stem cells succeeded in generating definitive endoderm (6)(7)(8). Numerous protocols have been published that have yielded insulin-producing cells from pluripotent stem cells (9)(10)(11).…”

Stem Cell Therapies for Type I Diabetes

“…Inherent variability in the behavior of individual ESC lines, in conjunction with these responses in the absence or presence of serum, could explain these results. Studies that identify the molecules released by developing pancreas could help to determine the main factors needed in ESC-IPC differentiation in vitro (Vaca, Martin et al 2006). These studies could also be applied in the development of synthetic molecules, such as indolactam V, IDE1 or IDE2, for easier production and control of murine and human ESC-IPC differentiation (Borowiak, Maehr et al 2009;Chen, Borowiak et al 2009).…”

Embryonic stem cells to beta-cells by understanding pancreas development

“…Studies have also recognized karyotypic instability of long term hESC cultures with recurrent chromosomal gain characteristic of some germ cell tumours (Draper et al 2004). These concerns are not alleviated by standard laboratory transplantation experiments; the length of time used to assess cell replacement in diabetic mice is too short for adequate appreciation of tumour risk in patients (Vaca et al 2006). …”