Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Insulin expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature β-cells. β-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide β-cells in vitro. We find that functional β-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

In an effort to regulate mammalian cell behavior in contact with solid material surfaces, we have functionalized surfaces with different ratios of both the putative cell binding (-Arg-Gly-Asp-) domain and a consensus heparan-binding domain. The peptide sequences -Arg-Gly-Asp- (-RGD-) and -Phe-His-Arg-Arg-Ile-Lys-Ala- (-FHRRIKA-) or mixtures of the two in the ratios of 75:25 (mimetic peptide surface I), 25:75 (mimetic peptide surface II), and 50:50 (mimetic peptide surface III) were immobilized on model surfaces using a heterobifunctional cross-linker to link the peptide(s) to amine-functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry, thickness of the overlayers, and surface density of immobilized peptides ( approximately 4-6 pmol/cm2). The degree of rat calvaria osteoblast-like cell spreading, focal contact formation, cytoskeletal organization, proliferation, and mineralization of the extracellular matrix (ECM) on model biomaterial surfaces was examined. Mimetic peptide surface II (MPS II) and MPS III supported the highest degree of cell spreading (p < 0.05), following 4 h of incubation, compared to MPS I, homogeneous -RGD-, and homogeneous -FHRRIKA- grafted surfaces. Furthermore, MPS I, MPS II, MPS III, and homogeneous -RGD- surfaces promoted the formation of focal contacts and stress fibers by attached bone cells. The strength of bone cell detachment following 30 min of incubation was significantly higher (p < 0.05) on MPS II surfaces compared to homogeneous -RGD- and -FHRRIKA-. However, the degree of cell proliferation on the peptide surfaces were not significantly different from each other (p > 0.1). Following 24 d in culture, the areas of mineralized ECM formed on MPS II and MPS III surfaces were significantly (p < 0.05) larger than those of other surfaces. These results demonstrate that utilizing peptide sequences incorporating both cell- and heparin-adhesive motifs can enhance the degree of cell surface interactions and influence the long-term formation of mineralized ECM in vitro.

Significance Human pluripotent stem cells (hPSCs) can be produced from any person and have the potential to differentiate into any cell type in the body. This study focuses on the generation of insulin-expressing cells from hPSCs and compares their gene expression, as assayed by transcriptional gene products, to that of insulin-expressing β cells from human fetal and adult samples. We employ a new method to isolate and profile insulin-expressing cells and conclude that several different hPSC lines generate very similar insulin-expressing cells, cells whose transcripts resemble fetal rather than adult β cells. This study advances the possibility of directing the differentiation of stem cells into functional β cells by comparing and cataloging differences between hPSC-derived insulin-expressing cells and human β cells.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes.

OBJECTIVEDifferentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells.RESEARCH DESIGN AND METHODSUsing a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed.RESULTSA high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass.CONCLUSIONSThese results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.

We develop a method to overcome previously documented restrictions on the differentiation propensities of pluripotent stem cells. Culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma protein, increases the proportion of cells in the early G1 phase of the cell cycle, and subsequently improves their competency for directed differentiation into multiple lineages in more than 25 stem cell lines. DMSO treatment also promotes terminal differentiation into functional derivatives.