Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing β β cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin-producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of β β cells for transplantation.We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells.The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers.Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells.Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca2+]i handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca2+ release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force–frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca2+ handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation–contraction coupling, resemble in part those of adult cardiomyocytes.

Objective: To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI). Methods: Undifferentiated hESCs (0.5-1610 6 ), human embryoid bodies (hEBs) (4-8 days; 0.5-1610 6 ), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2-4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining. Results: Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p,0.05). The transplanted hESCs and hEBs rarely created new vessels and did not form new myocardium. Transplantation of hESCM tissue into normal heart produced islands of disorganised myofibres, fibrosis and, in a single case, a teratoma. However, transplantation of hESCMs into the infarcted myocardium did prevent post-MI dysfunction and scar thinning. Conclusions: Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.

SUMMARY:Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development. (Lab Invest 2003, 83:1811-1820.