In vivo characterization of transplanted human embryonic stem cell-derived pancreatic endocrine islet cells.

OBJECTIVES: Islet-like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin-induced diabetic immuno-incompetent mice. MATERIALS AND METHODS: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). RESULTS: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C-peptide and glucagon, and for the ductal epithelial marker cytokeratin-19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas-specific cell markers was maintained for 70 days post-transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C-peptide in response to an oral bolus of glucose. CONCLUSIONS: hESC-derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C-peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin-producing cells for transplantation into patients with type 1 diabetes.

Insulin expressing cells from differentiated embryonic stem cells are not beta cells.

AIM/HYPOTHESIS: Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes. METHODS: ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks. RESULTS: Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state. CONCLUSIONS/INTERPRETATION: Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.

Raf-1 kinase activity predicts for paclitaxel resistance in TP53mut, but not TP53wt human ovarian cancer cells.

We have recently reported that there is a significant Raf-1 kinase dependency of paclitaxel resistance in human cervical tumor cell lines. In light of the possibility that Raf-1 kinase inhibitors could be used to enhance paclitaxel responsiveness in ovarian cancer, we have characterized the Raf-1 kinase dependency of paclitaxel resistance in ovarian cancer cells. The relationship between Raf-1 kinase activity and the sensitivity to clinically relevant paclitaxel concentrations was determined in four ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/WT and OAW42/WT). Furthermore, in recognition that such a drug combination would initially be used in patients whose tumors have recurred following cisplatin/paclitaxel treatment, we also determined the Raf-1 kinase dependency of paclitaxel cytotoxicity in cisplatin resistant variants of two of the ovarian cell lines (2780/CP and OAW42/CP). In the two cell lines (2780/WT and OAW42/WT) that possess a wild-type TP53 (TP53wt), the relationship between Raf-1 kinase activity and paclitaxel resistance was different from that observed in the cervical tumor cell lines. In these cell lines, paclitaxel-induced far more cell killing than would have been predicted from their Raf-1 kinase activity. However, in the ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/CP and OAW42/CP) that have a mutant TP53 (TP53mut), the cytotoxicity induced by 60 nM paclitaxel exhibited the same relationship to Raf-1 kinase activity as previously observed in cervical tumor cell lines. These data suggest that the therapeutic efficacy of paclitaxel in ovarian cancer patient whose tumors have TP53mut might be increased if it is administered in combination with Raf-1 kinase inhibitors, e.g., ISIS 5132.