Induction of functional islet-like cells from human iPS cells by suspension culture

Introduction: To complement islet transplantation for type1 diabetic patients, cell-based therapy using pluripotent stem cells such as ES cells and iPS cells is promising. Many papers have already reported the induction of pancreatic β cells from these cell types, but a suspension culture system has not usually been employed. The aim of this study is to establish a suspension culture method for inducing functional islet-like cells from human iPS cells. Methods: We used 30 ml spinner type culture vessels for human iPS cells throughout the differentiation process. Differentiated cells were analyzed by immunostaining and C-peptide secretion. Cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. Blood human C-peptide and glucagon levels were measured serially in mice, and grafts were analyzed histologically. Results: We obtained spherical pancreatic beta-like cells from human iPS cells and detected verifiable amounts of C-peptide secretion in vitro. We demonstrated reversal of hyperglycemia in diabetic model mice after transplantation of these cells, maintaining non-fasting blood glucose levels along with the human glycemic set point. We confirmed the secretion of human insulin and glucagon dependent on the blood glucose level in vivo. Immunohistological analysis revealed that grafted cells became α, β and δ cells in vivo. Conclusions: These results suggest that differentiated cells derived from human iPS cells grown in suspension culture mature and function like pancreatic islets in vivo. Keywords: iPS cells, Islet, Pancreatic β cel

Efficient induction of pancreatic alpha cells from human induced pluripotent stem cells by controlling the timing for BMP antagonism and activation of retinoic acid signaling.

Diabetes mellitus is caused by breakdown of blood glucose homeostasis, which is maintained by an exquisite balance between insulin and glucagon produced respectively by pancreatic beta cells and alpha cells. However, little is known about the mechanism of inducing glucagon secretion from human alpha cells. Many methods for generating pancreatic beta cells from human pluripotent stem cells (hPSCs) have been reported, but only two papers have reported generation of pancreatic alpha cells from hPSCs. Because NKX6.1 has been suggested as a very important gene for determining cell fate between pancreatic beta and alpha cells, we searched for the factors affecting expression of NKX6.1 in our beta cell differentiation protocols. We found that BMP antagonism and activation of retinoic acid signaling at stage 2 (from definitive endoderm to primitive gut tube) effectively suppressed NKX6.1 expression at later stages. Using two different hPSCs lines, treatment with BMP signaling inhibitor (LDN193189) and retinoic acid agonist (EC23) at Stage 2 reduced NKX6.1 expression and allowed differentiation of almost all cells into pancreatic alpha cells in vivo after transplantation under a kidney capsule. Our study demonstrated that the cell fate of pancreatic cells can be controlled by adjusting the expression level of NKX6.1 with proper timing of BMP antagonism and activation of retinoic acid signaling during the pancreatic differentiation process. Our method is useful for efficient induction of pancreatic alpha cells from hPSCs

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma

The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC), which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP) as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis

Expression of mutant mRNA and protein in pancreatic cells derived from MODY3- iPS cells.

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients' pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner

Encapsulated human islets in alginate fiber maintain long-term functionality

Maintenance of islet function after in vitro culture is crucial for both transplantation and research. Here we evaluated the effects of encapsulation in alginate fiber on the function of human islets which were distributed by the Alberta Islet Distribution Program. Encapsulated human islets from 15 deceased donors were cultured under 5.5 or 25 mM glucose conditions in vitro. The amounts of C-peptide and glucagon secreted from encapsulated islets into the culture media were measured periodically, and immunohistochemical studies were performed. Encapsulated islets maintained C-peptide and glucagon secretion for more than 75 days in 5 cases; in two cases, their secretion was also successfully detected even on day 180. α- and β-cell composition and β-cell survival in islets were unaltered in the fiber after 75 or 180 days of culture. The encapsulated islets cultured with 5.5 mM glucose, but not those with 25 mM glucose, exhibited glucose responsiveness of C-peptide secretion until day 180. We demonstrate that alginate encapsulation enabled human islets to maintain their viability and glucose responsiveness of C-peptide secretion after long-term in vitro culture, potentially for more than for 180 days