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 ß-cell composition and ß-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.

The functional maturity of grafted human pluripotent stem cell derived-islets (hSC-Islets) evaluated by the glycemic set point during blood glucose normalizing process in diabetic mice.

Human pluripotent stem cell (hPSCs) derived-pancreatic islets (hSC-islets) are good candidates for cell replacement therapy for patients with diabetes as substitutes for deceased donor-derived islets, because they are pluripotent and have infinite proliferation potential. Grafted hSC-islets ameliorate hyperglycemia in diabetic mice; however, several weeks are needed to normalize the hyperglycemia. These data suggest hSC-islets require maturation, but their maturation process in vivo is not yet fully understood. In this study, we utilized two kinds of streptozotocin (STZ)-induced diabetes model mice by changing the administration timing in order to examine the time course of maturation of hSC-islets and the effects of hyperglycemia on their maturation. We found no hyperglycemia in immune-compromised mice when hSC-islets had been transplanted under their kidney capsules in advance, and STZ was administered 4 weeks after transplantation. Of note, the blood glucose levels of those mice were stably maintained under 100 mg/dl 10 weeks after transplantation; this is lower than the mouse glycemic set point (120-150 mg/dl), suggesting that hSC-islets control blood glucose levels to the human glycemic set point. We confirmed that gene expression of maturation markers of pancreatic beta cells tended to upregulate during 4 weeks after transplantation. Periodical histological analysis revealed that revascularization was observed as early as 1 week after transplantation, but reinnervation in the grafted hSC-islets was not detected at all, even 15 weeks after transplantation. In conclusion, our hSC-islets need at least 4 weeks to mature, and the human glycemic set point is a good index for evaluating ultimate maturity for hSC-islets in vivo.

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids_4MACE2-TMPRSS2 and Spheroids_15M63ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS-CoV-2 agents, remdesivir and 5h (an inhibitor of SARS-CoV-2's main protease), inhibited the infectivity and replication of SARS-CoV-2 in a dose-dependent manner, suggesting that these human-derived induced spheroids should serve as valuable target cells for the evaluation of anti-SARS-CoV-2 activity. IMPORTANCE The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells. Thus, the addition of human iPSC-derived spheroids for anti-SARS-CoV-2 drug evaluation studies could provide the opportunity for more comprehensive interpretation of the antiviral activity of compounds against SARS-CoV-2.

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.

Establishment of a diabetes mellitus type 1 model in the common marmoset.

Common marmosets have attracted considerable attention as a small standard primate model in biomedical research. However, no marmoset diabetes model is available. Here, we established a marmoset diabetes model via the combination of partial pancreatectomy and intravenous streptozotocin (STZ). A partial pancreatectomy was performed in 11 common marmosets and multiple STZ doses were intravenously administered. Diabetes was diagnosed upon sustained hyperglycaemia (nonfasting blood glucose level >200 mg/dl). Blood glucose and biochemistry were periodically assessed, in addition to glucose tolerance testing, continual blood glucose determination using a continuous glucose monitoring system, urine testing and histological evaluation. In 8 of the 11 animals (73%), diabetes mellitus was induced. The diabetic marmosets also showed abnormal intravenous and oral glucose tolerance test results. Blood glucose levels decreased in response to human insulin administration. The hyperglycaemic state was irreversible and persisted for more than 3 months, and the animals' condition was manageable via daily insulin administration. Thus, diabetes can be successfully induced and maintained in the common marmoset via partial pancreatectomy and STZ administration. This protocol effectively generates a valuable animal model for studying disease pathogenesis, risk factors and therapeutic interventions, including islet transplantation.

The intraperitoneal space is more favorable than the subcutaneous one for transplanting alginate fiber containing iPS-derived islet-like cells.

INTRODUCTION: Although immunosuppressants are required for current islet transplantation for type 1 diabetic patients, many papers have already reported encapsulation devices for islets to avoid immunological attack. The aim of this study is to determine the optimal number of cells and optimal transplantation site for human iPS-derived islet-like cells encapsulated in alginate fiber using diabetic model mice. METHODS: We used a suspension culture system for inducing islet-like cells from human iPS cells throughout the islet differentiation process. Islet-like spheroids were encapsulated in the alginate fiber, and cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. We compared the efficacy of transplanted cells between intraperitoneal and subcutaneous administration of alginate fibers by measuring blood glucose and human C-peptide levels serially in mice. Grafts were analyzed histologically, and gene expression in pancreatic ß cells was also compared. RESULTS: We demonstrated the reversal of hyperglycemia in diabetic model mice after intraperitoneal administration of these fibers, but not with subcutaneous ones. Intraperitoneal fibers were easily retrieved without any adhesion. Although we detected human c-peptide in mice plasma after subcutaneous administration of these fibers, these fibers became encased by fibrous tissue. CONCLUSIONS: These results suggest that the intraperitoneal space is favorable for islet-like cells derived from human iPS cells when encapsulated in alginate fiber.

Definitive endoderm differentiation is promoted in suspension cultured human iPS-derived spheroids more than in adherent cells.

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.

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.

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 ß 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, ß 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.

Efficient generation of functional pancreatic ß-cells from human induced pluripotent stem cells.

BACKGROUND: Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic ß-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic ß-cells. METHODS: A six-stage protocol was established for the differentiation of human iPSCs to pancreatic ß-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3ß inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. RESULTS: Addition of CHIR99021 (3 µmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. CONCLUSION: Functional pancreatic ß-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.

Establishment of maturity-onset diabetes of the young-induced pluripotent stem cells from a Japanese patient.

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 13 disease genes have been identified. However, the pathogenesis of MODY is not fully understood, because the pancreatic ß-cells of the patients are inaccessable. Therefore, we attempted to establish MODY patient-derived induced pluripotent stem cells (MODY-iPS) cells to investigate the pathogenic mechanism of MODY by inducing pancreatic ß-cells. We established MODY5-iPS cells from a Japanese patient with MODY5 (R177X), and confirmed that MODY5-iPS cells possessed the characteristics of pluripotent stem cells. In the course of differentiation from MODY5-iPS cells into pancreatic ß-cells, we examined the disease gene, HNF1B messenger ribonucleic acid. We found that the amount of R177X mutant transcripts was much less than that of wild ones, but they increased after adding cycloheximide to the medium. These results suggest that these R177X mutant messenger ribonucleic acids are disrupted by nonsense-mediated messenger ribonucleic acid decay in MODY-iPS cells during the developmental stages of pancreatic ß-cells.

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.

Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture.

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.