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We have reported the effective 3D-culture system to differentiate insulin producing cell (IPC) from human adipose derived stem cell (hADSC) recombinant peptide micro-pieces (RCP μ-pieces, FUJIFILM, Tokyo, Japan) (Ikemoto T, et al. Pancreas. 2018, Ikemoto T, et al. Sci Rep 2019, Wada Y, et al. Sci Rep 2019). Although a successful short-term result was confirmed within 30 days with a single administration of IPCs, a long-term fate of IPCs remains unclear. Thus, the aim of this study is to clarify the long-term result of transplanted IPCs in mice. IPCs were generated from hADSCs, which were isolated from patient's subcutaneous adipose, with our original 2-steop protocol which was a xeno-antien free, 3D-culture system as mentioned above ( Pancreas. 2018, Sci Rep 2019). BALB/c nu-nu mice were induced to a diabetic state with 200mg/kg streptozotocin for the recipients. 2.0 × 106 IPCs were implanted to these mice under the kidney capsule (Tx group, n=4), same quantity normal saline were administered as control (Sham group, n=4). Non-fasting blood glucose and body weight were measured up to 180 days. Then transplanted mice were sacrificed for immunohistochemical evaluations. In Tx group, blood glucose levels in all recipients decreased below 200 mg/dl on day 10 and below 150 mg/dl around day 60 (100%), even though all mice in Sham group were dead in 40 days (100%). The normoglycemic state in Tx group continued until 180 days with IPCs single transplantation (Fig 1). No abnormal tumorous lesions or inflammations were observed at the transplanted sites. Transplanted IPCs were strongly stained by anti-insulin antibody, anti-C peptide antibody and anti-human leukocyte antigen class I antibody (Fig 2). Our IPCs can be applied to clinical trial as a promising strategy for type 1 DM patients and totally pancreatectomized patients. [ABSTRACT FROM AUTHOR]

We have reported the possibility of non-clinical PoC for generating insulin-producing cells (IPCs) differentiated from adipose-derived stem cells (ADSCs). However, whether ADSC isolated from type 1 diabetes mellitus (T1DM) patients has the differentiation ability or not is still unclear. Thus, we aimed to determine whether appropriately responsive IPCs could be generated from ADSCs isolated from actual T1DM patients. We isolated ADSCs from the discarded adipose tissue from four patients undergoing surgery at our institute and differentiated them into IPCs using our established two-step xeno-antigen free, three-dimensional culture method (UMIN000035546,IRB approved on Jan 10, 2018). Characters of isolated ADSCs, in vitro cell quality and cell function, programmed cell death ligand-1(PDL-1) expression and in vivo functional tests by transplantation to streptozotocin (STZ) induced diabetic nude mice were investigated. ADSCs from T1DM patients and commercially obtained ADSCs showed the same surface markers; CD31−CD34−CD45−CD90+CD105+CD146−. Moreover, the generated IPCs at day 21 demonstrated appropriate autonomous insulin secretion in vitro (stimulation index;3.5). The non-fasting blood glucose concentrations of STZ-induced diabetic nude mice were normal 30 days after IPC transplantation. The normalized rate of STZ-induced diabetic nude mice was significantly different to the sham-operated group (P<0.05). IPCs generated from T1DM adipose tissue demonstrated high levels of expression of PDL-1, even though freshly isolated ADSCs did not expressed. These findings suggest that IPCs obtained from the adipose tissue of T1DM patients are capable of secreting insulin long-term and achieving normoglycemia following a single auto-transplantation, and without the need for immunosuppression. [ABSTRACT FROM AUTHOR]

As a curative treatment for type 1 diabetes, we have established and reported a rapid and efficient protocol of insulin producing cell (IPC) differentiation from adipose derived stem cell (ADSC) which were isolated with a minimally invasive procurement (Wada Y, Ikemoto T, et al. Sci Rep. 2019). Experimental β cell transplantation is often performed under the kidney capsule in murine model, however this is not suitable for clinical application. We examined the optimal transplantation site for clinical transplantation of IPCs. ADSCs were differentiated into IPC using our developed xeno-antigen free, 3D cultured (scaffold; cellnest, provided by Fuji Film CO.,Ltd) and two-step protocol which we have reported (Ikemoto T, et al. Sci Rep. 2019). BALB/c nu-nu mice were induced to a diabetic state with 200mg/kg streptozotocin as the recipients. 2.0 × 106 generated IPCs were transplanted in the mesenteric space (n=4), under the kidney capsule (n=4), and subcutaneous space (n=2), respectively. Non-fasting blood glucose and body weight were measured and observed in every 3 days. Immunohistochemical evaluations were undergone after the transplanted mice were sacrificed sequentially. Under the kidney capsule group, blood glucose level decreased below 200mg/dl on day 10 and then gradually reached the normal range (4/4, 100%). In the mesenteric space group, blood glucose level approached the normal range more quickly and normalized below 150mg/dl around day 14 (4/4,100%). In both groups blood glucose level was normalized with single shot of IPC transplantation. As a preliminary data, the tendency of improving hyperglycemic state was observed in subcutaneous space group, however, that improvement did not reach the significance. The optimal site for IPC transplantation was considered to be in the mesenteric space from our investigations. Simultaneously, we plan to research the usefulness of subcutaneous transplantation with administration of angiogenetic factor, for achieving less invasive transplantation of IPCs. [ABSTRACT FROM AUTHOR]

The aim of this study is to clarify the effectiveness of a new 3D-culture system of hepatocyte like cells (HLCs) from human adipose derived mesenchymal stem cells (ADSCs). 2.0 × 104 human ADSCs with or without 0.02 mg of a scaffold-type extracellular matrix called compound X per well were seeded in 96-well U bottom plate, then our 3-step differentiation protocol was applied. At each step, cell morphology and gene expressions were investigated. Hepatocyte functions were evaluated with albumin secretion, CYP3A4 activity and ammonium metabolism. Those parameters were compared between 2D and 3D HLCs. In 2D culture, ADSCs had gradually changed those morphology, and HLCs had the shapes of hepatocytes. Definitive endoderm showed significantly higher Sox17 mRNA levels compared to undifferentiated ADSCs. Hepatic endoderm showed also higher AFP levels, and HLCs showed higher ALB levels. In 3D culture, HLCs made the forms of spheroids from day6, and the sizes were gradually increased. Gene expression levels of each step had significantly higher levels than 2D HLCs. 3D HLCs showed higher CYP3A4 activity and better ammonium metabolism compared with 2D HLCs. Our new 3D-culture differentiation protocol of HLCs from ADSCs might be effective and promising for clinical cell transplantation. [ABSTRACT FROM AUTHOR]