(B) FACS analysis of BMNCs after 6 days of priming with MIN-6 CM (remaining), INS-1 CM (middle), and MPs-depleted MIN-6 CM (right). from your -cells. primed BMNCs indicated the -cell-specific transcription factors, as well as insulin, and improved hyperglycemia and glucose intolerance after transplantation into the streptozotocin-induced diabetic mice. Furthermore, we have found that components of the CM which result in the differentiation were enclosed by or integrated into micro particles (MPs), rather than being secreted as soluble factors. Identification of these differentiation-directing factors might enable us to develop novel technologies required for the production of clinically relevant IPCs. Introduction Diabetes mellitus (DM) is usually characterized by chronic hyperglycemia resulting from the defects in insulin secretion, insulin action, or both. Type 1 DM results from autoimmune destruction of the -cells in the pancreatic islets1,2, whereas more common type 2 DM results from insulin Rabbit Polyclonal to DP-1 resistance in the peripheral tissues and subsequent -cell dysfunction3C5. Although islet transplantation can achieve better glycemic control than insulin therapy6,7, many complicated issues including shortage of islet donors and necessity of immune suppression, have hampered this treatments widespread use. During the last several decades, extensive research has been focused on the treatment of type 1 DM based on the generation of the surrogate insulin generating cells (IPCs) from your stem cells. Many research groups have developed stepwise differentiation protocols that mimic the developmental paradigms to differentiate the pluripotent stem cells (PSC) into the IPC progenitors that are capable of maturation priming with conditioned media (CM) prepared from your culture supernatants of the syngeneic or xenogeneic -cells under stress conditions can direct the BMNCs to express the -cell-specific proteins, including insulin, C-peptide, PDX-1, MafA, and Nkx6.1, within 6 days. Moreover, primed BMNCs improved hyperglycemia and glucose intolerance after systemic infusion in the diabetic mice. We also found that IPC differentiation was specifically mediated by the MPs shed from your -cells managed under stress conditions because priming with MP-depleted CM did not induce IPC generation. These results suggest that identification of the MP-associated differentiation-directing factors might enable us to establish novel technologies for the production of IPCs. Results differentiation of BMNCs into IPCs It has been previously reported that BMNCs significantly contribute to adult -cell renewal in mice27C31, but other reports have contradicted these findings32,33. In the beginning, we tested whether BMNCs can differentiate into IPCs. We generated chimeric C57BL/6 mice harboring BMNCs from your insulin promoter luciferase/GFP transgenic (MIP-Luc/GFP) mice and then treated the mice with streptozotocin (STZ) to eliminate the -cells, while control mice were treated with the same volume of vehicle (Fig.?1A). We then analyzed pancreatic sections by immunofluorescence staining with antibodies against GFP, insulin, and PDX-1 at different time points. It should be noted that this GFP-expressing cells began to appear approximately 24 days after STZ treatment, and the number of the GFP-positive cells increased up to 48 days after STZ treatment (Figs?1B; S1A; Table?S1). These results implied that this GFP and insulin double positive cells were differentiated from BMNCs ESI-05 that were mobilized from your bone marrow. We also detected the GFP and insulin double positive cells in the small intestine on day 18 in MIP-Luc/GFP mice treated with STZ (Figs?1C; S1B), consistent with an ESI-05 increase in the luciferase transmission in the intestine of these mice (Fig.?S1C). These phenomena are similar to previous reports that have exhibited heterotopic neogenesis of IPCs in diabetic animal models, such as STZ-treated mice34C36. We hypothesized that damaged -cells might shed some factors that direct the differentiation of BMNCs into IPCs. Thus, we prepared conditioned media (CM) from your culture supernatant of an insulinoma cell collection maintained under stress at low levels of glucose and serum. We mixed the CM with Matrigel at a ratio of 1 1:1 and transplanted the combination into the subcutaneous region of the healthy chimeric MIP-Luc/GFP mice. Immunofluorescence staining of the Matrigel platforms harvested on day 18 after transplantation revealed newly differentiated insulin and GFP double positive cells only in ESI-05 the Matrigel platforms made ESI-05 up of CM of syngeneic MIN-6 insulinoma cells (Fig.?1D). However, the CM-free Matrigel or the Matrigel platforms containing CM of a clonal endothelial bEND.3 cell line ESI-05 did not show any positive cells (Fig.?S1D). These results indicate that BMNCs were capable of differentiating into IPCs in response to the signals or components shed from your damaged -cells. Open in a separate window Physique 1 BMNCs differentiate into IPCs differentiation of BMNCs into IPCs We developed a simple and reproducible IPC-generating priming protocol (Fig.?2A) after extensive screening of various culture conditions. Priming with CM prepared from your culture supernatants of the syngeneic (MIN-6) or xenogeneic (INS-1) insulinoma cell lines directed the MIP-Luc/GFP derived BMNCs to express GFP within 6 days (Fig.?2B). However, microparticles (MPs)-depleted CM, in which large particles over 100?kDa have been.