Mice transplanted with free islets then rapidly lost normoglycemia and became diabetic: the median normoglycemia time was 6?days

Mice transplanted with free islets then rapidly lost normoglycemia and became diabetic: the median normoglycemia time was 6?days. measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. Results In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (gamma mouse model [12]. However, the functionality of human islets cotransplanted with human MSC into immunocompetent diabetic mice has, to our knowledge, not so far been investigated. In a setting of human to mouse transplantation, immune rejection is massive and needs to be overcome by immunosuppressive treatments. As a strategy to avoid such treatments, semipermeable microcapsules can be used to protect cells from the host immune reaction [13, 14]. Semipermeable microcapsules allow the exchanges of nutrients, oxygen, and small molecules (lower than 100?kDa) necessary for maintaining viability SYM2206 of cells [15]. Hence, for the human to mouse islet transplantation we used newly developed biomaterials enabling ionotropic interaction between alginate (Alg) molecules and covalent crosslinking between poly(ethylene glycol) (PEG) derivatives, which have higher mechanical resistance compared to Alg [16]. Further, the molecular mechanism leading to improved islet graft survival and function is unclear. Several studies attributed the beneficial effect to factors released by MSC [17] and also to regulatory effects on the host immune system [4]. It is not known whether direct cell contact between islets and MSC plays a role in the beneficial effects of MSC on islet function. However, adhesion molecules such as cadherins and integrins are expressed in human islets [18, 19] and play a role in regulating insulin secretion. Notably, cadherin interactions on beta cells play a role in increased insulin secretion after glucose stimulation [20] and are implicated in protecting islets from apoptosis [21]. Morphological analysis SYM2206 of human islets demonstrated dispersed alpha and beta cells [22], and only recently structures of subislets have?been identified, where alpha cells are organized around centrally located beta cells [23]. This cell set up, based on cell relationships between alpha cells and beta cells, and also with stromal cells around islets, is important for an ideal insulin response by beta cells [24]. Further, this particular arrangement is not present in so-called pseudoislets, which are islet cell aggregations acquired in vitro after enzymatic islet dissociation, but reappears after transplantation in mice [25]. The mechanism leading to this rearrangement remains unclear, but likely involves exogenous factors derived from the sponsor environment. Hence, in this study we analyzed the effect of MSC on islet function as well as their effect on morphology and function of pseudoislets, implying an increased cell contact between MSC and alpha and beta cells. We observed an increased insulin secretion from islets in contact with human being MSC in vitro, which prompted us to investigate a possible involvement of intercellular adhesion molecules, such as epithelial (E)-cadherin, neural cell adhesion molecule (NCAM), epithelial (Ep)CAM-1, vascular (V)CAM-1, N-cadherin, and intercellular (I)CAM-1. Furthermore, we targeted to assess the function of human being islets coencapsulated with MSC upon transplantation in diabetic mice. Methods Isolation and tradition of human being pancreatic islets and human being MSC Human being islets were SYM2206 isolated following a Ricordi protocol [26], and their purity was assessed after dithizone staining and determined using Metamorph (Common Imaging, Western Chester, SYM2206 PA, USA). Islets utilized for these experiments were 80C100% genuine, and no handpicking was performed. Human being islets were offered Rabbit polyclonal to ANG4 for research only when considered not suitable for medical transplantation, through the JDRF honor 1-RSC-2014-100-1-X, ECIT Islet for Basic Research program. The amount of islets was indicated in islet equivalents (IEQ), normalizing each islet to an average diameter of 150?m. We regarded as that 1 IEQ consists of 103 cells. Islets were cultured in HEPES-buffered CMRL1066 medium supplemented with 5.6?mmol/L glucose (Gibco-Invitrogen, Basel, Switzerland), 100?IU/ml penicillin, 100?mg/ml streptomycin (P-S; Gibco-Invitrogen), SYM2206 and 10% FCS (Gibco-Invitrogen) (total CMRL) at 37?C in humidified air flow containing 5% CO2. MSC were from the femoral head of patients undergoing total hip alternative as explained previously and characterized by FACS analysis and their ability to differentiate into osteoblasts, chondrocytes, and adipocytes [27]. Informed consent was given by all individuals and the experimental process was authorized by the local ethical committee of the University or college Private hospitals of Geneva (NAC 01-015). Briefly, MSC were purified from crushed bone marrow by gradient centrifugation, and then cultured in Iscoves revised Dulbeccos medium (Cambrex, Verviers, Belgium) supplemented with 10% FCS, P-S, and 10?ng/ml platelet-derived growth element BB (PDGF-BB; PeproTech EC Ltd, London, UK) [28]. MSC from three different donors between passages 2 and 4 were used and were cultured at 37?C in humidified.

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