Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study.

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy.

Transplantation of insulin-producing cells derived from human mesenchymal stromal/stem cells into diabetic humanized mice.

BACKGROUND: The purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice. METHODS: hAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6-8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR. RESULTS: Following STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR. CONCLUSION: Transplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.

Possible mechanisms for the renoprotective action of adipose-derived mesenchymal stem cells with CD44-targeted hyaluronic acid against renal ischemia.

The present study aimed to investigate the invitro preconditioning of adipose-derived mesenchymal stem cells (ADMSCs) with CD44-targeted hyalournic acid (HA) on ischemic kidney injury in rats. Ninety male Sprague Dawley rats were randomly allocated into the following groups; i) sham group, ii) control group: rats exposed to 45 min left renal ischemia with saline treatment, iii) HA group as control group but rats treated with HA, iv) ADMSCs group as control but rats treated with ADMSCs v) HA + ADMSCs group as ADMSCs but rats treated with ADMSCs preconditioned with CD44-tageted HA for 14 days. We found that treattment with either ADMSCs or HA + ADMSCs caused significant decrease in the elevated serum creatinine and BUN and malondialdehyde (MDA) concentrations and expression of TGF-ß1, fibronectin, collagen type I, inducible nitric oxide synthease (iNOS) and microRNAs (miR-21, miR-17-5p, miR-10a) in kidney and significant increase in creatinine clearance, superoxide dismutase (SOD), reduced glutathione (GSH) and the expression of Bcl2, vascular endothelial growth factor (VEGF), Wnt/ß-catenin pathway genes in kidney compared to control group (p < 0.05). Moreover, HA + ADMSCs group caused more significant improvement in these parameters than ADMSCs group (p < 0.05), while HA group did not cause any significant improvement in these parameters compared to control group. These results suggest that preconditioning of ADMSCs preconditioned with CD44-targted HA enhanced their cytoprotective effect against ischemic kidney injury. This renoprotective effect might be due to activation of angiogenesis, Wnt/ß-catenin pathway proteins, and suppression of oxidative stress, apoptosis, inflammation and fibrosis.