Indian Journal of Endocrinology and Metabolism (Jan 2012)
Ex vivo generation of glucose sensitive insulin secreting mesenchymal stem cells derived from human adipose tissue
Abstract
Background: Diabetics are incapable of producing insulin/have autoimmune mechanisms making it ineffective to control glucose secretion. We present a prospective study of glucose-sensitive insulin-secreting mesenchymal stem cells (IS-MSC) generated from human adipose tissue (h-AD) sans xenogenic material. Materials and Methods: Ten grams h-AD from donor anterior abdominal wall was collected in proliferation medium composed of a-Minimum Essential Media (a-MEM), albumin, fibroblast-growth factor and antibiotics, minced, incubated in collagenase-I at 37°C with shaker and centrifuged. Supernatant and pellets were separately cultured in proliferation medium on cell+ plates at 37°C with 5% CO 2 for 10 days. Cells were harvested by trypsinization, checked for viability, sterility, counts, flow-cytometry (CD45 - /90 + /73 + ), and differentiated into insulin-expressing cells using medium composed of DMEM, gene expressing up-regulators and antibiotics for 3 days. They were studied for transcriptional factors Pax-6, Isl-1, pdx-1 (immunofluorescence). C-peptide and insulin were measured by chemiluminescence. In vitro glucose sensitivity assay was carried out by measuring levels of insulin and C-peptide secretion in absence of glucose followed by 2 hours incubation after glucose addition. Results: Mean IS-AD-MSC quantum was 3.21 ml, cell count, 1.5 x10 3 cells/μl), CD45 - /90 + /73 + cells were 44.37% /25.52%. All of them showed presence of pax-6, pdx-1, and Isl-1. Mean C-Peptide and insulin levels were 0.36 ng/ml and 234 μU/ml, respectively, pre-glucose and 0.87 ng/ml and 618.3 μU/ml post-glucose additions. The mean rise in secretion levels was 2.42 and 2.65 fold, respectively. Conclusion: Insulin-secreting h-AD-MSC can be generated safely and effectively showing in vitro glucose responsive alteration in insulin and C-peptide secretion levels.
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