Generation of Insulin‐Producing Cells From Human‐Induced Pluripotent Stem Cells Using a Stepwise Differentiation Protocol Optimized With Platelet‐Rich Plasma

Abstract

Studies on patient-specific human-induced pluripotent stem cells (hiPSCs) as well as a series of autologous growth factors presumably will reveal their many benefits for cell base replacement therapy in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a multistep protocol by adding platelet-rich plasma (PRP) that induce the hiPSCs into insulin-producing cells (IPCs). We present here a differentiation protocol consisting of five stages in two groups including protocol with PRP and without PRP. Charac-teristics of derived IPCs in both groups were evaluated at the mRNA and protein levels, cell cycle and viability in the end stage of cell differentiation. The in vitro studies indicated the treatment of hiPSCs in the protocol with PRP resulting in differentiated cells with strong characteristics of IPCs including islet-like cells, the expression of mature and functional pancreatic beta cell specific marker genes. In addition to these pancreatic specific markers were detected by immunochemistry and Western blot. Our differentiated cells in two groups secreted insulin and C-peptide in a glucose stimulation test by ELISA showing in vitro functional. The results of the cell cycle assay confirmed that differentiation has been done. We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs. This study provides a new approach to investigate the role of PRP in pancreatic differentiation protocols and enhance the feasibility of using patient-specific iPSCs and autologous PRP for future beta cells replacement therapies for T1DM. J. Cell. Physiol. 232: 2878-2886, 2017. © 2016 Wiley Periodicals, Inc.

Generation of insulin‐producing cells from human adipose‐derived mesenchymal stem cells on PVA scaffold by optimized differentiation protocol

“…However, the results of this study indicated that the Glut‐2 gene was expressed in both experimental groups, but, the difference between the two experimental groups was not statistically significant. This result aligned with Enderami et al (). For this reason, the result (Figures a and b) showed that the difference of C‐peptide and insulin releasing in response to the low level of glucose (5.5 mM) concentration between the two experimental groups was not significant.…”

“…In the present study, insulin secretion in response the high level of glucose stimulation of IPCs on the PVA scaffold was significantly higher than the 2D culture system. In this study, the observed results of pancreatic specific markers expression in mRNA and protein level assessment were aligned with the other studies in the field of stem cell differentiation to IPCs (Enderami et al, ; Khorsandi et al, ; Zhang et al, ). One of the advantages of 3D culture system in the long process of differentiation is increasing cell proliferation and viability compared with the 2D culture.…”

“…In the second stage the ADSCs were then cultured in the differentiation medium II containing 1% nonessential amino acids (Invitrogen), 20 ng/ml epidermal growth factor (EGF, Sigma–Aldrich), 20 ng/ml basic fibroblast growth factor (bFGF, Sigma–Aldrich), 2 mmol/L Lglutamine (Sigma–Aldrich), 2% B27 (Invitrogen), 10 ng/ml exendine‐4 (Sigma–Aldrich), and 2% PRP for 6 days. In stage 3, the hADSCs were cultured for an additional 6 days in differentiation medium III containing 10 ng/ml betacellulin (Sigma–Aldrich), 2% B27, 10 mmol/ L nicotinamide (Sigma–Aldrich), 10 ng/ml activin A (Sigma–Aldrich), 10 ng/ml exendine‐4, and 1% PRP (Enderami et al, ). In the second and third stages, the differential media was replaced with fresh medium every 2 days.…”

“…The attachment, viability, and proliferation ability of hADSCs on PVA nanofibers were determined via MTT assay during 7 days. To do that, sterilized scaffolds were placed in cell culture plate, seeded with as many as 4 × 10 3 cells per cm 2 and incubated at 37°C, 5% CO 2 (Enderami et al, 2016 method was applied to analyze the data.…”

Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells

“…[44][45][46] Consistent with these reports, our cell cycle assay also showed that the majority of undifferentiated hiPSCs were in the proliferation phase (S phase), however, after treatment with activin A, as an specification inducing signal, the majority of hiPSCs cultured in both TCP and DWJS were entered in the G0/G1 phase, with the cells cultured on DWJS having more G0/G1 positive cells. These reports demonstrated that pluripotent stem cells have a short G0/G1 phase; however, when these cells are exposed to specification cues, their developmental programs are activated and their cell cycle remodels, so that the length of G1 phase increase.…”