We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.

Murine mesenchymal stem cells (mMSC) and the difficult task of isolation and purification of them have been the subject of rather extensive investigation. The present study sought to isolate these cells from two different mouse strains, one outbred and the other inbred, primarily through a relatively simple but novel approach, the most important feature of which was the low density primary culture of bone marrow cells. For this purpose, mononuclear cells from either NMRI or BALB/c bone marrow were plated at about 500 cells per well of 24-well plates and incubated for 7 days. At this point, the fibroblastic clones that had emerged were pooled together and expanded through several subcultures. To investigate the mesenchymal nature, we differentiated the cells into the osteoblastic, chondrocytic and adipocytic lineages in different subcultures up to passage 10. According to the results, 1 week after culture initiation, several clones each comprising several fibroblastic cells appeared in each plate. The cells from different passages were capable of differentiating into corresponding skeletal tissues. In the present investigation, the best culture condition for maximum proliferation and also the expression of certain surface marker on isolated cells were examined. In this term the two murine strains showed some differences.

No abstract

No abstract

Pancreatic tissue engineering as a therapeutic option for restoring and maintenance of damaged pancreas function has a special focus to using synthetic Scaffolds. This study was designed to evaluate pancreatic differentiation of human induced pluripotent stem cells (hiPSCs) on poly-L-lactic acid and polyvinyl alcohol (PLLA/PVA) scaffolds as 3 D matrix. During differentiation process, morphology of cells gradually changed and iPSCs derived insulin producing cells (iPSCs-IPCs) formed spherical shaped cell aggregation that was the typical shape of islets of pancreas. The highly efficient differentiation of iPSCs into a relatively homogeneous population of IPCs was shown by immunostaining. Real-time reverse transcription polymerase chain reaction (RT-PCR) results demonstrated that iPSCs-IPCs expressed pancreas-specific transcription factors (Pdx1, insulin, glucagon and Ngn3). The expressions of these transcription factors in PLLA/PVA scaffold were significantly higher than 2 D groups. Furthermore, we showed that concentration of insulin and C-peptide in PLLA/PVA scaffold and/or 2 D culture in response to various concentrations of glucose increased but the difference between them were not significant. Altogether the current results demonstrated that PLLA/PVA scaffold could provide the microenvironment that promotes the pancreatic differentiation of iPSCs, up-regulate pancreatic-specific transcription factors and improved metabolic activity.

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.

The studies have been done on patient-specific human adipose-derived from mesenchymal stem cells (hADSCs) like a series of autologous growth factors and nanofibrous scaffolds (3D culture) will probably have many benefits for regenerative medicine in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a polyvinyl alcohol (PVA) scaffold and a differentiation protocol by adding platelet-rich plasma (PRP) that induces the hADSCs into insulin-producing cells (IPCs). The characteristics of the derived IPCs in 3D culture were compared with conventional culture (2D) groups evaluated at the mRNA and protein levels. The viability of induced pancreatic cells was 14 days. The in vitro studies showed that the treatment of hADSCs in the 3D culture resulted in differentiated cells with strong characteristics of IPCs including pancreatic-like cells, the expression of the islet-associated genes at the mRNA and protein levels in comparison of 2D culture group. Furthermore, the immunoassay tests showed that these differentiated cells in these two groups are functional and secreted C-peptide and insulin in a glucose stimulation challenge. The results of our study for the first time demonstrated that the PVA nanofibrous scaffolds along with the optimized differentiation protocol with PRP can enhance the differentiation of IPCs from hADSCs. In conclusion, this study provides a new approach to the future pancreatic tissue engineering and beta cell replacement therapies for T1DM.