Mesenchymal stem cells as an appropriate feeder layer for prolonged in vitro culture of human induced pluripotent stem cells.

Feeder layers have been applied extensively to support the growth and stemness potential of stem cells for in vitro cultures. Mouse embryonic fibroblast and mouse fibroblast cell line (SNL) are common feeder cells for human induced pluripotent stem cells (hiPSCs) culture. Because of some problems in the use of these animal feeders and in order to simplify the therapeutic application of hiPSCs, we tested human adult bone marrow mesenchymal stem cells (hMSCs) as a potent feeder system. This method benefits from prevention of possible contamination of animal origin feeder systems. hiPSCs transferred onto mitotically inactivated hMSCs and passaged every 5 days. Prior to this culture, MSCs were characterized by flow cytometry of their surface markers and evaluation of their osteogenic and adipogenic differentiation potentials. The morphology, expressions of some specific pluripotency markers such as SSEA-3, NANOG and TRA-1-60, alkaline phosphates activity, formation embryoid bodies and their differentiation potentials of iPSCs on SNL and MSC feeder layers were evaluated. To investigate the prolonged maintenance of pluripotency, the quantitative transcriptions of some pluripotency markers including OCT4, SOX2, NANOG and REX1 were compared in the iPS clones on SNL or MSC feeders. Human iPSCs cultured on human MSCs feeder were slightly thinner and flatter than ones on the other feeder system. Interestingly MSCs supported the prolonged in vitro proliferation of hiPSCs along with maintenance of their pluripotency. Altogether our results suggest human mesenchymal stem cells as an appropriate feeder layer for human iPSCs culture for clinical applications and cell therapy.

Osteogenic Differentiation of MSCs on Fibronectin-Coated and nHA-Modified Scaffolds.

The increasing demand for biocompatible bone substitutes has made it a priority to tissue engineering and regenerative medicine scientists. Combination of minerals, growth factors, and extracellular matrix (ECM) proteins with nanofibrous scaffolds is a potential promising strategy for bone reconstruction and clinical applications. In this study, nanohydroxyapatite (nHA) was incorporated in electrospun nanofibrous polycaprolactone (PCL) scaffolds coated with fibronectin (Fn). The potential bone regeneration capacities of these scaffolds were evaluated in vitro and in vivo using mouse mesenchymal stem cells (mMSCs). The interconnected pores and proper mechanical characteristics of the fabricated electrospun PCL mats in combination with nHA and Fn provided suitable environment for cell attachment, proliferation, and enhanced osteogenic differentiation. The synergistic effect of Fn and nHA on the both in vitro and in vivo increase of calcium deposition was assessed by biochemical analysis. In addition, alkaline phosphatase (ALP) activity in nHA-incorporated PCL scaffold (PCL/nHA) and Fn-coated PCL/nHA (PCL/nHA/Fn) were significantly higher in comparison to the control group. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analyses of important bone-related genes (ALP, osteocalcin, osteopontin, and Runx2) revealed that Fn has additive effect on promoting the osteogenic differentiation. The aforementioned results indicated that nanofibrous PCL/nHA scaffold coated with Fn is a promising candidate for bone-tissue engineering applications.

Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.

The proliferation study of hips cell-derived neuronal progenitors on poly-caprolactone scaffold.

INTRODUCTION: The native inability of nervous system to regenerate, encourage researchers to consider neural tissue engineering as a potential treatment for spinal cord injuries. Considering the suitable characteristics of induced pluripotent stem cells (iPSCs) for tissue regeneration applications, in this study we investigated the adhesion, viability and proliferation of neural progenitors (derived from human iPSCs) on aligned poly-caprolactone (PCL) nanofibers. METHODS: Aligned poly-caprolactone nanofibrous scaffold was fabricated by electrospinning and characterized by scanning electron microscopy (SEM). Through neural induction, neural progenitor cells were derived from induced pluripotent stem cells. After cell seeding on the scaffolds, their proliferation was investigated on different days of culture. RESULTS: According to the SEM micrographs, the electrospun PCL scaffolds were aligned along with uniformed morphology. Evaluation of adhesion and viability of neural progenitor cells on plate (control) and PCL scaffold illustrated increasing trends in proliferation but this rate was higher in scaffold group. The statistical analyses confirmed significant differences between groups on 36h and 48h. DISCUSSION: Evaluation of cell proliferation along with morphological assessments, staining and SEM finding suggested biocompatibility of the PCL scaffolds and suitability of the combination of the mentioned scaffold and human iPS cells for neural regeneration.