Aneuploidy rate and Stemness in Low-level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel and Lapatinib.

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel and lapatinib was nontoxic at 0.01, 0.01, and 0.2µM concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells.

Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient's somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.

Efficient and cost-effective generation of hepatocyte-like cells through microparticle-mediated delivery of growth factors in a 3D culture of human pluripotent stem cells.

Biomedical application of human pluripotent stem cell-derived hepatocyte-like cells (hPSC-HLCs) relies on efficient large-scale differentiation, which is commonly performed by a suspension culture of three-dimensional (3D) multicellular spheroids in bioreactors. However, this approach requires large amounts of growth factors (GFs) and the need to overcome limited diffusional transport posed by the inherent 3D structure of hPSC spheroids. Here, we have hypothesized that localized delivery of GFs by incorporation of GF-laden degradable polymeric microparticles (MPs) within the hPSC spheroids would circumvent such limitations. In this study, GFs for hepatocytic differentiation were encapsulated in gelatin-coated poly (l-lactic acid)/poly (DL-lactic-co-glycolic acid) (PLLA/PLGA) MPs which were subsequently incorporated into the hPSC spheroids. Gene expression analyses demonstrated that MP delivery of the GFs resulted in similar expression levels of hepatocytic markers despite the use of 10-fold less total GFs. The differentiated HLCs in the MP group exhibited ultrastructure and functional characteristics comparable with the conventional soluble GF group. The generated HLCs in the MP group were successfully engrafted in an acute liver injury mouse model and maintained hepatocytic function after implantation. These results suggested that sustained and localized delivery of GFs using MPs might offer a novel approach towards scalable technologies for hepatocytic differentiation and engineer a better 3D microenvironment for cells.

Cloning, expression and functional characterization of in-house prepared human basic fibroblast growth factor.

OBJECTIVE: Human basic fibroblast growth factor (bFGF) plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). MATERIALS AND METHODS: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability. RESULTS: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo. CONCLUSION: The results show no significant difference in function between our in-house produced and commercialized bFGF.

Proteomic analysis of monkey embryonic stem cell during differentiation.

Proteome analyses of embryonic stem cells (ESCs) will help to uncover mechanisms underlying cellular differentiation, expansion, and self-renewal. We applied a 2-DE based proteomic approach coupled with mass spectrometry to identify genes controlling monkey ESCs proliferation and differentiation. We analyzed proteome of ESCs during proliferation and different stages of spontaneous differentiation (day 3, 6, 12, and 30) by embryoid body formation. Out of about 663 +/- 15 protein spots reproducible detected on gels, 127 proteins showed significant changes during differentiation. Mass spectrometry analysis of differentially expressed proteins resulted in identification of 95 proteins involved in cell cycle progression and proliferation, cell growth, transcription and chromatin remodeling, translation, metabolism, energy production and Ras signaling. In addition, we created protein interaction maps and distinctly different topology was observed in the protein interaction maps of the monkey ESC proteome clusters compared with maps created using randomly generated sets of proteins. Taken together, the results presented here revealed novel key proteins and pathways that are active during ESC differentiation.