A Xeno-Free Culturing Protocol for Pluripotent Stem Cell-Derived Oligodendrocyte Precursor Cell Production

Abstract

AIM: To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. MATERIALS & METHODS: The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. RESULTS: Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40-60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1-81 and Oct-4 -positive cells. CONCLUSIONS: This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.