Background: Elimination of all animal components during derivation and long-term culture of human embryonic stem cells (hESCs) is necessary for future applications of hESCs in clinical cell therapy.
Methods: In this study, we established the culture system of xeno-free human foreskin fibroblast feeders (XF-HFF) in combination with chemically defined medium (CDM). XF-HFF/CDM was compared with several conventional culture systems. The hESCs cultured in different media were further characterized through karyotype analysis, pluripotency gene expression, and cell differentiation ability.
Results: The hESCs in the XF-HFF/CDM maintained their characteristics including typical morphology and stable karyotype. In addition, hESCs were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. RT-PCR analysis shown that the stem cell markers OCT3/4, hTERT, SOX2, and Nanog were present in the cell line hESC-1 grown on XF-HFF/CDM. Furthermore, the results of cell growth and expression of bFGF, Oct-4, and hTERT indicated that XF-HFF/CDM had better performance than human serum-matrix/CDM and XF-HFF/human serum.
Conclusion: The comparison of different xeno-free culture conditions will facilitate clarifying the key features of self-renewal, pluripotency, and derivation and will shed light on clinic applications of hESCs.
Keywords: Chemically defined medium; Human embryonic stem cell; Human foreskin fibroblast cells; Xeno-free and serum-free culture system.