Abstract
BackgroundElimination 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.MethodsIn 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.ResultsThe 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.ConclusionThe 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.
Highlights
Elimination of all animal components during derivation and long-term culture of human embryonic stem cells is necessary for future applications of hESCs in clinical cell therapy
Characterizations of hESCs in the xeno-free human foreskin fibroblast feeders (XF-HFF)/chemically defined medium (CDM) culture system hESCs cultured in XF-HFF/CDM exhibit normal cell morphologies To determine whether hESCs grown in XF-HFF/CDM were maintained in an undifferentiated state, the morphology of hESC-1 cultured in XF-HFF/CDM was examined
The results showed that hESC-1 grown in XF-HFF/ CDM expressed the expected stem cell markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 2a)
Summary
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. Human embryonic stem cells (hESCs) can differentiate into various cell types and possess great potential for cellreplacement therapy including treatment for diabetes, cardiac infarction, and neurodegenerative diseases [1, 2]. The major challenge for the clinical application of hESCs are animal-derived products and undefined factors during invitro establishment and expansion of the cells. HESCs are conventionally cultured on feeder cell layers, of which mouse embryonic fibroblasts (MEFs) are the most common and present several unknown animalderived products [3]. Feeder-free cultures generally present a higher degree of spontaneous differentiation in comparison with conventional cultures, signifying that higher concentration of exogenous basic fibroblast growth factor (bFGF) is required and feeder-free derivation is not optimal for developing transplantable hESC derivatives [11]
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