Study of transforming growth factor alpha for the maintenance of human embryonic stem cells

Andy C H Chen,William S B Yeung,Y L Lee,Denise Y C Hou,Phillip C N Chiu,P C Ho,Qian Peng,Ronald T K Pang,S W Fong,Kai-Fai Lee

doi:10.1007/s00441-012-1476-7

Abstract

Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have self-regenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor α (TGFα) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFα in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFα maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFα treatment activated the p44/42 MAPK pathway but not the PI3K/Akt pathway. In addition, TGFα treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFα provides insights for the development of clinical grade hESCs for therapeutic applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-012-1476-7) contains supplementary material, which is available to authorized users.

Highlights

Human embryonic stem cells are derived from the inner cell mass of blastocysts
Results hFF-1 but not WI-38 supported the culture of Human embryonic stem cells (hESCs)
We compared the mRNA expressions of the pluripotent (NANOG, OCT4) and early differentiation (KRT8, KRT18) markers in these cells; we found that KRT8 and KRT18 were significantly higher in BG01V grown on WI-38 when compared with those growing on hFF-1 (Fig. 1d; P

Summary

Introduction

Human embryonic stem cells (hESCs) are derived from the inner cell mass of blastocysts. Human ESCs were first successfully cultured on mouse embryonic fibroblast (MEF) feeder cells (Reubinoff et al 2000; Thomson et al 1998). To avoid possible xenogeneic contamination with the use of mouse feeder cells (Martin et al 2005; Stacey et al 2006), human feeder cells such as foreskin fibroblasts (Hovatta et al 2003) and immortalized fibroblasts derived from hESCs (Xu et al 2004) were subsequently used. The use of human feeder cells still has the problem of viral transmission and is labor intensive (Stacey et al 2006). Feeder-free systems have been developed (Amit et al 2004; Draper et al 2004; Xu et al 2001) but some of these systems cause chromosome instabilities and impair the cellular behavior of hESCs (Catalina et al 2008; Maitra et al 2005)

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Results

Conclusion