Transcriptional expression profile of cultured human embryonic stem cells in vitro and in vivo

Abstract

The aims of this study were to analyze the spontaneous differentiation of human embryonic stem cells in vitro and in vivo and to investigate the influence of in vitro partial differentiation on in vivo teratoma formation in immunodeficient mice. Standardized methods are needed for long-term cultivation of undifferentiated stem cells and the multilineage cells that spontaneously differentiate from them. Accordingly, SA002 human embryonic stem cells were cultured on irradiated mouse embryonic fibroblasts cells, on irradiated human foreskin fibroblasts, or were cultured feeder-free using matrigel. Expression of marker protein transcripts was analyzed in undifferentiated and differentiated stem cells using real-time PCR, and both types of stem cells were transplanted subcutaneously into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice to test for teratoma formation. Teratoma histology and expression profiles were subsequently characterized. Cells cultured using different conditions and morphologically undifferentiated cells had comparable marker expression profiles, showing high expression levels of markers for pluripotency and low-to-moderate expression levels of germ layer markers. Cells showing spontaneous differentiation that were cultured in feeder-free conditions in the absence of basic fibroblast growth factor demonstrated slight upregulation of sex determining region Y-box 17, connexin 32, and albumin expression at early time points, as well as expression of octamer-binding transcription factor 4, proteoglycan epitopes on podocalyxin (Trafalgar), and alkaline phosphatase. At later time points, expression of hepatocyte nuclear factor-3-beta, and hepatocyte nuclear factor-4-alpha and alpha fetoprotein was upregulated, whereas beta-3-tubulin, chemokine receptor, nestin, sex-determining region Y-box 17, and connexin 32 were downregulated. Expression of pluripotency markers remained high, and hematopoetic markers were not expressed. SA002 cells that showed spontaneous partial differentiation in vitro had a low teratoma formation capacity in vivo. Cells that were partially differentiated led to slower growing teratomas with more uniform histology.