Establishment of a feeder and serum-free culture system for human embryonic stem cells

Abstract

SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

Video Bioinformatics Analysis of Human Pluripotent Stem Cell Morphology, Quality, and Cellular Dynamics

“…27,28 Because of their small cytoplasmic to nuclear ratio, the number of cells in hESC colonies is difficult to determine, but cell counting in colonies has been successfully done using label-free time-lapse images. 8 New PSC culture media are continually being developed and tested with hPSC, as well as with embryonic stem cells from other species, 29,30 and StemCellQC could be used to help evaluate the performance of such media. StemCellQC provides a novel video bioinformatics tool that will complement existing methods for the analysis of complex time-lapse PSC data.…”

Different transcriptional profiles of human embryonic stem cells grown in a feeder-free culture system and on human foreskin fibroblast feeder layers

“…However, feeder cell-dependent cultures are often problematic because animal-derived feeder cell layers pose the risk of zoonotic disease transmission and human-tissue-based feeder layers show a risk of contamination [ 4 ]. To overcome these problems, many feeder-free culture systems for supporting hESC propagation, such as mTeSR1 (Stem Cell Technologies, Vancouver, Canada), StemPro hESC SFM (Thermo Fisher, China) and HEScGRO (Millipore, China), have recently been developed [ 5 ].…”