Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7

Abstract

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual-and trypsinpassaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 ± 2.3 and 41.1 ± 3.1 beats / min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.

Automated, scalable culture of human embryonic stem cells in feeder‐free conditions

“…Recently we developed protocols to maintain hESCs on Matrigel in MEF-conditioned medium (CM; Denning et al, 2006) with trypsin-passaging at regular periodicities, and these conditions have since been used to successfully culture 14 hESC lines derived in five countries Braam et al, 2008;Burridge et al, 2007). We have also demonstrated transfer of manually cultured human mesenchymal stem cells to the completely automated CompacT SelecT cell culture platform, allowing statistical analysis of process performance and systematic process improvement (Thomas et al, 2008).…”

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…placenta-derived feeder cells); culturing in nutrient medium preconditioned by other type of cells (e.g. END-2 cells or mouse embryonic fibroblasts (MEF) [88][89][90][91] or by human cardiac fibroblasts [92].…”

Random Mitotic Activities Across Human Embryonic Stem Cell Colonies

“…We found this passaging method was extremely gentle, which resulted in a very high recovery rate. We usually passaged cells at the split ratio of 1 to 22 every 5 to 7 days, in contrast to 1 to 6 used by most enzymatic or mechanical dissociation methods [11,[14][15][16]. The cell growth was closely monitored by cell morphology and colony shape.…”