title: 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 trypsin-passaging, 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.
authors.affiliation: Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, UK. chris.denning@nottingham.ac.uk
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
authors.forename: Cinzia
authors.initials: C
authors.lastname: Allegrucci
authors.forename: Helen
authors.initials: H
authors.lastname: Priddle
authors.forename: Maria D
authors.initials: MD
authors.lastname: Barbadillo-Muñoz
authors.forename: David
authors.initials: D
authors.lastname: Anderson
authors.forename: Tim
authors.initials: T
authors.lastname: Self
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Smith
authors.forename: C Tony
authors.initials: CT
authors.lastname: Parkin
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Young
mesh_terms: D015415:Biomarkers; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection
chemical_list: D015415:Biomarkers