title: A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells.
abstract: Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.
authors.affiliation: 1 Stem Cell & Regenerative Medicine Consortium, LKS Faculty of Medicine, The University of Hong Kong , Pokfulam, Hong Kong .
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Weng
authors.forename: Chi-Wing
authors.initials: CW
authors.lastname: Kong
authors.forename: Lihuan
authors.initials: L
authors.lastname: Ren
authors.forename: Ioannis
authors.initials: I
authors.lastname: Karakikes
authors.forename: Lin
authors.initials: L
authors.lastname: Geng
authors.forename: Jiaozi
authors.initials: J
authors.lastname: He
authors.forename: Maggie Zi Ying
authors.initials: MZ
authors.lastname: Chow
authors.forename: Chong Fai
authors.initials: CF
authors.lastname: Mok
authors.forename: Harvey Y S
authors.lastname: Chan
authors.forename: Sarah E
authors.lastname: Webb
authors.forename: Wendy
authors.initials: W
authors.lastname: Keung
authors.forename: Howard
authors.initials: H
authors.lastname: Chow
authors.forename: Andrew L
authors.lastname: Miller
authors.forename: Anskar Y H
authors.initials: AY
authors.lastname: Leung
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Hajjar
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
authors.forename: Camie W
authors.initials: CW
authors.lastname: Chan
mesh_terms: D019149:Bioreactors; D055415:Bone Morphogenetic Protein 4 / Q000096:biosynthesis; D002454:Cell Differentiation / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology*; D006352:Heart Ventricles / Q000166:cytology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology*
chemical_list: D055415:Bone Morphogenetic Protein 4