Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.
Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.
Huber
Irit
I
Sohnis Family Research Laboratory for the Regeneration of Functional Myocardium and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Itzhaki
Ilanit
I
Caspi
Oren
O
Arbel
Gil
G
Tzukerman
Maty
M
Gepstein
Amira
A
Habib
Manhal
M
Yankelson
Lior
L
Kehat
Izhak
I
Gepstein
Lior
L
D002454:Cell Differentiation; D002460:Cell Line; D002999:Clone Cells; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006321:Heart / Q000196:embryology / Q000502:physiology*; D006801:Humans; D032342:Muscle Cells / Q000166:cytology / Q000502:physiology*; D009206:Myocardium / Q000166:cytology; D014162:Transfection
D017931:DNA Primers; D049452:Green Fluorescent Proteins