Identification and selection of cardiomyocytes during human embryonic stem cell differentiation

Abstract

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.

Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state

“…Technically, however, the purification of chamber-specific derivatives for proteomic experiments remain a major challenge. During the course of this study, Huber et al reported a genetic way to identify hESCderived ventricular derivatives by expressing GFP under the transcriptional control of the myosin light chain (MLC)-2v promoter [33]; while this promoter employed is indeed ventricular-restricted in adult, it remains unclear whether such is also the case in early CMs such as those derived from ESCs (as reviewed by Moorman et al) [34]. Successful selection methods for hESC-derived atrial and pacemaker CMs have not been reported to date.…”

Stem cell therapy for cardiac disease

“…eGFP-expressing cells derived from the ventricular MLC2 (MLC2v) transgenic line formed stable intracardiac cell grafts following transplantation (57). Injection of neomycin resistance-selected hEBs into the hind limb muscles of SCID mice resulted in no teratoma formation after 23 wk (58).…”

Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function

“…25,26 Our group has also given a contribution to this field: we have constructed a cardiacspecific LVV in which expression of the transgene is driven by a short fragment of the cardiac troponin I promoter (TNNI3) with a human cardiac a-actin enhancer (hEnAct). 27 Using an enhanced green fluorescence protein reporter, the TNNI3-LVV has been demonstrated to be effective in tracking cardiac lineage induction during differentiation in both mouse and human embryonic stem cells; moreover, the addition of hEnAct conferred a further increase in expression specificity to the TNNI3-LVV in human embryonic stem cells.…”