Embryonic Stem Cells Facilitate the Isolation of Persistent Clonal Cardiovascular Progenitor Cell Lines and Leukemia Inhibitor Factor Maintains Their Self-Renewal and Myocardial Differentiation Potential in vitro

Abstract

Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines.

Biogrid—a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates

“…The process recapitulates many aspects of cell differentiation in early development (Kurosawa, 2007). Other models of spontaneous or directed in vitro differentiation can also be used to specifically determine the potential of cells to form different progeny such as neural (Chambers et al, 2009) and cardiac (Hoebaus et al, 2013) stem/progenitor cells.…”

Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with thenkx2.5gene

“…As a positive control, we repeated these experiments with a Mesp1 specific antibody and confirmed the already reported interaction of Mesp1 with the MCE and PE of the nkx2.5 gene ( Bondue et al, 2008 ). Desmin was also present in a TF complex bound to the PE in differentiating cardiovascular progenitor cells (CVPCs), isolated as phenotypically stable cell lines from hearts of newborn mice ( Hoebaus et al, 2013 ), and in primary heart cells from newborn mice ( Fig. 4 F).…”

“…10T1/2 fibroblasts and C2C12 myoblasts were maintained in DMEM supplemented with 2 mM L-glutamine, 0.05 mg/ml streptomycin, 0.03 mg/ml penicillin, and 10% (v/v) fetal bovine serum (FBS) from Gibco (M10). Murine des +/+ (AB2.2 and W4) ( Lauss et al, 2005 ), des +/+ des ect (DC6) ( Hofner et al, 2007 ), des −/− ( Weitzer et al, 1995 ) , des Δ1-48/Δ1-48 ( Höllrigl et al, 2002 ) ESCs and A5 cardiovascular progenitor cells (CVPCs) ( Hoebaus et al, 2013 ) were maintained on SNL76/7 feeder cells in the same medium but supplemented with 15% (v/v) FBS (Hyclone, SH3007001) (M15) instead. Differentiation of ESCs and CVPCs was achieved by aggregation to embryoid bodies (EBs) and cardiac bodies as previously described ( Hoebaus et al, 2013 ; Hofner et al, 2007 ).…”

VUT-MK142 : a new cardiomyogenic small molecule promoting the differentiation of pre-cardiac mesoderm into cardiomyocytes

“…To determine whether VUT-MK142 not only promotes expression of genes involved in cardiomyogenesis but also promotes cardiomyogenesis in a way which results in the increased commitment of mesodermal precursors to the cardiomyogenic lineage or the increased differentiation of committed precursors of cardiomyocytes into rhythmically contracting adult-like cardiomyocytes, we investigated the influence of VUT-MK142 on myocardial differentiation of murine cardiovascular progenitor cells (CVPCs). 15 Therefore, CVPCs were aggregated to form cardiac bodies (CBs) and cardiomyogenesis was monitored in these CBs for 20 days by determining the percentage of CBs with beating cardiomyocytes and the number of cardiomyocyte clusters per CB. The addition of VUT-MK142 at a time when CVPCs become committed to myocardial differentiation between days 0 and 5 after aggregation of cells 16 resulted in a two-fold increase in the number of CBs with beating cardiomyocytes (Fig.…”