Chamber‐specific differentiation of Nkx2.5‐positive cardiac precursor cells from murine embryonic stem cells

Abstract

Embryonic stem (ES) cells are a useful system to study cardiac differentiation in vitro. It has been difficult, however, to track the fates of chamber-specific cardiac lineages, since differentiation is induced within the embryoid body. We have established an in vitro culture system to track Nkx2.5(+) cell lineages during mouse ES cell differentiation by using green fluorescent protein (GFP) as a reporter. Nkx2.5/GFP(+) cardiomyocytes purified from embryoid bodies express sarcomeric tropomyosin and myosin heavy chain and heterogeneously express cardiac troponin I (cTnI), myosin light chain 2v (MLC2v) and atrial natriuretic peptide (ANP). After 4-week culture, GFP(+) cells exhibited electrophysiological characteristics specific to sinoatrial (SA) node, atrial, or ventricular type. Furthermore, we found that administration of 10(-7) M retinoic acid (RA) to embryoid bodies increased the percentage of MLC2v(-)ANP(+) cells; this also increased the expression of atrial-specific genes in the Nkx2.5/GFP(+) fraction, in a time- and dose-dependent fashion. These results suggest that Nkx2.5(+) lineage cells possess the potential to differentiate into various cardiomyocyte cell types and that RA can modify the differentiation potential of Nkx2.5(+) cardiomyocytes at an early stage.

Excessive O‐GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells

“…We previously established the ES cell line, Nkx2.5GFP, to track cardiac cell lineages during mouse ES cell differentiation. In this cell line, the green fluorescent protein (GFP) gene is knocked in to the Nkx2.5 locus [15]. Nkx2.5 is a homeobox-containing cardiac transcription factor that is expressed in the heart primordium throughout the course of development as well as in cardiomyocytes.…”

Future Treatment of Heart Failure Using Human iPSC-Derived Cardiomyocytes

“…Current procedures for eliminating such contamination and boosting cardiomyocyte enrichment involve genetic modification [14,15] and nongenetic methods using a mitochondrial dye [16] or antibodies to specific cell-surface markers [17]. However, none of these methods are ideal for the therapeutic application of PSC-derived cardiomyocytes due to insufficient stability, genotoxicity, and the use of fluorescence-activated cell sorting (FACS).…”

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

“…16,17 Therefore, the creation of transgenic cell lines harbouring either a reporter gene or exhibiting antibiotic resistance under the control of a cardiac-restrictive promoter have been proposed; these methods have been demonstrated to be effective and easily applicable in mouse systems. [18][19][20][21][22] Unfortunately, the translation of these efforts to the clinic is not possible because in human cells, homologous recombination events are infrequent and cloning efficiency is extremely low. 23 As a result, alternative strategies are needed for obtaining stable, long-term gene expression in the human setting, and LVVs may play a part in this requirement.…”