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Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells¹

KYOKO HIDAKA^*, JONG-KOOK LEE, HOE SUK KIM^*, CHUN HWA IHM^*, AKIO IIO^*, MINETARO OGAWA, SHIN-ICHI NISHIKAWA, ITSUO KODAMA and TAKAYUKI MORISAKI^*,,2

Departments of
^* Bioscience, National Cardiovascular Center Research Institute, and
Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Fujishiro-dai, Suita, Osaka, Japan;
Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Chikusa-ku, Nagoya, Japan; and
Department of Molecular Genetics, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan

²Correspondence: Departments of Bioscience, National Cardiovascular Center Research Institute, and Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, 5–7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail: morisaki{at}ri.ncvc.go.jp

SPECIFIC AIMS

Diversification of cardiomyocytes is believed to occur at a very early stage in cardiac development; however, little is known regarding chamber-specific differentiation of embryonic stem (ES) cell-derived cardiomyocytes. As embryoid bodies (EBs) contain cardiomyocytes in different stages with different chamber specificities, it has been difficult to evaluate the diversification of ES cell-derived cardiomyocytes. To circumvent these problems, we have established an ES cell line [Nkx2.5 green fluorescent protein (GFP) ES] in which GFP is knocked into the Nkx2.5 locus. This study seeks to 1) establish a system to track the differentiation of Nkx2.5(+) cardiomyocytes purified from EBs; 2) determine if the Nkx2.5(+) cell lineage diversifies into various cardiac cell types, including sinoatrial (SA) node-, atrial-, or ventricular-type cells; and 3) test if retinoic acid (RA) affects chamber-specific differentiation of ES cell-derived cardiomyocytes.

PRINCIPAL FINDINGS

1. Purification and differentiation of Nkx2.5(+) cardiomyocytes derived from EBs
A small fraction of GFP(+) cells derived from Nkx2.5 GFP ES cells was recognized by flow cytometry around d6, just before the initiation of spontaneous beating; this fraction became more prominent at d7. We sorted the Nkx2.5/GFP(+) cardiomyocytes from d8 EBs and cultured them for N days on plastic plates (designated as d8+N). Shortly after sorting (d8+1) >98% of GFP(+) cells stained positively for sarcomeric tropomyosin or myosin heavy chain (MHC), compared with only <1% of GFP(–) cells. Conversely, some Nkx2.5/GFP(+) cells weakly expressed cardiac troponin I (cTnI) at d8 + 1 (36±11%). The population of cTnI(+) cells increased over time and the majority of GFP(+) cells strongly expressed cTnI at d8 + 8 (89±6%).

2. Chamber-specific differentiation of the Nkx2.5(+) cardiomyotes
At d10 + 7, 17 out of 20 cells (85%) showed continuous, spontaneous beating. At d10 + 28, 2 out of 15 cells (13%) showed spontaneous beating. Two cells demonstrated prominent pacemaker depolarization and slow upstroke phase, as characteristic of SA node cells (Fig. 1 A). To examine the quiescent cells, the effect of CCh (10 µM), a muscarinic agonist, was tested. In 6 out of 13 quiescent cells (Fig. 1B ), the resting membrane potential was hyperpolarized by 14 ± 2.5 mV in response to CCh. In the remaining 7 cells, the action potential configuration was not affected (Fig. 1C ). We also examined the immunoreactivity of purified and cultured Nkx2.5/GFP(+) cells at early stages (d8+4) using anti-myosin light chain 2v (MLC2v) and anti-atrial natriuretic peptide (ANP) antibodies (Fig. 2 A). The Nkx2.5/GFP(+) cell fraction contained MLC2v(+)ANP(–), MLC2v(–)ANP(+), and MLC2v(+)ANP(+)cells, suggesting that some degree of differentiation, indicated by immunological markers, occurs before electrophysiological properties become evident.

View larger version (20K): [in this window] [in a new window]   Figure 1. (Fig. 5 in the full text) Electrophysiological properties of Nkx2.5/GFP(+) cells. Action potential waveforms and sensitivity to carbamyl choline (CCh; 10 µM) were examined. Representative traces of action potentials recorded in A) spontaneously beating (SA node-type), B) quiescent CCh-sensitive (atrial-type), and C) quiescent CCh-insensitive (ventricular-type) cells. Action potentials were recorded under 1.0 Hz stimulation. CCh was applied to the bath solution for 5 min after recording at the control state. D) Proportions of SA node-, atrial-, and ventricular-type cells defined by automaticity and sensitivity to CCh (n=15). W-O, wash out.

View larger version (61K): [in this window] [in a new window]   Figure 2. (Fig. 7 in the full text) Effect of RA on cardiac diversification of EBs. A) Differentiation of Nkx2.5/GFP(+) cells into ANP- and/or MLC2v-positive cells. GFP(+) cells sorted from d8 EBs were cultured for 4 days and stained with anti-MLC2v (c, d) and anti-ANP (e, f) antibodies. Original bars, 100 ìm. B) RA modifies the percentage of MLC2v(–)ANP(+) cells. EBs were treated with RA or disulfiram (DS) at various concentrations for different periods as shown. Nkx2.5/GFP(+) cells were sorted at d8 and analyzed at d8 + 4. Values are given as means ± SE from three independent experiments. The results are summarized at left. Untreated (U) EBs or 10-6 M DS-treated EBs gave rise to slightly more MLC2v(+) than MLC2v(–)ANP(+) cells (A<V). Treatment of EBs with 10-7 M RA (d4–d8) greatly increased the percentage of MLC2v(–)ANP(+) cells (A>>V). This effect was weaker with 10-8 M RA (A>V). Treatment with 10-7 M RA (d4–d5) or 10-5 M DS gave rise to MLC2v(+) cells as frequently as MLC2v(–)ANP(+) (A=V). Treatment with 10-6 M RA (d4–d8) or 10-7 M RA (d3–d8) inhibited generation of GFP(+) cells (data not shown). C) RA increases the expression of atrial-specific genes. EBs were treated with RA or DS from d4 to d8. Nkx2.5/GFP(+) cells were sorted at d8 for real-time reverse transcriptase-polymerase chain reaction (PCR). Relative amounts of the PCR products were determined as ratios to those of a control sample (unsorted d8 EBs). Values are given as means ± SE from three independent experiments.

3. RA affects chamber-specific differentiation of EBs
To examine the effects of RA on developing EBs, RA was administrated to EBs before sorting the Nkx2.5/GFP(+) cells, and the purified cells were analyzed at d8 + 4. When we treated EBs with 10^-7 M RA between d4 and d8 (d4–d8), the percentage of MLC2v(–)ANP(+) cells became significantly larger than that derived from untreated EBs (Fig. 2B ). Treatment with 10^-8 M RA also increased the percentage of MLC2v(–)ANP(+) cells to a lesser extent. We also found that the expression levels of atrial genes, Hrt1, Coup-tfII, Mhca, and Mlc2a, were increased on treatment of EBs with 10^-7 M RA (Fig. 2C ). The effect of DS, an inhibitor of retinaldehyde dehydrogenase, on EBs was smaller than that of RA.

CONCLUSIONS

Using Nkx2.5 GFP ES cells, we could successfully collect MHC(+) or troponin (+) cardiomyocytes from EBs at d8 or later. These cells expressed cTnI at a low frequency, but a majority of them expressed cTnI after a 1-week culture. As cTnI is a late-stage marker of cardiogenesis, this suggests that the Nkx2.5/GFP(+) cells further differentiated in vitro after isolation from EBs. Moreover, after long-term culture (d10+28), we identified spontaneously beating SA node-like cells and CCh-sensitive and -insensitive quiescent cells in the Nkx2.5/GFP(+) fraction (Fig. 1D ). The CCh-sensitive and the -insensitive quiescent cells were referred to as atrial- and ventricular-type, respectively. These results indicate that Nkx2.5/GFP(+) cells have the potential to differentiate into various types of cardiac cells, including SA node, atrial, and ventricular cells, during long-term culture. In fact, diversification appears to occur even earlier, as demonstrated by the presence of differential staining for the markers MLC2v and ANP at d8 + 4.

Chamber-specific differentiation is closely related to positional information along the embryonic anterior-posterior axis. Ventricular chambers originate from the anterior cardiac mesoderm, and the atria arise from the posterior. In chick or mouse embryo, exogenous administration of RA results in posteriorization of the heart tube, and a lack of RA signaling results in its anteriorization. We observed that administration of 10^-7 M RA to developing EBs increased the percentage of MLC2v(–)ANP(+) cells while inducing the expression of atrial-specific genes. These results suggest that RA promotes differentiation into the atrial lineage. Thus, the effect of RA on EBs seems to be similar to that on chick or mouse embryos. In contrast, the effect of DS on EBs was not as prominent as that on embryos, suggesting that endogenous production of RA may not be important in the diversification of cardiomyocytes in EBs, at least in our system.

In summary, we have established an in vitro system to track the fate of Nkx2.5(+) cardiomyocytes derived from ES cells. They indeed diversify into various types of cardiomyocyte, and exogenous RA can modify their differentiation and diversification potential. Thus, this system can be used to elucidate the mechanism of cardiomyocyte differentiation and diversification at a cellular level and will be a valuable tool to manipulate and control in vitro cardiac differentiation from multipotent stem cells.

View larger version (24K): [in this window] [in a new window]   Figure 3. (Summary Figure) Diversification of ES cell-derived Nkx2.5(+) cardiac cells. Purification of the Nkx2.5/GFP(+) cells from EBs identifies diversification of cardiomyocytes that differentiate into SA node-, atrial-, or ventricular-type cells. Thus, Nkx2.5(+) cells represent cardiac precursors for major types of cardiomyocytes in the heart. Administration of RA to EBs at early stages of cardiogenesis modifies the direction of differentiation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0104fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0104fje

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