Kyoko Hidaka

Developmental stage-specific biphasic roles of Wnt/β-catenin signaling in cardiomyogenesis and hematopoiesis

Although Wingless (Wg)/Wnt signaling has been implicated in heart development of multiple organisms, conflicting results have been reported regarding the role of Wnt/β-catenin pathway in cardiac myogenesis: Wg/armadillo signaling promotes heart development in Drosophila, whereas activation of Wnt/β-catenin signaling inhibits heart formation in avians and amphibians. Using an in vitro system of mouse ES cell differentiation into cardiomyocytes, we show here that Wnt/β-catenin signaling exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis. Activation of the Wnt/β-catenin pathway in the early phase during embryoid body (EB) formation enhances ES cell differentiation into cardiomyocytes while suppressing the differentiation into hematopoietic and vascular cell lineages. In contrast, activation of Wnt/β-catenin signaling in the late phase after EB formation inhibits cardiomyocyte differentiation and enhances the expression of hematopoietic/vascular marker genes through suppression of bone morphogenetic protein signaling. Thus, Wnt/β-catenin signaling exhibits biphasic and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis, depending on the stage of development.

Nongenetic method for purifying stem cell-derived cardiomyocytes.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

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

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.

RNA‐binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21‐dependent and ‐independent regulatory pathways

Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post‐transcriptional regulation of the gene expression of myogenic proteins, as well as cell‐cycle related proteins. RNA‐binding proteins bind to specific sequences of target RNA and regulate gene expression in a post‐transcriptional manner. However, few tissue‐specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell‐cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over‐expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation‐RT‐PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over‐expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.

Expression pattern of novel chick T-box gene, Tbx20.

Abstract. Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, we attempted to isolate the chick H15-related T-box gene and analyze its expression pattern during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression was initially detected in the posterior lateral mesoderm, after which it expanded to the anterior and was intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm.

The Cellular Prion Protein Identifies Bipotential Cardiomyogenic Progenitors

Rationale: The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell–based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation. Objective: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors. Methods and Results: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential. Conclusions: PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)&agr;, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFR&agr;+ (PRa) cells. The Myhc− PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc− at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc− PRa cell–derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.

RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation.

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast‐to‐myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA‐binding proteins bind to specific target RNA sequences and regulate gene expression in a post‐transcriptional manner. This study demonstrates that RNA‐binding motif protein 24 (Rbm24) interacts with the 3′‐untranslated region of myogenin mRNA and affects its half‐life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24‐knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin‐dependent post‐transcriptional regulatory pathway.

Expression of ErbB receptors in ES cell-derived cardiomyocytes

To explore the role of ErbB-mediated signaling in cardiogenesis of ES cells, we examined the expression of ErbB receptors as well as effects of a ligand and inhibitors using Nkx2.5GFP ES cells, in which the GFP gene was knocked-in to the Nkx2.5 locus to monitor cardiac differentiation. Although all ErbB receptors were expressed in developing embryoid bodies, expression of ErbB4 was almost exclusively found in differentiated cardiomyocytes. Heregulin beta1, a ligand of ErbB receptors, enhanced the generation of Nkx2.5/GFP(+) cardiomyocytes in embryoid bodies, while AG1478 and PD153035, inhibitors of ErbBs, drastically blocked the generation of Nkx2.5/GFP(+) cardiomyocytes. These results suggest that the signaling pathway mediated by ErbBs is important in the induction and differentiation of cardiomyocytes from ES cells.

Cardiac ischemia activates vascular endothelial cadherin promoter in both preexisting vascular cells and bone marrow cells involved in neovascularization

Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin–expressing cells function in postnatal neovascularization. To trace VE-cadherin–expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter–reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin–mediated cell adhesion between cells may be involved not only in homing of bone marrow–derived cells to ischemic area but also mobilization from bone marrow.

Expression of MEF2 Genes during Human Cardiac Development.

To better understand the regulatory mechanisms in gene expression of human cardiomyocytes, we studied the expression of MEF2 genes encoding transcription factors during the course of cardiac development. Expression of all four MEF2 transcripts (MEF2A, MEF2B, MEF2C, and MEF2D) were detected in all developmental stage of the human heart, while Mef2b transcripts were down-regulated in mouse heart development. Although none of the MEF2 genes, besides mouse Mef2b, exhibited any remarkable quantitative change in their transcripts, qualitative changes in MEF2 transcripts were found during the course of cardiac development. In particular, MEF2D transcripts showed prominent changes by alternative splicing in the perinatal period. MEF2D transcripts containing the 21-base exon (exon b) were predominantly expressed after birth. At the same time, transcripts of the alpha myosin heavy chain (alphaMHC) gene increased after birth, as the splicing pattern in transcripts of the cardiac troponin T (cTnT) gene changed to decrease the transcripts of cTnT1 after birth. These changes seemed to be correlated with the alternative splicing changes of MEF2 genes, especially MEF2D. The alternative splicing as well as transcriptional regulation in MEF2 genes might be important for regulating the alphaMHC gene and the maturation of cardiomyocytes.