Satoshi Yamanaka

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.

Pluripotency of embryonic stem cells

Embryonic stem (ES) cells derived from pre-implantation embryos have the potential to differentiate into any cell type derived from the three germ layers of ectoderm (epidermal tissues and nerves), mesoderm (muscle, bone, blood), and endoderm (liver, pancreas, gastrointestinal tract, lungs), including fetal and adult cells. Alone, these cells do not develop into a viable fetus or adult animal because they do not retain the potential to contribute to extraembryonic tissue, and in vitro, they lack spatial and temporal signaling cues essential to normal in vivo development. The basis of pluripotentiality resides in conserved regulatory networks composed of numerous transcription factors and multiple signaling cascades. Together, these regulatory networks maintain ES cells in a pluripotent and undifferentiated form; however, alterations in the stoichiometry of these signals promote differentiation. By taking advantage of this differentiation capacity in vitro, ES cells have clearly been shown to possess the potential to generate multipotent stem and progenitor cells capable of differentiating into a limited number of cell fates. These latter types of cells may prove to be therapeutically viable, but perhaps more importantly, the studies of these cells have led to a greater understanding of mammalian development.

Cardiomyogenic Stem and Progenitor Cell Plasticity and the Dissection of Cardiopoiesis

Cell-based therapies hold promise of repairing an injured heart, and the description of stem and progenitor cells with cardiomyogenic potential is critical to its realization. At the vanguard of these efforts are analyses of embryonic stem cells, which clearly have the capacity to generate large numbers of cardiomyocytes in vitro. Through the use of this model system, a number of signaling mechanisms have been worked out that describes at least partially the process of cardiopoiesis. Studies on adult stem and on progenitor cells with cardiomyogenic potential are still in their infancy, and much less is known about the molecular signals that are required to induce the differentiation to cardiomyocytes. It is also unclear whether the pathways are similar or different between embryonic and adult cell-induced cardiomyogenesis, partly because of the continued controversies that surround the stem cell theory of cardiac self-renewal. Irrespective of any perceived or actual limitations, the study of stem and progenitor cells has provided important insights into the process of cardiomyogenesis, and it is likely that future research in this area will turn the promise of repairing an injured heart into a reality.

Proliferation of mouse embryonic stem cell progeny and the spontaneous contractile activity of cardiomyocytes are affected by microtopography.

The niche in which stem cells reside and differentiate is a complex physicochemical microenvironment that regulates cell function. The role played by three‐dimensional physical contours was studied on cell progeny derived from mouse embryonic stem cells using microtopographies created on PDMS (poly‐dimethyl‐siloxane) membranes. While markers of differentiation were not affected, the proliferation of heterogeneous mouse embryonic stem cell‐derived progeny was attenuated by 15 μm‐, but not 5 μm‐high microprojections. This reduction was reversed by Rho kinase and myosin light chain kinase inhibition, which diminishes the tension generating ability of stress fibers. Purified cardiomyocytes derived from embryonic stem cells also showed significant blunting of proliferation and increased beating rates compared with cells grown on flat substrates. Thus, proliferation of stem cell‐derived progeny appears to be regulated by microtopography through tension‐generation of contractility in the third‐dimension. These results emphasize the importance of topographic cues in the modulation of stem cell progeny behavior. Developmental Dynamics 238:1964–1973, 2009.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

BACKGROUND Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. METHODS Differentiating mouse ES cells were treated with suramin (500 µM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. RESULTS Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. CONCLUSION We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.

Long-Term Improvement in Postinfarct Left Ventricular Global and Regional Contractile Function Is Mediated by Embryonic Stem Cell–Derived Cardiomyocytes

Background—Pluripotent stem cells represent one promising source for cellular cardiomyoplasty. In this study, we used cardiac magnetic resonance to examine the ability of highly enriched cardiomyocytes (CMs) derived from murine embryonic stem cells (ESC) to form grafts and improve contractile function of infarcted rat hearts. Methods and Results—Highly enriched ESC-CMs were obtained by inducing cardiac differentiation of ESCs stably expressing a cardiac-restricted puromycin resistance gene. At the time of transplantation, enriched ESC-CMs expressed cardiac-specific markers and markers of developing CMs, but only 6% of them were proliferating. A growth factor–containing vehicle solution or ESC-CMs (5 to 10 million) suspended in the same solution was injected into athymic rat hearts 1 week after myocardial infarction. Initial infarct size was measured by cardiac magnetic resonance 1 day after myocardial infarction. Compared with vehicle treatment, treatment with ESC-CMs improved global systolic function 1 and 2 months after injection and significantly increased contractile function in initially infarcted areas and border zones. Immunohistochemistry confirmed successful engraftment and the persistence of &agr;-actinin–positive ESC-CMs that also expressed &agr;-smooth muscle actin. Connexin-43–positive sites were observed between grafted ESC-CMs but only rarely between grafted and host CMs. No teratomas were observed in any of the animals. Conclusions—Highly enriched and early-stage ESC-CMs were safe, formed stable grafts, and mediated a long-term recovery of global and regional myocardial contractile function after infarction.

Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are >98% pure, and at least 85% of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint and maturation of heart cells. We conclude that this in vitro model will facilitate the analysis of cell cycle control mechanisms of CMs.

The Distribution of Cool Spots as Microrefugia in a Mountainous Area.

It has recently been proposed that microrefugia played an important role in species survival during past climate change events. However, the current distributions of microrefugia remain largely unknown. Wind-hole sites are areas affected by preferential flows of cool air generated in interstitial spaces created by rock fragments or colluvia. Alpine plant species occurring in lowland wind-hole sites isolated from alpine zones may be relicts of the last glacial period. Hokkaido, northern Japan, is known to contain many wind-hole sites in which alpine plant species can occur. Here we surveyed 55 wind-hole sites in the Kitami region, eastern Hokkaido, and observed two alpine plant species (lingonberry, Vaccinium vitis-idaea, and Labrador tea, Rhododendron groenlandicum ssp. diversipilosum var. diversipilosum) in 14 wind-hole sites. Statistical modeling showed that wind-hole sites are likely to occur in areas with high maximum slope angles and volcanic rock cover, and concave surfaces. Our predictions of wind-hole site distributions suggest that such topographic conditions are common in our study area, and that many undiscovered wind-hole sites exist. Ignoring microhabitats may greatly underestimate species distributions in topographically complex regions, and dispersed cool spots may also function as stepping stones and temporal habitats for cold-adapted species. Because these localized unique habitats usually occur in economically unproductive sites, identifying and protecting potential microrefugia (cool spots) would be a robust and cost-effective mitigation of climate change impacts.

Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells

Numerous groups have documented that Ascorbic Acid (AA) promotes cardiomyocyte differentiation from both mouse and human ESCs and iPSCs. AA is now considered indispensable for the routine production of hPSC-cardiomyocytes (CMs) using defined media; however, the mechanisms involved with the inductive process are poorly understood. Using a genetically modified mouse embryonic stem cell (mESC) line containing a dsRED transgene driven by the cardiac-restricted portion of the ncx1 promoter, we show that AA promoted differentiation of mESCs to CMs in a dose- and time-dependent manner. Treatment of mPSCs with AA did not modulate total SMAD content; however, the phosphorylated/active forms of SMAD2 and SMAD1/5/8 were significantly elevated. Co-administration of the SMAD2/3 activator Activin A with AA had no significant effect, but the addition of the nodal co-receptor TDGF1 (Cripto) antagonized AA’s cardiomyogenic-promoting ability. AA could also reverse some of the inhibitory effects on cardiomyogenesis of ALK/SMAD2 inhibition by SB431542, a TGFβ pathway inhibitor. Treatment with BMP2 and AA strongly amplified the positive cardiomyogenic effects of SMAD1/5/8 in a dose-dependent manner. AA could not, however, rescue dorsomorphin-mediated inhibition of ALK/SMAD1 activity. Using an inducible model system, we found that SMAD1, but not SMAD2, was essential for AA to promote the formation of TNNT2+-CMs. These data firmly demonstrate that BMP receptor-activated SMADs, preferential to TGFβ receptor-activated SMADs, are necessary to promote AA stimulated cardiomyogenesis. AA-enhanced cardiomyogenesis thus relies on the ability of AA to modulate the ratio of SMAD signaling among the TGFβ-superfamily receptor signaling pathways.