Makoto Sahara

A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.

Programming and reprogramming a human heart cell

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the “programming” and “reprogramming” of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart.

Expansion of cardiac progenitors from reprogrammed fibroblasts as potential novel cardiovascular therapy

The human heart has an unremitting and laborious job, to continuously provide all the organs in the body with oxygen and nutrients. An insult occurring to the muscular organ in the form of an ischemic injury, such as myocardial infarction (MI) reduces heart function by causing irreversible damage. Severe injuries to the heart can lead to heart failure (HF) and death. Unfortunately, the human heart has very little ability to repair itself upon injury, predominantly due to the inherent quiescent state of the adult cardiomyocytes (CMs), the major “power house” cell type of the heart. Currently, heart transplants remain one of the most successful therapeutic options for patients in end stage HF. However, even if the extensive recipient list could be met with matched available donors, complications arise in the form of graft dysfunction, immune rejection and infection. Therefore there is a pressing need to develop novel cardiac therapies.

A specified therapeutic window for neuregulin-1 to regenerate neonatal heart muscle

The most prominent cause of impaired cardiac function in adults is ischemic heart disease such as myocardial infarction, whereas decreased heart function in children is generally the consequence of congenital heart disease (CHD), structural malformations of the heart that are present at birth. Heart defects are the most common birth defect in humans—and the most deadly too, boasting the infamous title of leading cause of birth defect-related morbidity and mortality (1). One in 100 newborns display signs of minor CHD, but in one out of 1,000 cases the malformations are severe enough to necessitate surgical intervention. The therapeutic conundrum is that surgical repair is often life-saving, but may not be enough by itself to guarantee the child a carefree existence later on. The human heart has virtually no inherent regenerative capability and despite long-standing efforts, no valid cardiac regenerative therapy proven to be clinically effective exists to date. Since scarred and dysfunctional regions of the heart cannot be replaced, children with CHD are at greater risk of developing chronic heart failure—even after surgical correction of the malformation (2,3). Unfortunately, currently available pharmacological therapies for heart failure were developed with the adult patient in mind and proved ineffective in pediatric trials, highlighting the need for child-specific heart failure therapies (4).

Cardiac Progenitor Cells in Basic Biology and Regenerative Medicine

Major cardiovascular events including myocardial infarction (MI) continue to dominate morbidity rates in the developed world. Although multiple device therapies and various pharmacological agents have been shown to improve patient care and reduce mortality rates, clinicians and researchers alike still lack a true panacea to regenerate damaged cardiac tissue. Over the previous two to three decades, cardiovascular stem cell therapies have held great promise. Several stem cell-based approaches have now been shown to improve ventricular function and are documented in preclinical animal models as well as phase I and phase II clinical trials. More recently, the cardiac progenitor cell has begun to gain momentum as an ideal candidate for stem cell therapy in heart disease. Here, we will highlight the most recent advances in cardiac stem/progenitor cell biology in regard to both the basics and applied settings.

Lnc’ed in to Cardiogenesis

Despite the continuous discovery of long noncoding RNAs (lncRNAs) with critical developmental roles, our knowledge of lncRNAs that control cardiac lineage commitment is still limited. In this issue, Guo etxa0al. (2018) report a novel lncRNA-mediated multiprotein complex assembly that directly regulates the key transcriptional programs of murine cardiogenesis.

Response to the letter by Guo et al., “Endothelial progenitor cells therapy: From bench to bedside”

Article history: Received 26 November 2015 Accepted 12 December 2015 Available online 17 December 2015 ative effects, will have to be addressed [3]. Aside from the strategies of progenitor transplantation for ischemic cardiovascular diseases, approaches to improve endogenous EPC function by pharmacotherapy or non-pharmacotherapy may be alternative ways for therapeutic angiogenesis, as Dr. Guo et al. suggested [1]. Indeed, it has been demonstrated that endogenous EPC levels and func-