Cardiac progenitors and paracrine mediators in cardiogenesis and heart regeneration

The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration.</p

Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNASeq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.Swedish Research Council for Health, Working Life and Welfare (Forte)Knut and Alice Wallenberg Foundation, KAW 2013.0028Swedish Research Council, 541-2013-8351, 539‐2013‐7002European Research Council Advanced Research Grant Award, AdG743225Publishe

Population and single-cell analysis of human cardiogenesis reveals unique LGR5 ventricular progenitors in embryonic outflow tract.

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.The Knut and Alice Wallenberg Foundation (KAW Dnr 2013.0028)Swedish Research Council Distinguished Professor Grant Dnr 541-2013-8351AstraZeneca PharmaceuticalsKarolinska InstitutetSwedish Heart Lung Foundation No. 20150421EMBO long-term fellowship (ALTF 620-2014)European Research Council Advanced Research Grant Award (AdG743225)Publishe

Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.</p

Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.</p

Human ISL1+ ventricular progenitors self-assemble into an in vivo functional heart patch and preserve cardiac function post infarction

The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm x 3 mm x 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.ERC AdG743225Swedish Research Council Distinguished Professor Grant Dnr 541-2013-8351The Knut and Alice Wallenberg Foundation (KAW Dnr 2013.0028)Horizon 2020 research and innovation programme grant agreement No 647714Publishe

Single-cell analysis of mammalian cardiogenesis elucidating an essential role of outflow tract progenitors

The heart is the first organ to form and start to function during mammalian embryogenesis. This complex organ system is constructed by a diverse set of cell types, involving mesodermal precursors and heart progenitors, at the earliest embryonic stages. These progenitor cells contribute to the formation of the distinct heart regions, such as atria, right and left ventricle, and outflow tract (OFT). However, it is still controversial and undefined which specific progenitors and paracrine molecular cues are responsible to form each of the distinct heart regions (e.g., OFT), requiring the rigorous analysis at higher resolution to identify the detailed cellular and molecular pathways on developing hearts. To tackle this problem, we applied a wide variety of the state-of-the-art biotechnologies and assays, including the in vitro cardiac differentiation system of human embryonic stem cells (hESCs), handling of the murine and human embryonic hearts, CRISPR/Cas9 gene editing, single-cell RNA sequencing (RNA-seq), and a mouse lineage tracing approach. We provide a comprehensive gene expression resource, characterizing the transcriptional dynamics of cardiac lineage specification and identifying novel markers of developing cardiac derivatives from multipotent progenitors to mature cardiac cells. Importantly, we have discovered the uniquely stage- and region-specific mesodermal precursors and/or heart progenitors that are essential on mammalian cardiogenesis. In Paper I, to determine the key regulators for cardiac linage specification and commitment, we first established a genome-wide CRISPR/Cas9 knockout screen platform using the in vitro hESC differentiation where we monitored the two distinct stage markers, an early cardiac mesodermal marker MESP1 and a heart progenitor marker ISL1. From the screen output, we compiled a list of 15 candidate genes and finally identified ZIC2 as an essential gene for early cardiac mesoderm formation. Interestingly, RNA-seq profiles of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single-cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps during the in vitro hESC cardiogenesis. In Paper II, through population and single-cell analysis of the in vitro hESC cardiac differentiation and the in vivo human embryonic/fetal hearts, we chart the developmental landscape of human cardiac formation at the cellular and molecular basis. Importantly, we have discovered a uniquely human subset of the OFT region-specific heart progenitors, marked by LGR5. The LGR5+ progenitors emerge specifically in the proximal OFT of human embryonic hearts (4 to 5 weeks of fetal age) and likely contribute to the OFT formation and alignment. Our results provide a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations. In Paper III, to obtain a whole picture of transcriptional and epigenetic regulation in the mesoderm lineage on the developing hearts, we first established Mesp1+ mesodermal lineage tracing mice. Mesp1 gene encodes a transcription factor of the b-HLH family, which is expressed broadly in the mesodermal cells and critical for the cardiovascular development in mammals. Using the CRISPR/Cas9 system and an IRES2-Cre cassette, we generated a Mesp1- IRES2-Cre knock-in mouse line and cross-bred them with reporter mice (Rosa26-tdTomato). On the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), we observed that more than 95% of the atria and ventricular cells in the hearts on an embryonic day 10.5 are the Mesp1+ mesodermal lineage. Interestingly, less percentage (<90%) of the OFT cells are positive for Mesp1, while the rest OFT cells (»10%) are positive for a neural crest marker, SOX10. We showed developmental dynamics of the Mesp1+ mesodermal lineage on murine embryonic hearts using the advanced light sheet microscopic images. Further, we showcased the singlecell RNA and chromatin sequencing analysis data of the Mesp1+ lineage on murine cardiogenesis. In summary, our studies in this thesis provide a comprehensive gene expression resource of developing cardiac derivatives including novel mesodermal precursors and heart progenitors. Therefore, the current works contribute to a better understanding of mammalian heart developmen