A genome-wide screen for essential genes that controls the formation of human heart progenitors

The heart is a complex organ system composed of multiple types of tissues. These tissues are produced by a diverse set of muscle and non-muscle cells, originated from a few pools of progenitors. During the heart development, these progenitors are able to expand and differentiate in a tightly controlled manner, generating diversified heart cell lineages. The progenies from these progenitors interact with each other and ultimately integrate into distinct heart tissues. The foundation of a healthy and functional heart stems from the state of its progenitor pools. Any errors that occurred during the formation, proliferation, differentiation, and assembly of these progenitors are the potential causes of many congenital heart diseases. To investigate the cellular mechanisms of human heart development and their implications in congenital heart diseases, we face many challenges, two of them are: 1) generation of progenitor cells that can self-assemble into mature cardiac tissue that faithfully resembles native mature adult cardiac tissue; 2) identification of regulators that controls the formation, proliferation and differentiation of these progenitors. In paper I, we reported the large-scale generation of an enriched pool of human pluripotent stem cells (hPSCs) derived human ventricular progenitors (HVPs). These HVPs can build a function ventricular heart muscle in vivo via a cell autonomous pathway, including controlled proliferation followed by normal growth, maturation, and self-assembly. This tissue generation process of HVPs recapitulates one of the earliest and most essential steps of organogenesis. With these properties, HVPs highly likely resembles the progenitors that contribute to the ventricular cardiac muscle tissue during human cardiogenesis. In the study, we also explore the therapeutic potential of HVPs in heart failure. As a resource for further analyzing the genetic and molecular pathways of HVPs, we also documented the transcriptomic transitions of the progenitor formation and subsequent differentiation via sequential RNA-Seq. With the success of generating HVPs, we next try to identify regulators, specifically, the ones that control the formation of HVPs. In paper II, we used CRIPSR-Cas9 system to target β-catenin (encoded by CTNNB1), a central component of the canonical WNT signaling pathway. The WNT signaling is a major player in cardiogenesis. By temporal modulating the WNT/β-catenin signaling pathway with small molecules, high differentiation efficiency (>90%) can be achieved. With CTNNB1 mutated hPSCs, we found that Wnt/β-catenin signaling is neither required for hPSC self-renewal, nor for neuroectoderm formation. However, Wnt/β-catenin signaling is absolutely essential for mesendoderm lineage, including cardiac progenitors and cardiomyocytes. This study pinpoints the β-catenin as the master switch of the human cardiogenesis. Another set of important signaling pathways in cardiogenesis are the TGFβ superfamily signaling pathways. Due to the complicate interaction between WNT/β-catenin and the TGFβ superfamily signaling pathways, it is difficult to define the roles of TGFβ superfamily signaling pathways from chemical inhibition studies. In paper III, we used CRIPSR-Cas9 system to target SMAD4, a central component in the whole superfamily. With SMAD4 mutated hPSCs, we confirmed the dispensable role of SMAD4 for hPSC self-renewal in vitro. Furthermore, we demonstrated the essential requirement of SMAD4 in the formation of human cardiac mesodermal precursor cell. By transcriptome analysis, we identified that SMAD4 mutants failed to differentiate into cardiac mesoderm and, after 6 days, switched to neuroectoderm. Primitive steak (PS) genes were expressed in both the wild type and the mutant cells on day 1. And interestingly, on day 1, the only active ligand in the TGFβ superfamily signaling pathways is NODAL, which specifies the pathway in the family as NODAL/SMAD4 pathway. Together, these data suggest that during human mesoderm induction, the WNT/β-catenin is responsible for triggering the expression of PS genes, while NODAL/SMAD4 is responsible for the feedback enhancement for PS gene expression. This study highlights the essential roles of NODAL/SMAD4 signaling pathway in human cardiac mesodermal induction. In order to unbiasedly uncover the regulators that control the formation of HVPs, in paper IV, we developed a genome-wide CRISPR screen based on cardiac differentiation from hPSCs. From the screen output, we compiled a list of 15 candidate genes. After validating 7 of these, 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 has greatly reduced. Transcriptome profiling reveals that they have switched to an alternative mesodermal cell fate. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of genome-wide unbiased CRISPR screens to identify key steps in heart progenitor fate determination during human cardiogenesis with hPSC model systems. In summary, we have generated HVPs, which can self-assemble into human ventricular muscle tissue and further identified CTNNB1, SMAD4, and ZIC2 as the essential regulators that controlled the formation of HVPs

Unraveling the Relationship between Co-Authorship and Research Interest

Co-authorship in scientific research is increasing in the past decades. There are lots of researches focusing on the pattern of co-authorship by using social network analysis. However, most of them merely concentrated on the properties of graphs or networks rather than take the contribution of authors to publications and the semantic information of publications into consideration. In this paper, we employ a contribution index to weight word vectors generated from publications so as to represent authors’ research interest, and try to explore the relationship between research interest and co-authorship. Result of curve estimation indicates that research interest couldn’t be employed to predict co-authorship. Therefore, graph-based researcher recommendation needs further examination

Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.The Knut and Alice Wallenberg Foundation (KAW Dnr 2013.0028)Croucher Foundation, Hong KongSwedish Research Council Grant no 541-2013-8351 and 539‐2013‐7002European Research Council Advanced Research Grant Award (AdG743225)Publishe

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

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

Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ.

Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by "superoxide flash" activity recorded with time lapse scanning.Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or "superoxide flashes" occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm(2)*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases

Absence of physiological Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation

Abstract Background Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. Methods A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. Results We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. Conclusions The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle

Cytokine-stimulated superoxide flashes in chondrocytes <i>in situ</i>.

<p><b>A</b><b>B</b>, Time-lapse views of a typical superoxide flash <i>in situ</i> in the red boxed region of 2.5 µm×5 µm. Time course plot of the superoxide flash is shown at the bottom. Note the swelling of the mitochondrion during the flash. <b>C</b>, Activity and properties of chondrocyte superoxide flashes i<i>n vitro</i> (510 events as in Figure. 2) and <i>in situ</i> (30 events). Data are reported as the mean ± SEM values. *, <i>p</i><0.05; **, <i>p</i><0.01. <b>D</b>, Time course of the superoxide flash response to cytokine stimulation. Data are reported as the mean ± SEM values. n = 21–29 imaging planes. *, <i>p</i><0.05; **, <i>p</i><0.01 versus the respective control.</p

Imaging chondrocytes and mitochondria <i>in situ</i>.

<p><b>A</b>, Confocal or two-photon-excitation imaging of chondrocyte mitochondria in femoral head cartilage <i>in situ</i>. Insert shows an enlarged view of the femoral head. <b>B</b>, Mitochondrial distribution in intact cartilage. The femoral head was sliced lengthwise along the midcoronal panel, labeled with TMRM and imaged by a confocal microscope. Arrows mark the cartilage surface and arrowheads mark the growth plate. <b>C</b>, Two-photon excitation images of TMRM-stained mitochondria (excitation at 850 nm, red) in chondrocytes at different depths from the cartilage surface. Layer thickness: 0.77 µm, XYZ of 3D: 100*100*100 µm³ (Z axis shows depth). <b>D</b>, Profiles of averaged mitochondrial (Mito, green) and chondrocyte (Cell, blue) cross-section areas and their ratios (Mito/Cell, red) as a function of distance from the surface. Right panels shows representative images from superficial, middle and deep cartilage zones.</p