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Analysis of differentiated hESCs by image scanning flow cytometry. hESCs allowed to differentiate on S17 stromal cells were analyzed by image scanning flow cytometry to provide concurrent data on cell phenotype and morphology. Cells were stained for (A) CD34 and CD31 and (B) CD34 and CD45 with cell size distribution of cells gated for CD34 bright CD31 ϩ and CD34 dim CD45 ϩ cells as indicated. Mean signal intensity for each cell population is shown.
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Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both h...
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... This novel instrument allows concurrent analysis for expression of specific cell surface antigens and individual cellular morphology in a high throughput manner. Population analysis using image scanning flow cytometry confirmed analysis by standard flow cytometry, where CD34 CD31 cells could be divided into both bright and dim CD34-expressing cells (Figure 2A), whereas CD34 CD45 cells were exclusively CD34 dim ( Figure 2B). One nontrivial result of these studies is to clearly demonstrate that phenotypic double- positive cell populations are not due to cell doublets or other aggregates. ...
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... This novel instrument allows concurrent analysis for expression of specific cell surface antigens and individual cellular morphology in a high throughput manner. Population analysis using image scanning flow cytometry confirmed analysis by standard flow cytometry, where CD34 CD31 cells could be divided into both bright and dim CD34-expressing cells (Figure 2A), whereas CD34 CD45 cells were exclusively CD34 dim ( Figure 2B). One nontrivial result of these studies is to clearly demonstrate that phenotypic double- positive cell populations are not due to cell doublets or other aggregates. ...
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... since the frequency of CD34 CD45 cells can be relatively low at early time points during differentiation of hESCs, this finding that is typically assumed, but not always definitively demonstrated by standard flow cytometric analysis, becomes especially important. Image scanning flow cytometry also clearly demonstrates by histograms based on cell size distribution that cells within the CD34 bright CD31 population had a higher mean cell size compared with cells within the CD34 dim CD31 and CD34 dim CD45 cell populations, providing a novel means to depict morphologic differences between the CD34 bright and CD34 dim hESC-derived cells (Figure 2A,B). Individual cell images generated from image scanning flow cytometry confirmed that the 2 cell populations also are morphologi- cally distinct. ...
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... cell images generated from image scanning flow cytometry confirmed that the 2 cell populations also are morphologi- cally distinct. Specifically, the CD34 bright cells have a large cell size, complex cytoplasm, and irregular cell shape, which is morphologi- cally consistent with endothelial cells ( Figure 2C). In contrast, the CD34 dim cells display a smaller cell size, less complex cytoplasm, and a homogeneous spherical/round cell shape, corresponding to a phenotype for hematopoietic stem and progenitor cells ( Figure 2D). ...
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... the CD34 bright cells have a large cell size, complex cytoplasm, and irregular cell shape, which is morphologi- cally consistent with endothelial cells ( Figure 2C). In contrast, the CD34 dim cells display a smaller cell size, less complex cytoplasm, and a homogeneous spherical/round cell shape, corresponding to a phenotype for hematopoietic stem and progenitor cells ( Figure 2D). 31 Comparison of the 2 cell populations finds Flk1 and CD45 cells are exclusively expressed on the CD34 bright and CD34 dim cells, respectively. ...
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... analysis of the endothelial cells generated from CD34 bright CD31 Flk1 cells demonstrates that they express CD31 and von Willebrand factor, are able to take up acetylated LDL, and form vascular tube network in matrigel ( Figure 4C-F). More importantly, they also generated CD45 cells and clusters of hematopoietic cells when cultured in hematopoietic-inducing conditions ( Figure 4G), as well as forming myeloid colonies in a standard methylcellulose assay ( Figure S2). Therefore, this CD34 bright CD31 Flk1 cell popula- tion is thus likely very similar to the CD45 neg PVF and CD34 CD45 CD31 hESC-derived cells from EB-mediated differentiation, a population with characteristics of hematogenic endothelium observed during mammalian development. ...
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... frequency of hematopoietic cells is similar to what was observed for hESC-derived hemangioblast cells generated by EB differentia- tion. 16 As expected, a higher frequency of hematopoietic potential was observed for CD34 dim CD31 Flk1 cells, as these cells contain more committed hematpoietic progenitor cells with little to no endothelial potential (Figures S2,S3). ...
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... By image scanning flow cytometry, we can further distinguish these CD34 bright CD31 Flk1 hematogenic endothelial cells from CD34 dim CD45 hESC-derived cells re- stricted to the hematopoietic lineage, and show that they are morphologically distinct. Although not done routinely, we also show that VE-cadherin is expressed on both CD34 bright and CD34 dim cells (Figure 2C,D). As VE-cadherin is a trypsin/Ca 2 - sensitive cell surface antigen and requires more laborious digestion protocols for identification by standard flow cytometry, the pheno- typic hematogenic endothelial cells described here can be isolated more efficiently and reliably. ...
Citations
... Interestingly, the WNT signaling pathway contributes to the fate of mesoderm cells into distinct lineages [40]. The continuous activation of WNT signaling is implicated in the formation of endothelial cells and other mesodermal lineages [41][42][43]. However, inhibiting the WNT signaling pathway promotes cardiomyocyte progenitor lineages post-mesoderm differentiation [16,17,24,26]. ...
The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe the “WNT Switch” method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious.
... Interestingly, the WNT signaling pathway contributes to the fate of mesoderm cells into distinct lineages [40]. The continuous activation of WNT signaling is implicated in the formation of endothelial cells and other mesodermal lineages [41][42][43]. However, inhibiting the WNT signaling pathway promotes cardiomyocyte progenitor lineages post-mesoderm differentiation [16,17,24,26]. ...
The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe a “WNT Switch” method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious.
... Several factors and signaling pathways have been identified as essential regulators for HSPC generation (Bigas et al., 2013;Orkin and Zon, 2008). Among these signals, Wnt/β-catenin signaling is an evolutionarily conserved signaling pathway that is involved in the regulation of embryonic HSPC development (Bigas et al., 2013;Gertow et al., 2013;Grainger et al., 2019Grainger et al., , 2016Richter et al., 2017;Woll et al., 2008). In the Wnt/β-catenin signaling (referred to as the canonical Wnt pathway) transduction process, Wnt ligands bind to Frizzled receptors, leading to β-catenin release from constitutive degradation, therefore promoting its stabilization and accumulation in cytoplasm. ...
In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/β-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding β-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances β-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/β-catenin signaling is precisely regulated at the post-transcriptional level.
... Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring potential clinical applications. Modulation of canonical Wnt signaling in hiPSCs was previously shown to induce mesendoderm, cardiogenesis, and the formation of vascular cells (Brade, Manner, & Kuhl, 2006;Sumi, Tsuneyoshi, Nakatsuji, & Suemori, 2008;Woll et al., 2008). Using this knowledge, a rapid and efficient differentiation protocol was developed to generate hiPSCderived ECs and vascular SMCs. ...
Congenital heart disease (CHD) represents a major class of birth defects worldwide and is associated with cardiac malformations that often require surgical intervention immediately after birth. Despite the intense efforts from multicentric genome/exome sequencing studies that have identified several genetic variants, the etiology of CHD remains diverse and often unknown. Genetically modified animal models with candidate gene deficiencies continue to provide novel molecular insights that are responsible for fetal cardiac development. However, the past decade has seen remarkable advances in the field of human induced pluripotent stem cell (hiPSC)‐based disease modeling approaches to better understand the development of CHD and discover novel preventative therapies. The iPSCs are derived from reprogramming of differentiated somatic cells to an embryonic‐like pluripotent state via overexpression of key transcription factors. In this review, we describe how differentiation of hiPSCs to specialized cardiac cellular identities facilitates our understanding of the development and pathogenesis of CHD subtypes. We summarize the molecular and functional characterization of hiPSC‐derived differentiated cells in support of normal cardiogenesis, those that go awry in CHD and other heart diseases. We illustrate how stem cell‐based disease modeling enables scientists to dissect the molecular mechanisms of cell–cell interactions underlying CHD. We highlight the current state of hiPSC‐based studies that are in the verge of translating into clinical trials. We also address limitations including hiPSC‐model reproducibility and scalability and differentiation methods leading to cellular heterogeneity. Last, we provide future perspective on exploiting the potential of hiPSC technology as a predictive model for patient‐specific CHD, screening pharmaceuticals, and provide a source for cell‐based personalized medicine. In combination with existing clinical and animal model studies, data obtained from hiPSCs will yield further understanding of oligogenic, gene–environment interaction, pathophysiology, and management for CHD and other genetic cardiac disorders.
... The Wnt-b-catenin pathway has been well studied in the context of endothelial differentiation (Wang et al., 2006a;Sumi et al., 2008;Woll et al., 2008;Patsch et al., 2015;Harding et al., 2017;Shao et al., 2019). In vitro, this insight has been used to modulate differentiation through the use of small-molecule Wnt activators (Lian et al., 2014;Bao et al., 2015). ...
The extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and β-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active β-catenin.
... Several embryoid body formation and 2D culture protocols, including serum-and feeder-free protocols, have been developed for hematopoietic differentiation of hESCs (Kennedy et al., 2007(Kennedy et al., , 2012Chadwick et al., 2003;Zambidis et al., 2005;Salvagiotto et al., 2011;Niwa et al., 2011;Pick et al., 2007;Uenishi et al., 2014;Ng et al., 2008;Shah et al., 2020). To mimic blood development from an early embryonic stage, these protocols typically rely on BMP4, activin A, WNT, and FGF2 to induce mesoderm formation, VEGF and FGF2 to support hemogenic endothelium, and hematopoietic cytokines to support development of emerging hematopoietic myeloid colony-forming cells (CFCs) and lymphoid progenitors (Chadwick et al., 2003;Zambidis et al., 2005;Kennedy et al., 2007;Salvagiotto et al., 2011;Niwa et al., 2011;Pick et al., 2007;Uenishi et al., 2014;Ng et al., 2008;Zhang et al., 2008;Wang and Nakayama, 2009;Wang et al., 2012;Woll et al., 2008;Cerdan et al., 2004;Liu et al., 2015;Kardel and Eaves, 2012). As an alternative approach, blood generation from iPSCs can be achieved by overexpression of master transcription factors using lentiviruses or modified mRNA (Elcheva et al., 2014;Brok-Volchanskaya et al., 2019;Lange et al., 2020;Moreau et al., 2016). ...
Cellular therapies combined with genetic engineering technologies have emerged as increasingly powerful tools for immunotherapies of cancers and viral infections including HIV infection. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of blood progenitors and terminally differentiated cells can further expand applicability of cellular immunotherapies by offering “off-the-shelf” therapeutic products to fit specific clinical needs for a broad group of patients. In this review, we discuss current advances in lymphoid and myeloid cell differentiation of iPSCs along with challenges in de novo generation of hematopoietic stem cells (HSCs) and allogeneic iPSC products for “off-the-shelf” immunotherapies.
... Since Wnt signaling promotes CM [2,38] and EC [39,40] differentiation from hPSCs, SpGel may serve as a platform for hiPSC differentiation into ECs and CMs. Previous studies have suggested that ECM and substrate stiffness affect the differentiation of MSCs into cells such as osteogenic cells, neural cells, and adipocytes via various signaling pathways, such as the ROCK, FAK, Src, ERK, and MAP kinase pathways [41][42][43][44]. ...
Cell therapy has been a promising strategy for cardiac repair after myocardial infarction (MI), but a poor ischemic environment and low cell delivery efficiency remain significant challenges. The spleen serves as a hematopoietic stem cell niche and secretes cardioprotective factors after MI, but it is unclear whether it could be used for human pluripotent stem cell (hiPSC) cultivation and provide a proper microenvironment for cell grafts against the ischemic environment. Herein, we developed a splenic extracellular matrix derived thermoresponsive hydrogel (SpGel). Proteomics analysis indicated that SpGel is enriched with proteins known to modulate the Wnt signaling pathway, cell-substrate adhesion, cardiac muscle contraction and oxidation-reduction processes. In vitro studies demonstrated that hiPSCs could be efficiently induced into endothelial cells (iECs) and cardiomyocytes (iCMs) with enhanced function on SpGel. The cytoprotective effect of SpGel on iECs/iCMs against oxidative stress damage was also proven. Furthermore, in vivo studies revealed that iEC/iCM-laden SpGel improved cardiac function and inhibited cardiac fibrosis of infarcted hearts by improving cell survival, revascularization and remuscularization. In conclusion, we successfully established a novel platform for the efficient generation and delivery of autologous cell grafts, which could be a promising clinical therapeutic strategy for cardiac repair and regeneration after MI.
... The Wnt genes encode a class of secreted glycoprotein ligands with approximately 350-400 amino acids in length, including 23-24 conserved cysteines with one or more N-terminal glycosylation sites and highly conserved sequence regions in different species [1]. Signaling pathways invoked by Wnt molecules are involved in the regulation of diverse biological processes that play crucial roles in the development of living organisms, including differentiation of cell morphology, maintenance of homeostasis as well as embryonic axis formation, regeneration of tissues, and regulation of immune responses [2][3]. ...
Background: Bohai Red, a new Argopecten scallop strain selected from the hybrids between the Peruvian scallop, Argopecten purpuratus and the bay scallop northern subspecies, A. irradians irradians, is now one of the most cultured scallop strains in northern China. As one of a series of studies focusing on adaptation of Bohai Red scallops to fluctuations in environmental factors, this study aimed to examine the expression profile of Wnt genes in response to different temperature stresses in Bohai Red.
Results: As Bohai Red scallops were originated from the hybrids between the Peruvian scallop and the bay scallop northern subspecies, we first identified all Wnt genes from the genomes of the Peruvian scallop and the bay scallop northern subspecies, as well as the bay scallop southern subspecies, A. i. concentricus. Twelve Wnt members were identified from the two subspecies of bay scallop, and 13 Wnt genes were found in the genome of the Peruvian scallop. Protein structure analyses showed that most Wnt genes poses all 5 conserved motifs except Wnt 1, Wnt 2, Wnt 6 and Wnt 9 in the bay scallops and Wnt2 and Wnt9 in the Peruvian scallop. Unexpectedly, Wnt8 gene was present while Wnt3 was absent in both the bay scallops and the Peruvian scallop. Phylogenetic analysis revealed that Wnt3 may have disappeared in the early evolution of mollusks. The expression profile of Wnt genes in Bohai Red exposed to different temperatures were examined by qRT-PCR. The results showed that expression of Wnt genes responded differentially to temperature changes. The Wnt genes such as Wnt1, Wnt6, Wnt7, Wnt11 and WntA that responded slowly to low and high temperature stresses may be related to the maintenance of basic homeostasis. Other Wnt genes such as Wnt4, Wnt9, Wnt5 and Wnt2 that responded rapidly to low temperature may play an important role in organismal protection against low temperature stress. And yet some Wnt genes including Wnt10, Wnt16, and Wnt8 that responded quickly to high temperature stress may play key roles in response to organismal stress provoked by high temperature stress.
Conclusions: Wnt genes are well conserved in Argopecten scallops, as in other bivalves. Wnt genes may play important roles in adaptation of Bohai Red scallops to changing temperatures. The results in this study will provide new insights into the evolution and function of Wnt genes in bivalves and eventually benefit culture of Bohai Red scallops.
... We chose ex ovo culture of chick embryos wherein developmental milestones could be directly monitored. We were aware that amnion is the major extraembryonic source of BMP4 and Wnt3 during the period that corresponds to closure of the neural tube (Sasaki et al., 2016), and that BMP4 and Wnt3 are key to the expansion of yolk sac blood islands (Goldman et al., 2009;Sadlon et al., 2004;Woll et al., 2008) in the period that corresponded to neural tube closure. Accordingly, microsurgical removal of amnion (partial and full) between Hamburger-Hamilton stage 9 and 10 reduced the linear density of subventricular yolk sac vasculature in the neural tube by z95% by elimination of the hematopoietic signals of amnionic origin ( Figure 4F). ...
Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation β-catenin.
... Wnt signaling encompasses at least three distinct and widely deployed developmental signaling cascades that influence several aspects of early heart formation, dependent on the developmental timing of its deployment and the involved ligands and receptors [122][123][124][125][126]. Canonical Wnt signaling, also referred to as Wnt/β-catenin signaling due to its key downstream transcriptional effector β-catenin, promotes LPM by supporting ventral and lateral fates during gastrulation. ...
The heart is the first functional organ to form during vertebrate development. Congenital heart defects are the most common type of human birth defect, many originating as anomalies in early heart development. The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease. Although composed of only two chambers compared with the four-chambered mammalian heart, the zebrafish heart integrates the core processes and cellular lineages central to cardiac development across vertebrates. The rapid, translucent development of zebrafish is amenable to in vivo imaging and genetic lineage tracing techniques, providing versatile tools to study heart field migration and myocardial progenitor addition and differentiation. Combining transgenic reporters with rapid genome engineering via CRISPR-Cas9 allows for functional testing of candidate genes associated with congenital heart defects and the discovery of molecular causes leading to observed phenotypes. Here, we summarize key insights gained through zebrafish studies into the early patterning of uncommitted lateral plate mesoderm into cardiac progenitors and their regulation. We review the central genetic mechanisms, available tools, and approaches for modeling congenital heart anomalies in the zebrafish as a representative vertebrate model.
