scholarly journals Single-cell RNA sequencing reveals that BMPR2 mutation regulates right ventricular function via ID genes

2021 ◽  
pp. 2100327
Author(s):  
Mingxia Du ◽  
Haibin Jiang ◽  
Hongxian Liu ◽  
Xin Zhao ◽  
Yu Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Heart Development ◽  
Pulmonary Regurgitation ◽  
Embryonic Stem ◽  
Cardiac Differentiation ◽  
Double Knockout ◽  
Morphogenetic Protein ◽  
Downstream Effectors ◽  
Induced Pluripotent

Mutations in bone morphogenetic protein type II receptor (BMPR2) have been found in patients with congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH). Our study aimed to clarify whether deficient BMPR2 signalling acts through downstream effectors, inhibitors of DNA-binding proteins (IDs), during heart development to contribute to the progress of PAH in CHD patients. To confirm that IDs are downstream effectors of BMPR2 signalling in cardiac mesoderm progenitors (CMPs) and contribute to PAH, we generated Cardiomyocytes (CMs)-specific Id 1/3 knockout mice (Ids cDKO), and 12/25 developed mild PAH with altered haemodynamic indices and pulmonary vascular remodelling. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) human embryonic stem cells that recapitulated the BMPR2 signalling deficiency of CHD-PAH iPSCs. CMs differentiated from induced pluripotent stem cells (iPSCs) derived from CHD-PAH patients with BMPR mutations exhibited dysfunctional cardiac differentiation and reduced Ca2+ transients, as evidenced by confocal microscopy experiments. Smad1/5 phosphorylation and ID1 and ID3 expression were reduced in CHD-PAH iPSCs and in Bmpr2+/– rat right ventricles. Moreover, Ultrasound revealed that 33% of Ids cDKO mice had detectable defects in their ventricular septum and pulmonary regurgitation. CMs isolated from the mouse right ventricles also showed reduced Ca2+ transients and shortened sarcomeres. Single-cell RNA(scRNA)-seq analysis revealed impaired differentiation of CMPs and downregulated USP9X expression in IDs KO cells compared with wild-type (WT) cells. We found that BMPR2 signals through IDs and USP9X to regulate cardiac differentiation, and the loss of ID1 and ID3 expression contributes to CM dysfunction in CHD-PAH patients with BMPR2 mutations.

scholarly journals Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Qin Lu ◽  
Yinan Liu ◽  
Yang Wang ◽  
Weiping Wang ◽  
Zhe Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Early Stage ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Cardiac Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
Cardiac Transcription Factors ◽  
20 Nm

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10−4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata4, α-Mhc, β-Mhc, and Tnnt2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp1, Brachyury T, Eomes, Isl1, Gata4, Nkx2.5, Tbx5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor (Fgf8), Fgf10, and Nodal at early stage, and bone morphogenetic protein 2 (Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin.

Dppa2/4 as a trigger of signaling pathways to promote zygote genome activation by binding to CG-rich region

2020 ◽  
Author(s):  
Hanshuang Li ◽  
Chunshen Long ◽  
Jinzhu Xiang ◽  
Pengfei Liang ◽  
Xueling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathways ◽  
Embryonic Stem ◽  
Proximal Promoter ◽  
Human Embryos ◽  
Double Knockout ◽  
Rich Region ◽  
Induced Pluripotent ◽  
Zygote Genome Activation ◽  
Genome Activation

Abstract Developmental pluripotency-associated 2 (Dppa2) and developmental pluripotency-associated 4 (Dppa4) as positive drivers were helpful for transcriptional regulation of zygotic genome activation (ZGA). Here, we systematically assessed the cooperative interplay of Dppa2 and Dppa4 in regulating cell pluripotency and found that simultaneous overexpression of Dppa2/4 can make induced pluripotent stem cells closer to embryonic stem cells (ESCs). Compared with other pluripotency transcription factors, Dppa2/4 can regulate majorities of signaling pathways by binding on CG-rich region of proximal promoter (0–500 bp), of which 85% and 77% signaling pathways were significantly activated by Dppa2 and Dppa4, respectively. Notably, Dppa2/4 also can dramatically trigger the decisive signaling pathways for facilitating ZGA, including Hippo, MAPK and TGF-beta signaling pathways and so on. At last, we found alkaline phosphatase, placental-like 2 (Alppl2) was completely silenced when Dppa2 and 4 single- or double-knockout in ESC, which is consistent with Dux. Moreover, Alppl2 was significantly activated in mouse 2-cell embryos and 4–8 cells stage of human embryos, further predicted that Alppl2 was directly regulated by Dppa2/4 as a ZGA candidate driver to facilitate pre-embryonic development.

scholarly journals Analysis of cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription

2017 ◽  
Author(s):  
Clayton E Friedman ◽  
Quan Nguyen ◽  
Samuel W Lukowski ◽  
Han Sheng Chiu ◽  
Abbigail Helfer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Heart Development ◽  
Time Course ◽  
Cardiac Differentiation ◽  
Transcriptional Networks ◽  
Hypertrophic Signaling

AbstractDifferentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. Unsupervised clustering and lineage prediction algorithms were used to map fate choices and transcriptional networks underlying cardiac differentiation. We leveraged this resource to identify strategies for controlling in vitro differentiation as it occurs in vivo. HOPX, a non-DNA binding homeodomain protein essential for heart development in vivo was identified as dys-regulated in in vitro derived cardiomyocytes. Utilizing genetic gain and loss of function approaches, we dissect the transcriptional complexity of the HOPX locus and identify the requirement of hypertrophic signaling for HOPX transcription in hPSC-derived cardiomyocytes. This work provides a single cell dissection of the transcriptional landscape of cardiac differentiation for broad applications of stem cells in cardiovascular biology.

scholarly journals Single-cell reconstruction of differentiation trajectory reveals a critical role of ETS1 in human cardiac lineage commitment

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Hang Ruan ◽  
Yingnan Liao ◽  
Zongna Ren ◽  
Lin Mao ◽  
Fang Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Heart Development ◽  
Cardiac Differentiation ◽  
Lineage Commitment ◽  
Rna Seq ◽  
Molecular Features ◽  
Molecular Events ◽  
Cardiac Lineage

Abstract Background Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration. Compared to the large body of studies investigating cardiac maturation and cardiomyocyte subtype-specific induction, molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized. Results In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation, we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features. Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions, we revealed crosstalk between cardiac progenitor cells and endoderm cells, which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5. In addition, computational analyses of single-cell RNA-Seq data unveiled ETS1 (ETS Proto-Oncogene 1) activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells. Consistent with the findings from single-cell analysis, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against ETS1 revealed genomic occupancy of ETS1 at cardiac structural genes at day 9 and day 14, whereas ETS1 depletion dramatically compromised cardiac differentiation. Conclusion Together, our study not only characterized the molecular features of different cell types and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state, but may also have important implications for understanding human heart development at early embryonic stage, as well as directed manipulation of cardiac differentiation in regenerative medicine.

scholarly journals Activin A Modulates CRIPTO-1/HNF4α+ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
Robin Duelen ◽  
Guillaume Gilbert ◽  
Abdulsamie Patel ◽  
Nathalie de Schaetzen ◽  
Liesbeth De Waele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Heart Development ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Activin A ◽  
Embryoid Bodies ◽  
Cardiac Differentiation ◽  
Potential Applications

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.

TAp63 Is Important for Cardiac Differentiation of Embryonic Stem Cells and Heart Development

Stem Cells ◽  
2011 ◽  
Vol 29 (11) ◽  
pp. 1672-1683 ◽  
Author(s):  
Matthieu Rouleau ◽  
Alain Medawar ◽  
Laurent Hamon ◽  
Shoham Shivtiel ◽  
Zohar Wolchinsky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Heart Development ◽  
Embryonic Stem ◽  
Cardiac Differentiation

scholarly journals Dppa2/4 target chromatin bivalency enabling multi-lineage commitment

2019 ◽  
Author(s):  
Mélanie A. Eckersley-Maslin ◽  
Aled Parry ◽  
Marloes Blotenburg ◽  
Christel Krueger ◽  
Valar Nila Roamio Franklin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Polymerase ◽  
Single Cell ◽  
Embryonic Stem ◽  
Lineage Commitment ◽  
Double Knockout ◽  
Pluripotent Cells ◽  
Low Expression

AbstractBivalent chromatin marks developmental promoters in pluripotent cells, yet their targeting and precise impact on lineage commitment remains unclear. We uncover Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as epigenetic priming factors, establishing chromatin bivalency. Single-cell transcriptomics and differentiation assays reveal Dppa2/4 double knockout embryonic stem cells fail to exit pluripotency and differentiate efficiently. Dppa2/4 associate with COMPASS and Polycomb complexes and are required to recruit and maintain their binding at a subset of developmentally important bivalent promoters which are characterised by low expression and poised RNA polymerase. Consequently, upon Dppa2/4 knockout, these dependent promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel targeting principle for bivalency to developmental promoters, poising them for future lineage specific activation.

scholarly journals Apoptotic Find-me Signals are an Essential Driver of Stem Cell Conversion To The Cardiac Lineage

2021 ◽  
Author(s):  
Loic Fort ◽  
Vivian Gama ◽  
Ian G Macara
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Epithelial Mesenchymal Transition ◽  
Embryonic Stem ◽  
Cardiac Differentiation ◽  
Apoptotic Cells ◽  
Knock Out ◽  
Mesenchymal Transition ◽  
Cell Conversion ◽  
Induced Pluripotent

Pluripotent stem cells can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) undergo widespread apoptosis upon 49 hrs of Wnt activation, followed by a synchronous epithelial-mesenchymal transition (EMT). The EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including EOMES, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting the involvement of soluble factors from apoptotic cells in mesoderm conversion. Treatment with a purinergic P2Y receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can, unexpectedly, function as essential paracrine signals in mesoderm specification.

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