ALPK2 Promotes Cardiogenesis in Zebrafish and Human Pluripotent Stem Cells.
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ABSTRACT:
SUBMITTER: Hofsteen P
PROVIDER: S-EPMC5993047 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transformation of pluripotent stem cells into cardiac tissue is the hallmark of cardiogenesis, yet pro-cardiogenic signals remain partially understood. Preceding cardiogenic induction, a surge in CXCR4 chemokine receptor expression in the early stages of stem cell lineage specification coincides with the acquisition of pre-cardiac profiles. Accordingly, CXCR4 selection, in conjunction with mesoderm-specific VEGF type II receptor FLK-1 co-expression, segregates cardiogenic populations. To assess the functionality of the CXCR4 biomarker, targeted activation and disruption were here exploited in the context of embryonic stem cell-derived cardiogenesis. Implicated as a cardiogenic hub through unbiased bioinformatics analysis, induction of the CXCR4/SDF-1 receptor-ligand axis triggered enhanced beating activity in stem cell progeny. Gene expression knockdown of CXCR4 disrupted spontaneous embryoid body differentiation and blunted the expression of cardiogenic markers MEF2C, Nkx2.5, MLC2a, MLC2v, and cardiac-MHC. Exogenous SDF-1 treatment failed to rescue cardiogenic-deficient phenotype, demonstrating a requirement for CXCR4 expression in mediating SDF-1 effects. Thus, a pro-cardiogenic signaling role for the CXCR4/SDF1 axis is herein revealed within pluripotent stem cell progenitors, exposing a functional target to promote lineage-specific differentiation.
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Project description:Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation, but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify ?-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore, treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous ?-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing.
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Project description:BACKGROUND:Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. RESULTS:Here, we report ARID1A, a DNA-binding subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Knockout-of-ARID1A (ARID1A-/-) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A -/- hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. CONCLUSIONS:We uncover the opposite roles by ARID1A to govern both early cardiac and neural development from pluripotent stem cells. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A, possibly through ARID1A-REST/NRSF interaction.
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Project description:WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/?-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.
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Project description:BACKGROUND: Adipose tissue is a source of multipotent adult stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic cells and adipogenic cells. Several reports have shown adipose-derived stem cells (ASCs) have the ability to undergo cardiomyogenesis. Studies have shown 5-azacytidine can successfully drive stem cells such as bone marrow derived stem cells to differentiate into cardiomyogenic cells. Therefore, in this study, we investigated the effect 5-azacytidine on the cardiogenic ability of ASCs. METHODS: The cardiogenic potential of ASCs was analysed by studying the morphological changes after induction, the changes in the cardiogenic genes expression i.e. GATA4, MLC-2v, MLC-2a, NKX2.5, ?-MHC, ?-MHC, Atrial natriuretic peptide (ANP), Connexin 43, Cardiac Troponin C, Cardiac Troponin I and myocyte enhancer factor (MEF2C) and the changes of embryonic stem cells genes expression at P5 and P10 using quantitative PCR. RESULTS: Our results showed that the induced ASCs did not show significant morphological difference compared to the non-induced ASCs. While quantitative PCR data indicated that most cardiogenic genes and stemness genes expression level decreased after induction at P5 and P10. CONCLUSION: 5-azacytidine is insufficient for the cardiogenic induction of the ASCs.
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Project description:BackgroundStudying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction.MethodsWe examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.ResultsThe combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway.ConclusionsVPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.
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Project description:Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Using the well-established mouse ESC culture, we demonstrated that HDAC inhibitors, both trichostatin A (TSA,50? nmol/L) and sodium butyrate (NaB, 200 ?µmol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs in response to oxidant stress. HDAC inhibition reduced the cleaved caspases 3, 6, 9, PARP, and TUNEL positive ESCs, which were abrogated with MG132 (0.5 ?µmol/L), a specific proteasome inhibitor. Furthermore, HDAC inhibition stimulates the growth of embryoid bodies (EB), which are associated with a faster spontaneous rhythmic contraction. HDAC inhibition increases the up-regulation of GATA4, MEF2C, Nkx2.5, cardiac actin, and ?-SMA mRNA and protein levels that were abrogated by MG132. TSA and NaB resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows for the activation of MEF2. Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicits the survival pathway.
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