Guided Differentiation of Pluripotent Stem Cells for Cardiac Cell Diversity
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE230587 | GEO | 2025/04/04
REPOSITORIES: GEO
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Project description:Guided Differentiation of Pluripotent Stem Cells for Cardiac Cell Diversity
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Project description:We apply short-read RNA sequencing technology to identify transcripts expressed during four time points of a human induced pluripotent stem cell derived cardiomyocyte differentiation protocol, corresponding to pluripotent, mesoderm, early cardiomyocyte, and cardiomyocyte cell stages. The RNA-seq reads are used to generate custom protein sequence database for proteogenomic applications and downstream mass spectrometry analysis. We demonstrate that this custom RNA-seq-guided proteomics approach can be used to identify protein isoforms that are differentially regulated during cardiac differentiation.
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Project description:Cardiac maturation is an important developmental phase where there are profound biological and functional changes after birth in mammals. Herein, we use our profiling of human heart maturation in vivo to identify key drivers of maturation in our human cardiac organoid (hCO) model. After screening of various metabolism modulating factors, we established a directed maturation (DM) protocol to induce mature cardiac expression and compared the proteomic changes to our original serum free (SF) protocol. In this dataset, we compared 4 replicates of DM to 4 replicates of SF derived cardiac organoids using global DIA-MS/MS.
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Project description:MALDI-imaging-guided Transcriptomics
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Project description:Cardiac maturation is an important developmental phase where there are profound biological and functional changes after birth in mammals. Herein, we use our profiling of human heart maturation in vivo to identify key drivers of maturation in our human cardiac organoid (hCO) model. After screening of various metabolism modulating factors, we established a directed maturation protocol to induce mature cardiac expression. We next compared directed maturation treatment to electrical pacing using phosphoproteomics in order to assess the similarities in the induction of maturation. The electrical pacing protocol utilized a custom platform, where we added Heart-Dyno inserts into C-pace system in 24-well plates enabling 120 bpm pacing for 5 minutes without causing toxicity. In this dataset, we compared 3 replicates of CTRL (our original serum free organoid protocol), electrical pacing (the standard protocol for maturation of cardiac stem cells), and directed maturation protocol (DM, new protocol) through phosphoproteomics.
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Project description:The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The CARMEN locus, which is known to dictate specification towards the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced lncRNAs. Here, we identify one of these isoforms, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA.
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Project description:In hESCs, Wnt3/β-catenin activity is low and Activin/SMAD signaling ensures NANOG expression to sustain pluripotency. In response to exogenous Wnt3 effectors, Activin/SMADs switch to cooperate with β-catenin and induce mesendodermal differentiation genes. We show here that the HIPPO effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. In the absence of YAP, Activin signaling is sufficient to induce expression of the endogenous Wnt3 cytokine, which stabilizes β-catenin and selectively activates genes required for cardiac mesoderm (ME) formation. Interestingly, Activin-stimulated YAP-knockout hESCs strongly express β-catenin-dependent cardiac mesoderm markers (BAF60c and HAND1), but unlike WT hESCs, fail to express cardiac inhibitor genes (CDX2, MSX1). Accordingly, YAP-/- cells treated with Activin alone can differentiate efficiently to beating cardiomyocytes in culture, bypassing the need for sequential treatment with exogenous Wnt ligand and Wnt inhibitors. Similarly, Activin in combination with small-molecule YAP inhibitors generates beating cardiomyocytes from wild-type hESCs following a one- step protocol. Our findings highlight an unanticipated role of YAP as an upstream regulator of WNT3 to maintain hESC pluripotency in the presence of Activin, and uncover a direct route for the development of human embryonic cardiac mesoderm.
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