Transcriptome profiling of MIR148A-TKO and wild-type hESC-derived cells on day 4 of cardiac differentiaton
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PROVIDER: PRJNA533655 | ENA |
REPOSITORIES: ENA
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Project description:To gain global insights into the transcriptomic response associated with MIR148A family knockout, we performed RNA sequencing (RNA-Seq) on hESC-differentiated cells on day 4 of cardiac differentiation for both WT and MIR148A-TKO groups. RNA-Seq analysis identified a total of 1837 differentially expressed genes in MIR148A-TKO and wild-type hESC-derived cells on day 4 of cardiac differentiaton. Furthermore, Gene Ontology enrichment analysis indicated knockout of all MIR148A family members inhibited lateral mesodermal and cardiac differentiation.
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Project description:A hESC MESP1-MCHERRY reporter line was used to isolate and study the molecular character of MESP1 expressing pre-cardiac progenitors, derived from hESC. MESP1 is a key-transcription factor for pre-cardiac mesoderm and is marking the progenitor for almost all cells of the heart. This reporter line was used to study cardiac differentiation and the derivation of early cardiac progenitors in vitro.
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Project description:Control or KD hESC were differentiated into organoids as decribed in Methods. RNA was extracted from individual organoids at day 20 after commencing the STEMdiff™ Cerebral Organoid Kit protocol. Day 0 was designated as the day of transfer of hESC to a 96-well plate for differentiation into embryoid bodies .
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Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.
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Project description:FGF-MEK-ERK pathway has been implicated in diverse contexts in development. The pathway involves many autoinhibitory loops, resulting in pulse-like activity patterns. Here, we modulated the ERK activity during cardiac differentiation at the lateral plate mesoderm stage by PD0325901, a potent inhibitor of MEK, which is an upstream effector of ERK. We collected the resulting cell population, both in 2D and 3D, after cardiac specification to analyse how the ERK inhibition during induction affects the cell fate choice.
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