Abstract 276: NOTCH1 is Essential for Ventricular Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Mingtao Zhao ◽  
Shiqiao Ye ◽  
Joe Zhang ◽  
Ningyi Shao ◽  
Chun Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Left Ventricular ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Pathogenic Variants ◽  
Induced Pluripotent

Pathogenic variants in NOTCH1 have been implicated in multiple types of congenital heart defects, such as bicuspid aortic valve, Tetralogy of Fallot, and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which NOTCH1 pathogenic variants cause abnormalities in human embryonic heart development are largely unknown. Here, we used CRISPR/Cas9-mediated genome editing to genetically delete NOTCH1 in human induced pluripotent stem cells (iPSCs). We found that NOTCH1 was dispensable for mesodermal and vascular endothelial differentiation of human iPSCs. Disruption of NOTCH activity promoted venous-specific gene expression but suppressed arterial-specific gene expression in iPSC-derived endothelial cells (iPSC-ECs). Intriguingly, NOTCH1 deletion significantly impaired the cardiac differentiation efficiency. In NOTCH1 homozygous knockout ( NOTCH1 -/- ) iPSC-derived cardiomyocytes (iPSC-CMs), atrial-specific genes ( NR2F2, KCNJ3 , and MYL7 ) were upregulated whereas ventricular-specific genes ( MYL2, IRX4 , and MYH7 ) were downregulated. Electrophysiological analysis by patch clamp and optical mapping indicated that atrial-like cardiomyocytes were dominant whereas the percentage of ventricular-like iPSC-CMs was dramatically reduced (<1%) in NOTCH1 -/- iPSC-CMs. In addition, mitochondrial respiration was reduced in NOTCH1 deficient iPSC-CMs compared to wild-type controls, which was likely attributed to the reduction of ventricular cardiomyocytes in NOTCH1 -/- iPSC-CMs. As NOTCH1 is primarily expressed in endothelial cells rather than cardiomyocytes, we conclude that NOTCH1 affects ventricular cardiomyocyte lineage commitment possibly through controlling cell fate determination of cardiac progenitors during human iPSC differentiation. Our study may provide novel insights into the mechanisms by which NOTCH1 mutations lead to left ventricular hypoplasia in HLHS patients.

scholarly journals Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Feikun Yang ◽  
Dean W. Richardson
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Mechanical Stretch ◽  
Specific Gene ◽  
Induced Pluripotent

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-β3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-β3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-β3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration.

Abstract 97: Epigenetic Signatures Contribute to the Superior Endothelial Cell Identity in Human Induced Pluripotent Stem Cells Derived From Endothelial Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Mingtao Zhao ◽  
Shijun Hu ◽  
Rajini Srinivasan ◽  
Fereshteh Jahaniani ◽  
Ning-Yi Shao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Early Passage ◽  
Tissue Of Origin ◽  
Induced Pluripotent ◽  
Epigenetic Signatures

Human induced pluripotent stem cells (iPSCs) can be derived from multiple types of somatic cells by transient overexpression of four Yamanaka factors. Epigenetic memory of the tissue of origin is seen in early passage iPSCs, which may interfere the directed differentiation towards target lineages in disease modeling and drug discovery. Here we derived human iPSC from three types of somatic cells of the same individuals: fibroblast (FB-iPSCs), endothelial cells (EC-iPSCs) and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into endothelial cells by using sequential administration of Activin, BMP4, bFGF and vEGF. EC-iPSCs show higher EC differentiation propensity and EC-specific markers (PECAM1 and NOS3) gene expression in early passage iPSCs than FB-iPSCs and CPC-iPSCs. In vivo, EC-iPSC-ECs display significantly greater revascularization capacity than those of FB-iPSCs and CPC-iPSCs when transplanted to the hindlimb ischemic mice. In addition, transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31+ population and higher EC-specific markers (PECAM1, KDR and ICAM) gene expression by using single cell qPCR. In vitro, EC-iPSC-ECs exhibit better endothelial cell character maintenance along with extensive culturing and passaging. Several chromatin signatures, including H3K27ac, H3K4me1 and p300 were found highly enriched in ECs and EC-iPSCs, but not in human embryonic stem cells (ESCs). Gene ontology analysis indicates that the differentially enriched regions are primarily associated with angiogenesis and vascular development, reflecting the residual epigenetic signatures in EC-iPSCs. Finally EC-specific enhancer markers undergo dynamic changes during the process of EC fate commitment and differentiation, though the majority of them sustain conserved pattern in EC-iPSCs, CPC-iPSCs and FB-iPSCs. In conclusion, these results highlight that the residual epigenetic signatures of tissue of origin may affect lineage differentiation propensity in early-passage human iPSCs.

Abstract 330: Expression of Glycolytic Enzymes in Induced Pluripotent Stem Cells, Derived Megakaryocytes, and Endothelial Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Arun Chandra ◽  
Dhananjay Vaidya ◽  
Rui Chang ◽  
Josh Knowles ◽  
Ivan Carcamo-oribe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Glycolytic Enzyme ◽  
Glycolytic Enzymes ◽  
Mixed Effect ◽  
Expression Levels ◽  
Induced Pluripotent

Introduction: Induced pluripotent stem cells (iPSC) have a significant capacity for self-renewal and can differentiate into any somatic cell type, such as megakaryocytes (MK) and vascular endothelial cells (EC). We have previously shown that iPSC-derived MKs (IPSC-MK) upregulate the gene expression levels of irreversible glycolytic enzymes committing glucose to glycolysis in comparison to their parental iPSC lines (Vaidya et al., 2016). This pattern differed from past comparisons between somatic source cells and iPSCs (Chung et al., 2007; Varum et al., 2011; Zhang et al., 2011). To further corroborate our data, we extended our analysis to iPSC-derived ECs (iPSC-EC). Methods: We studied expression of glycolytic enzyme genes in 30 human iPSC lines and paired iPSC-EC and iPSC-MK lines using RNA-seq. iPSC-EC to iPSC expression ratios for 11 glycolytic enzymes were estimated using a multilevel mixed-effect model regression, and a significance threshold of p < 0.05/11 = 0.0045 was used. A bivariate statistical analysis was used to assess the correlation between iPSC-EC and iPSC-MK data. Results: Of the 11 glycolytic enzymes studied, 4 were highly expressed in iPSC-ECs (Log2(iPSC-EC/iPSC) ≥ 1) while 1 was highly expressed in iPSCs (Log2(iPSC/iPSC-EC) ≥ 1). Transcripts for functionally irreversible enzymes including ADPGK, PFKP, and PKLR displayed higher expression levels in iPSC-ECs compared to iPSCs. Similarly, 6 glycolytic genes demonstrated higher expression in iPSC-MKs, while 2 were more highly expressed in iPSCs. The functionally irreversible enzymes HK1, ADPGK, and PFK exhibited higher expression levels in iPSC-MKs. The ratios of gene expression (derived-lines:iPSCs) for all 37 genes in the iPSC-MK results correlated with those of the iPSC-EC results: Pearson’s r = 0.52, p = 0.001. Conclusion: Both iPSC-ECs and iPSC-MKs upregulate the expression of glycolytic enzymes compared to their parental iPSCs. In light of data showing that differentiated cells are more metabolically active and have more mitochondria than iPSCs (Chandra et al., 2020), these results suggest that differentiation of iPSCs is accompanied by an increase in both glycolytic and TCA cycle activities, with glycolytic generation of pyruvate used for mitochondrial production of ATP.

scholarly journals T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S234-S234
Author(s):  
Lorna Farrelly ◽  
Shuangping Zhang ◽  
Erin Flaherty ◽  
Aaron Topol ◽  
Nadine Schrode ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Gene Expression Pattern ◽  
Early Gene ◽  
Label Free ◽  
Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

scholarly journals Automated Deep Learning-Based System to Identify Endothelial Cells Derived from Induced Pluripotent Stem Cells

2018 ◽  
Vol 10 (6) ◽  
pp. 1687-1695 ◽  
Author(s):  
Dai Kusumoto ◽  
Mark Lachmann ◽  
Takeshi Kunihiro ◽  
Shinsuke Yuasa ◽  
Yoshikazu Kishino ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Deep Learning ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent
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