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.

Abstract P412: Mitochondrial Metabolic Transcriptome Profiles During Cardiomyocyte Differentiation From Mouse And Human Pluripotent Stem Cells

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Sung Woo Cho ◽  
Hyoung Kyu Kim ◽  
Jin Han ◽  
Ji-Hee Sung
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Oxidative Metabolism ◽  
Time Dependent ◽  
Lineage Commitment ◽  
Simultaneous Increase ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Cardiac Lineage ◽  
Mitochondrial Oxidative Metabolism

Simultaneous increase of myofibrils and mitochondria is a key process of cardiomyocyte differentiation from pluripotent stem cells (PSCs). Specifically, development of mitochondrial oxidative energy metabolism in cardiomyocytes is essential to providing the beating function. Although previous studies reported that mitochondrial oxidative metabolism have some correlation with the differentiation of cardiomyocytes, the mechanism by which mitochondrial oxidative metabolism is regulated and the link between cardiomyogenesis and mitochondrial function are still poorly understood. In the present study, we performed transcriptome analysis on cells at specific stages of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected highly upregulated mitochondrial metabolic genes at cardiac lineage commitment and time-dependent manner during cardiomyocyte differentiation and identified the protein-protein interaction network connecting between mitochondrial metabolic and cardiac developmental genes. We found several mitochondrial metabolic regulatory genes at cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpa, Cdkn2a in mESC-derived cells and CCK, NOS3 in hiPSC-derived cells) and time-dependent manner during cardiomyocyte differentiation (Eno3, Pgam2, Cox6a2, Fabp3 in mESC-derived cells and PGAM2, SLC25A4 in hiPSC-derived cells). Notably, mitochondrial metabolic proteins are highly interacted with cardiac developmental proteins time-dependent manner during cardiomyocyte differentiation rather than cardiac lineage commitment. Furthermore, mitochondrial metabolic proteins are mainly interacted with cardiac muscle contractile proteins rather than cardiac transcription factors in cardiomyocyte. Therefore, mitochondrial metabolism is critical at cardiac maturation rather than cardiac lineage commitment.

Abstract 65: Latrophilin-2 is a Specific Cell-surface Marker for Cardiac Progenitor Cells and Specifies Cardiac Lineage Commitment and Development

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Hyun-Jai Cho ◽  
Choon-Soo Lee ◽  
Jin-Woo Lee ◽  
Jung-Kyu Han ◽  
Han-Mo Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Cardiac Development ◽  
Cardiac Differentiation ◽  
Specific Cell ◽  
Specific Marker ◽  
Cardiac Progenitor Cells ◽  
Cardiac Lineage

Backgrounds: The identification of a lineage-specific marker plays a pivotal role in understanding developmental process and is utilized to isolate a certain cell type with high purity for the therapeutic purpose. We here report a new cardiac-specific marker, and demonstrate its functional significance in the cardiac development. Methods and Results: When mouse pluripotent stem cells (ES and iPS cells) were stimulated with BMP4, Activin A, bFGF and VEGF, they differentiated into cardiac cells. To screen cell-surface expressing molecules on cardiac progenitor cells compared to undifferentiated mouse iPS and ES cells, we isolated Flk1+/PDGFRa+ cells at differentiation day 4 and performed microarray analysis. Among candidates, we identified a new G protein-coupled receptor, Latrophilin-2 (LPHN2) whose signaling pathway and its effect on cardiac differentiation is unknown. In sorting experiments under cardiac differentiation condition, LPHN2+ cells derived from pluripotent stem cells strongly expressed cardiac-related genes (Mesp1, Nkx2.5, aMHC and cTnT) and exclusively gave rise to beating cardiomyocytes, as compared with LPHN2- cells. LPHN2-/- mice revealed embryonically lethal and huge defects in cardiac development. Interestingly, LPHN2+/- heterozygotes were alive and fertile. For the purpose of cardiac regeneration, we transplanted iPS-derived LPHN2+ cells into the infarcted heart of adult mice. LPHN2+ cells differentiated into cardiomyocytes, and systolic function of left ventricle was improved and infarct size was reduced. We confirmed LPHN2 expression on human iPS and ES cell-derived cardiac progenitor cells and human heart. Conclusions: We demonstrate that LPHN2 is a functionally significant and cell-surface expressing marker for both mouse and human cardiac progenitor and cardiomyocytes. Our findings provide a valuable tool for isolating cardiac lineage cells from pluripotent stem cells and an insight into cardiac development and regeneration.

YAP/TEAD3 signal mediates cardiac lineage commitment of human‐induced pluripotent stem cells

2019 ◽  
Vol 235 (3) ◽  
pp. 2753-2760 ◽  
Author(s):  
Zhenbo Han ◽  
Ying Yu ◽  
Benzhi Cai ◽  
Zihang Xu ◽  
Zhengyi Bao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Lineage Commitment ◽  
Cardiac Lineage ◽  
Induced Pluripotent

scholarly journals Uncoupling p53 from an Embryonic Regulome Exhausts Quiescent CML Stem Cells through Inhibition of a HIF1alpha Molecular Program

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1541-1541
Author(s):  
Mary T Scott ◽  
Wei Liu ◽  
Rebecca Mitchell ◽  
Cassie Clarke ◽  
Hassan Almasoudi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Research Funding ◽  
Embryonic Stem ◽  
Rna Seq ◽  
Combination Drug ◽  
Human Cd34 ◽  
Molecular Features ◽  
Cd34 Cells

Abstract Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML). By integrating known ES regulatory circuitry with transcriptomics datasets, including deep single-cell RNA-seq profiling of 15,670 LSC from five patients with CML, we identified a core ESC regulome in the LSC containing 1243 genes. The significant majority of this regulome (1102 genes) was up-regulated in cycling LSC, whilst quiescent LSC showed up-regulation of a characteristic set of 101 genes, unique to cells with high ESC identity and with regulatory circuitry enriched for c-Myc and Nanog modules. Membership of the ESC regulome included the TP53 gene which was transcriptionally repressed and detected at a lower frequency in quiescent LSC compared to cycling ones (11.8% vs 43.6%). We also demonstrated that tyrosine kinase inhibitors (TKI) repress the ESC regulome and TP53 expression in LSC, suggesting that the regulome safeguards against high levels of TP53 expression, thus promoting survival of quiescent LSC in the presence of TKI. We hypothesized that overcoming the influence of the regulome on TP53 expression would provide an opportunity to eradicate quiescent LSC. To this end, we used an MDM2 inhibitor (MDM2i), RG7388 (idasanutlin) or RG7112, to stabilize the p53 protein, examining its potential in combination with nilotinib (NIL) to eradicate CML LSC in vitro and in vivo, with RG7388 being the most optimized and furthest in development. The combination of NIL plus MDM2i in vitro was more effective at targeted LSC from primary patient samples than NIL treatment alone, as evidenced by reduced CFC and LTC-IC outputs (p<0.05, 0.01 respectively). Intriguingly, the combination of NIL plus MDM2i did not result in significant reductions in the number of LSC compared to NIL only, when we quantified them at the end of drug treatments in pre-clinical mouse models. Instead, we observed a functional decline of the LSC as evidenced by diminished engraftment potential in 2 o recipient mice (p<0.05; SCLtTA x BCR-ABL1 transgenic model) or diminished colony-plating potential (p<0.05). This was followed by near complete depletion of the LSC population (p<0.05) 28 days after cessation of combination drug treatment (patient-derived xenografts/PDX in immunocompromised mice). In order to understand the molecular events underpinning these drug effects on LSC, we performed RNA-seq analysis of drug-treated CD34 + cells in vitro (bulk cells), or of human CD34 + cells obtained from PDX (single cell RNA-seq). CD34 + cells treated with NIL plus MDM2i in vitro showed evidence of increased p53 stabilization and activation of p53 target genes, and this was accompanied by repression of the ESC regulome beyond that normally observed with NIL only. Similarly, in PDX we observed increased repression of the ESC regulome in human CD34 + cells exposed to the combination of NIL plus MDM2i that included repression of HIF1alpha and a signature of genes required for cellular adaptations to hypoxia, and growth factor-mediated resistance to TKI therapy. Further, single cell analysis of differentiated human CD45 + cells from our PDX model, provided compelling evidence that acquisition of this repressive signature in the LSC, through combined NIL plus MDM2i treatment, re-wires them towards a basophilic fate, consistent with functional exhaustion of the LSC compartment. In conclusion, we have identified an ESC regulome in CML LSC and demonstrate that a combination of a TKI plus an MDM2i leads to p53 upregulation which antagonizes this regulome, providing a highly effective strategy to target near complete loss of functional LSC in pre-clinical models. Our study has revealed a new therapeutic paradigm to examine in other cancer stem cell populations that utilize ESC regulatory programs. Disclosures Higgins: Roche/Genentech: Current Employment, Current equity holder in publicly-traded company. Copland: Astellas: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Jazz: Honoraria, Speakers Bureau.

scholarly journals A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

2020 ◽  
Vol 21 (9) ◽  
pp. 3229 ◽  
Author(s):  
Kejin Hu
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Rna Seq ◽  
Transcriptional Responses ◽  
Molecular Events ◽  
Gene Response ◽  
Yamanaka Factors ◽  
Reprogramming Factors ◽  
Negative Impacts ◽  
Human Ipsc

Yamanaka reprogramming is revolutionary but inefficient, slow, and stochastic. The underlying molecular events for these mixed outcomes of induction of pluripotent stem cells (iPSC) reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming and are compromised by the fact that only a rare population proceeds towards pluripotency, and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 h, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 h, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted upreprogramming or downreprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.

scholarly journals Cardiac Differentiation of Pluripotent Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Kristiina Rajala ◽  
Mari Pekkanen-Mattila ◽  
Katriina Aalto-Setälä
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Structural Characteristics ◽  
Cardiac Differentiation ◽  
Pacemaker Cells ◽  
Adult Cardiomyocytes ◽  
Strong Focus ◽  
Significant Interest ◽  
Cardiac Lineage

The ability of human pluripotent stem cells to differentiate towards the cardiac lineage has attracted significant interest, initially with a strong focus on regenerative medicine. The ultimate goal to repair the heart by cardiomyocyte replacement has, however, proven challenging. Human cardiac differentiation has been difficult to control, but methods are improving, and the process, to a certain extent, can be manipulated and directed. The stem cell-derived cardiomyocytes described to date exhibit rather immature functional and structural characteristics compared to adult cardiomyocytes. Thus, a future challenge will be to develop strategies to reach a higher degree of cardiomyocyte maturationin vitro, to isolate cardiomyocytes from the heterogeneous pool of differentiating cells, as well as to guide the differentiation into the desired subtype, that is, ventricular, atrial, and pacemaker cells. In this paper, we will discuss the strategies for the generation of cardiomyocytes from pluripotent stem cells and their characteristics, as well as highlight some applications for the cells.

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 Discovery and characterization of variance QTLs in human induced pluripotent stem cells

2018 ◽  
Author(s):  
Abhishek K. Sarkar ◽  
Po-Yuan Tung ◽  
John D. Blischak ◽  
Jonathan E. Burnett ◽  
Yang I. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Genetic Variation ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Human Tissues ◽  
Rna Seq ◽  
Induced Pluripotent ◽  
Expression Variance ◽  
Gene Expression Levels

AbstractQuantification of gene expression levels at the single cell level has revealed that gene expression can vary substantially even across a population of homogeneous cells. However, it is currently unclear what genomic features control variation in gene expression levels, and whether common genetic variants may impact gene expression variation. Here, we take a genome-wide approach to identify expression variance quantitative trait loci (vQTLs). To this end, we generated single cell RNA-seq (scRNA-seq) data from induced pluripotent stem cells (iPSCs) derived from 53 Yoruba individuals. We collected data for a median of 95 cells per individual and a total of 5,447 single cells, and identified 241 mean expression QTLs (eQTLs) at 10% FDR, of which 82% replicate in bulk RNA-seq data from the same individuals. We further identified 14 vQTLs at 10% FDR, but demonstrate that these can also be explained as effects on mean expression. Our study suggests that dispersion QTLs (dQTLs) which could alter the variance of expression independently of the mean can have larger fold changes, but explain less phenotypic variance than eQTLs. We estimate 424 individuals as a lower bound to achieve 80% power to detect the strongest dQTLs in iPSCs. These results will guide the design of future studies on understanding the genetic control of gene expression variance.Author summaryCommon genetic variation can alter the level of average gene expression in human tissues, and through changes in gene expression have downstream consequences on cell function, human development, and human disease. However, human tissues are composed of many cells, each with its own level of gene expression. With advances in single cell sequencing technologies, we can now go beyond simply measuring the average level of gene expression in a tissue sample and directly measure cell-to-cell variance in gene expression. We hypothesized that genetic variation could also alter gene expression variance, potentially revealing new insights into human development and disease. To test this hypothesis, we used single cell RNA sequencing to directly measure gene expression variance in multiple individuals, and then associated the gene expression variance with genetic variation in those same individuals. Our results suggest that effects on gene expression variance are smaller than effects on mean expression, relative to how much the phenotypes vary between individuals, and will require much larger studies than previously thought to detect.

scholarly journals Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells Using Monolayer Culture

2015 ◽  
Vol 10s1 ◽  
pp. BMI.S20050 ◽  
Author(s):  
Ivan Batalov ◽  
Adam W. Feinberg
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Heart Development ◽  
Cardiac Tissue ◽  
Cell Types ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Differentiation ◽  
Heart Repair ◽  
External Stimuli

Human pluripotent stem cells (PSCs) are a promising cell source for cardiac tissue engineering and cell-based therapies for heart repair because they can be expanded in vitro and differentiated into most cardiovascular cell types, including cardiomyocytes. During embryonic heart development, this differentiation occurs under the influence of internal and external stimuli that guide cells to go down the cardiac lineage. In order to differentiate PSCs in vitro, these or similar stimuli need to be provided in a controlled manner. However, because it is not possible to completely recapitulate the embryonic environment, the factors essential for cardiac differentiation of PSCs in vitro need to be experimentally determined and validated. Since PSCs were first developed, significant progress has been made in optimizing techniques for their differentiation toward cardiomyocytes. In this review, we will summarize recent advances in these techniques, with particular focus on monolayer-based methods that have improved the efficiency and scalability of cardiomyocyte differentiation.

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