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

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.

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P3479Sequential stimulation and inhibition of lysophosphatidic acid receptor 4 are critical for cardiac differentiation and repair

European Heart Journal ◽

10.1093/eurheartj/ehz745.0349 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

H J Cho ◽

J W Lee ◽

C S Lee ◽

Y R Ryu ◽

H S Kim ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Progenitor Cells ◽

Lysophosphatidic Acid ◽

Functional Significance ◽

Early Stage ◽

Cardiac Differentiation ◽

Cardiac Progenitor Cells ◽

Acid Receptor ◽

Downstream Signaling

Abstract Background The clinical application of cell therapy to repair the damaged heart needs to understand the precise differentiation process of stem cells and the characteristics of cardiac progenitor cells. Purpose We examined the cardiac-specific markers that expressed on the cell surface and determined their functional significance during cardiac differentiation. Methods and results We screened cell-surface expressing proteins on cardiac progenitor cells at differentiation day 3 compared to undifferentiated pluripotent stem cells (PSCs). Among candidates, we identified lysophosphatidic acid receptor 4 (LPAR4) that is a G protein-coupled receptor. During in vitro differentiation of mouse PSCs toward cardiac cells, LPAR4 expression peaked for 3–5 days and then and declined immediately. Also in vivo, LPAR4 was specifically expressed in the early stage of heart development in embryos and disappeared completely in adults, suggesting that stimulatory signal of LPAR4 at an early stage should be shut off for further progression of differentiation. We next have identified the LPAR4 downstream signaling molecule, p38MAPK, by comparing PSCs and LPAR4 knockdown PSCs. In both mouse and human PSCs, ODP (LPAR4 specific agonist) followed by p38MAPK blocker (SB203580) treatment significantly increased cardiac differentiation efficiency. Furthermore, we investigated whether LPAR4 is the maker for adult cardiac progenitor cells. We found that LPAR4-positive cells were rarely present in normal adult mouse hearts, but LPAR4-positive cells were increased when the heart was damaged. LPAR4-positive cells from adult hearts differentiated into cardiomyocytes. After myocardial infarction (MI), the sequential stimulation and inhibition of LPAR4 with ODP and p38MAPK blocker resulted in the reduction of infarct size and improvement of left ventricular dysfunction. Conclusion We demonstrated that LPAR4 is a cardiac progenitor-specific marker and its functional significance during cardiac differentiation and regeneration. Our findings provide a new insight in cell-free cardiac repair by the modulation of progenitor-specific downstream signaling. Acknowledgement/Funding Grants from “Strategic Center of Cell and Bio Therapy” (grant number: HI17C2085) and “Korea Research-Driven Hospital” (HI14C1277)

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Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

Cell Research ◽

10.1038/cr.2011.195 ◽

2011 ◽

Vol 22 (1) ◽

pp. 219-236 ◽

Cited By ~ 136

Author(s):

Nan Cao ◽

Zumei Liu ◽

Zhongyan Chen ◽

Jia Wang ◽

Taotao Chen ◽

...

Keyword(s):

Stem Cells ◽

Ascorbic Acid ◽

Induced Pluripotent Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Cardiac Differentiation ◽

Cardiac Progenitor Cells ◽

Induced Pluripotent

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Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

Development ◽

10.1242/dev.132209 ◽

2016 ◽

Vol 143 (9) ◽

pp. 1475-1481 ◽

Cited By ~ 26

Author(s):

Derek T. Peters ◽

Christopher A. Henderson ◽

Curtis R. Warren ◽

Max Friesen ◽

Fang Xia ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Surface Marker ◽

Asialoglycoprotein Receptor ◽

Cell Surface Marker ◽

Specific Cell ◽

Specific Cell Surface

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Cardiac Differentiation of Pluripotent Stem Cells

Stem Cells International ◽

10.4061/2011/383709 ◽

2011 ◽

Vol 2011 ◽

pp. 1-12 ◽

Cited By ~ 66

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.

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Single-cell reconstruction of differentiation trajectory reveals a critical role of ETS1 in human cardiac lineage commitment

BMC Biology ◽

10.1186/s12915-019-0709-6 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 8

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.

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LncRNA HBL1 is required for genome-wide PRC2 occupancy and function in cardiogenesis from human pluripotent stem cells

Development ◽

10.1242/dev.199628 ◽

2021 ◽

Author(s):

Juli Liu ◽

Sheng Liu ◽

Lei Han ◽

Yi Sheng ◽

Yucheng Zhang ◽

...

Keyword(s):

Stem Cells ◽

Gene Transcription ◽

Pluripotent Stem Cells ◽

Cardiac Development ◽

Chromatin Modification ◽

Human Pluripotent Stem Cells ◽

Cardiac Differentiation ◽

Genome Wide ◽

And Function

Polycomb Repressive Complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of RNA- PRC2 interaction in human cardiogenesis remains elusive. Here, we found human-specific Heart Brake LncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss-of-JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. Additionally, ChIP-seq revealed reduced EED-occupancy on 62 overlapped cardiogenic genes in HBL1−/- and JARID2−/- hPSCs, indicating the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurred, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved “microRNA-1-JARID2” axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis.

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Identification of Differentially Regulated Pathways in Cardiac Development and Cardiac Gene Expression during In Vitro Cardiac Differentiation of HLHS-derived Human Induced Pluripotent Stem Cells using Transcriptome Analysis

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0038-1628043 ◽

2018 ◽

Vol 66 (S 01) ◽

pp. S1-S110

Author(s):

M. Dreßen ◽

B. Northoff ◽

S. Laue ◽

I. Neb ◽

Z. Zhang ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Transcriptome Analysis ◽

Pluripotent Stem Cells ◽

Cardiac Development ◽

Cardiac Differentiation ◽

Cardiac Gene Expression ◽

Cardiac Gene ◽

Induced Pluripotent

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Efficient Cardiac Differentiation of Human Amniotic Fluid-Derived Stem Cells into Induced Pluripotent Stem Cells and Their Potential Immune Privilege

International Journal of Molecular Sciences ◽

10.3390/ijms21072359 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2359

Author(s):

Yi-Hsien Fang ◽

Saprina P.H. Wang ◽

Zi-Han Gao ◽

Sheng-Nan Wu ◽

Hsien-Yuan Chang ◽

...

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Mhc Class Ii ◽

Pluripotent Stem Cells ◽

Therapeutic Potential ◽

Troponin T ◽

Electrophysiological Study ◽

Cardiac Differentiation ◽

Specific Marker ◽

Human Amniotic Fluid

Mature mammalian hearts possess very limited regenerative potential. The irreversible cardiomyocyte loss after heart injury can lead to heart failure and death. Pluripotent stem cells (PSCs) can differentiate into cardiomyocytes for cardiac repair, but there are obstacles to their clinical application. Among these obstacles is their potential for post-transplant rejection. Although human amniotic fluid-derived stem cells (hAFSCs) are immune privileged, they cannot induce cardiac differentiation. Thus, we generated hAFSC-derived induced PSCs (hAFSC-iPSCs) and used a Wnt-modulating differentiation protocol for the cardiac differentiation of hAFSC-iPSCs. In vitro studies using flow cytometry, immunofluorescence staining, and patch-clamp electrophysiological study, were performed to identify the characteristics of hAFSC-iPSC-derived cardiomyocytes (hAFSC-iPSC-CMs). We injected hAFSC-iPSC-CMs intramuscularly into rat infarcted hearts to evaluate the therapeutic potential of hAFSC-iPSC-CM transplantation. At day 21 of differentiation, the hAFSC-iPSC-CMs expressed cardiac-specific marker (cardiac troponin T), presented cardiomyocyte-specific electrophysiological properties, and contracted spontaneously. Importantly, these hAFSC-iPSC-CMs demonstrated low major histocompatibility complex (MHC) class I antigen expression and the absence of MHC class II antigens, indicating their low immunogenicity. The intramyocardial transplantation of hAFSC-iPSC-CMs restored cardiac function, partially remuscularized the injured region, and reduced fibrosis in the rat infarcted hearts. Therefore, hAFSC-iPSCs are potential candidates for the repair of infarcted myocardium.

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