Abstract 14: The Cytoskeletal Architecture of Myocytes Derived from Human Cardiac Progenitor Cells

The stem cell antigen c-kit characterizes a heterogeneous pool of human cardiac progenitor cells (hCPCs) that exhibit a remarkable degree of regenerative potential and are currently employed in clinical trials. While this hCPC pool contains distinct subpopulations of c-kit+ cells that preferentially differentiate into muscular or vascular cardiac cells, we hypothesize that hCPCs may be coerced to specify only along the cardiomyogenic lineage by manipulating the Wnt/β-catenin pathway. We report that pharmacological inhibition of the non-canonical Wnt pathway facilitated the commitment of more than >95% c-kit+ hCPCs to the cardiomyocyte lineage after 4 days in-vitro: this constitutes a substantially more homogeneous population than previously reported with dexamethasone treatment. The hCPC-derived myocytes stained positive for Nkx2.5, a transcription factor that orchestrates cardiomyogenic differentiation, and for the contractile protein sarcomeric α-actin. To test if we could push the cells towards a more mature phenotype, we mimicked the cyclic modulation of the Wnt pathway observed during development. While activation of Wnt signaling resulted in widespread cell death and reduction in cell size, subsequent Wnt inhibition prompted the spared cells to proliferate. With this protocol, hCPC-derived myocytes increased in size and displayed more mature cytoskeletal architectures. In contrast with dexamethasone treated cells, where the localization of α-sarcomeric actinin is mostly diffuse in the cytoplasm, here we observed both Z-bodies and Z-disks like structures. The latter exhibited a periodicity of ~1.6 um and were clustered in larger, more aligned actin bundles. This finding suggests that the tension developed along these cytoskeletal components may play a role in the recruitment of sarcomeric proteins. In conclusion, Wnt signaling inhibition in hCPCs may be sufficient to obtain a homogeneous population of cells with features of myocytes, characterized by improved cytoskeletal organization than dexamethasone treated cells and similar to that observed in myocytes derived from human induced pluripotent stem cells.

Download Full-text

Abstract 217: Long Non-coding RNA Myocardial Infarction-associated Transcript (MIAT) Protects Cardiac Progenitor Cells From Oxidative Stress-induced Cell Death

Circulation Research ◽

10.1161/res.119.suppl_1.217 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Liu Yang ◽

Yang Yu ◽

Baron Arnone ◽

Chan Boriboun ◽

Jiawei Shi ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Infarction ◽

Cell Death ◽

Progenitor Cells ◽

Cardiac Cells ◽

Border Zone ◽

Mouse Heart ◽

Cardiac Progenitor Cells

Background: Long non-coding RNAs (lncRNAs) are an emerging class of RNAs with no or limited protein-coding capacity; a few of which have recently been shown to regulate critical biological processes. Myocardial infarction-associated transcript (MIAT) is a conserved mammalian lncRNA, and single nucleotide polymorphisms (SNPs) in 6 loci of this gene have been identified to be strongly associated with the incidence and severity of human myocardial infarction (MI). However, whether and how MIAT impacts on the pathogenesis of MI is unknown. Methods & Results: Quantitative RT-PCR analyses revealed that MIAT is expressed in neonatal mouse heart and to a lesser extent in adult heart. After surgical induction of MI in adult mice, MIAT starts to increase in 2 hours, peaks at 6 hours in atria and 12 hours in ventricles, and decreases to baseline at 24 hours. Fluorescent in situ hybridization (FISH) revealed a slight increase in the number of MIAT-expressing cells in the infarct border zone at 12 hours post-MI. Moreover, qRT-PCR analyses of isolated cardiac cells revealed that MIAT is predominantly expressed in cardiosphere-derived cardiac progenitor cells (CPCs). Treatment of CPCs with H 2 O 2 led to a marked upregulation of MIAT, while knockdown (KD) of MIAT resulted in a significantly impaired cell survival in vitro with H 2 O 2 treatment and in vivo after administered in the ischemic/reperfused heart. Notably, bioinformatics prediction and RNA immunoprecipitation identified FUS (fused in sarcoma) as a novel MIAT-interacting protein. FUS-KD CPCs displayed reduced cell viability and increased apoptosis under oxidative stress. Furthermore, MIAT overexpression enhanced survival of WT CPCs but not FUS-KD CPCs, suggesting that the protective role of MIAT is mediated by FUS. Conclusions: MIAT interacts with FUS to protect CPCs from oxidative stress-induced cell death.

Download Full-text

Cardiac Progenitor Cells from Stem Cells: Learning from Genetics and Biomaterials

Cells ◽

10.3390/cells8121536 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1536 ◽

Cited By ~ 2

Author(s):

Sara Barreto ◽

Leonie Hamel ◽

Teresa Schiatti ◽

Ying Yang ◽

Vinoj George

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Genetic Factors ◽

Induced Pluripotent Stem Cell ◽

Significant Shift ◽

Cardiac Progenitor Cells ◽

Recent Developments ◽

A Cell ◽

Induced Pluripotent

Cardiac Progenitor Cells (CPCs) show great potential as a cell resource for restoring cardiac function in patients affected by heart disease or heart failure. CPCs are proliferative and committed to cardiac fate, capable of generating cells of all the cardiac lineages. These cells offer a significant shift in paradigm over the use of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes owing to the latter’s inability to recapitulate mature features of a native myocardium, limiting their translational applications. The iPSCs and direct reprogramming of somatic cells have been attempted to produce CPCs and, in this process, a variety of chemical and/or genetic factors have been evaluated for their ability to generate, expand, and maintain CPCs in vitro. However, the precise stoichiometry and spatiotemporal activity of these factors and the genetic interplay during embryonic CPC development remain challenging to reproduce in culture, in terms of efficiency, numbers, and translational potential. Recent advances in biomaterials to mimic the native cardiac microenvironment have shown promise to influence CPC regenerative functions, while being capable of integrating with host tissue. This review highlights recent developments and limitations in the generation and use of CPCs from stem cells, and the trends that influence the direction of research to promote better application of CPCs.

Download Full-text

Abstract 238: Role of Erythropoietin Signaling in the Biology of Mouse Cardiac Progenitor Cells

Circulation Research ◽

10.1161/res.111.suppl_1.a238 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Maria P Zafiriou ◽

Claudia Noack ◽

Michael Didie ◽

Bernhard Unsoeld ◽

Ali El-Armouche ◽

...

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Cell Activation ◽

Ischemia Reperfusion ◽

Cardiac Cells ◽

Functional Improvement ◽

Cardiac Progenitor Cells ◽

Epor Expression

Erythropoietin (Epo) was shown to improve cardiac function following ischemia reperfusion mainly via neo-angiogenesis and anti-apoptotic mechanisms. We found EpoR expression to be particularly high in adult cardiac progenitor cells (CPCs). Thus, we reasoned that Epo may play a role in the biology of these cells. We isolated CPCs from adult C57BL/6 hearts by enzymatic digestion and filtration (pore size: 30 µm). By means of immunofluorescence microscopy (IF) and flow cytometry (FC) we analyzed EpoR expression in the CPCs. 24±3% of the investigated cardiac cells were positive for EpoR with 3±2% of these being c-kit+ and 28%±2% Sca-1+. 52% of the EpoR+ cells expressed endothelial cell markers (40±2% CD34+, 9±2% FLK1+). 42±4% expressed myocyte markers (αMHC+, cTNT+). IF revealed a progenitor-like population with immature cell morphology and proliferation potential (ki67+). Cell cycle analysis showed an enrichment of αMHC+ EpoR+ cells in S and G2 phase (49±7%, n=3) as compared to the αMHC- EpoR- population (13±3%, n=3). Moreover, we tested the effect of Epo in the biology of these CPCs in vitro. At d14 we observed a two-fold increase of GATA4+ and cTnT+ cardiac cells in the co-cultures treated with Epo (n=3). CPC cycle arrest abrogated the aforementioned effects, suggesting that Epo influences mainly CPC proliferation. Finally, we tested the potential of Epo to protect against ischemia by inducing the proliferation of these αMHC+ CPCs in vivo in a myocardial infarction (MI) model. 4 weeks post MI, echocardiography did not reveal a significant functional improvement of the Epo receiving mice (2x, 2U/g Epo i.p). Nevertheless, FC analysis of the progenitor pool showed a significant augmentation of αMHC+ and cTnT+ cells (Sham: 19±3% vs Epo 35±3%, n=5; MI: 10.6±2.3%, n=6 vs Epo 20.3±1.9%, n=8). These data suggest an activation of myogenic progenitors by Epo, despite the lack of apparent regeneration under the investigated conditions. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic progenitor cell pool in the adult heart. Epo drives their proliferation in vitro and in vivo even upon acute cardiac injury. We are currently investigating the long-term consequences of the observed progenitor cell activation in models of chronic ischemic injury.

Download Full-text

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells

Blood ◽

10.1182/blood-2011-07-369199 ◽

2012 ◽

Vol 119 (7) ◽

pp. 1683-1692 ◽

Cited By ~ 64

Author(s):

Michiko Ichii ◽

Mark Barton Frank ◽

Renato V. Iozzo ◽

Paul W. Kincade

Keyword(s):

Wnt Signaling ◽

Progenitor Cells ◽

Stromal Cells ◽

Wnt Pathway ◽

Hematopoietic Stem ◽

Kit Ligand ◽

Flow Cytometric ◽

Extracellular Matrix Components ◽

Canonical Wnt ◽

Angiopoietin 1

Abstract Considerable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

Download Full-text

Novel Identified Circular Transcript of RCAN2, circ-RCAN2, Shows Deviated Expression Pattern in Pig Reperfused Infarcted Myocardium and Hypoxic Porcine Cardiac Progenitor Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22031390 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1390

Author(s):

Julia Mester-Tonczar ◽

Patrick Einzinger ◽

Johannes Winkler ◽

Nina Kastner ◽

Andreas Spannbauer ◽

...

Keyword(s):

Myocardial Infarction ◽

Progenitor Cells ◽

Regulatory Networks ◽

Circular Rnas ◽

Cardiac Progenitor Cells ◽

Tissue Samples ◽

Healthy Myocardium ◽

Acute Mi

Circular RNAs (circRNAs) are crucial in gene regulatory networks and disease development, yet circRNA expression in myocardial infarction (MI) is poorly understood. Here, we harvested myocardium samples from domestic pigs 3 days after closed-chest reperfused MI or sham surgery. Cardiac circRNAs were identified by RNA-sequencing of rRNA-depleted RNA from infarcted and healthy myocardium tissue samples. Bioinformatics analysis was performed using the CIRIfull and KNIFE algorithms, and circRNAs identified with both algorithms were subjected to differential expression (DE) analysis and validation by qPCR. Circ-RCAN2 and circ-C12orf29 expressions were significantly downregulated in infarcted tissue compared to healthy pig heart. Sanger sequencing was performed to identify the backsplice junctions of circular transcripts. Finally, we compared the expressions of circ-C12orf29 and circ-RCAN2 between porcine cardiac progenitor cells (pCPCs) that were incubated in a hypoxia chamber for different time periods versus normoxic pCPCs. Circ-C12orf29 did not show significant DE in vitro, whereas circ-RCAN2 exhibited significant ischemia-time-dependent upregulation in hypoxic pCPCs. Overall, our results revealed novel cardiac circRNAs with DE patterns in pCPCs, and in infarcted and healthy myocardium. Circ-RCAN2 exhibited differential regulation by myocardial infarction in vivo and by hypoxia in vitro. These results will improve our understanding of circRNA regulation during acute MI.

Download Full-text

Human Induced Pluripotent Stem Cell‐Derived Neural Progenitor Cells Produce Distinct Neural 3D In Vitro Models Depending on Alginate/Gellan Gum/Laminin Hydrogel Blend Properties

Advanced Healthcare Materials ◽

10.1002/adhm.202100131 ◽

2021 ◽

pp. 2100131

Author(s):

Julia Kapr ◽

Laura Petersilie ◽

Thomas Distler ◽

Ines Lauria ◽

Farina Bendt ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Pluripotent Stem Cell ◽

Neural Progenitor Cells ◽

Induced Pluripotent Stem Cell ◽

Neural Progenitor ◽

In Vitro Models ◽

Gellan Gum ◽

Induced Pluripotent

Download Full-text

Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽

10.1161/circ.132.suppl_3.18845 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Pratik A Lalit ◽

Max R Salick ◽

Daryl O Nelson ◽

Jayne M Squirrell ◽

Christina M Shafer ◽

...

Keyword(s):

Progenitor Cells ◽

Disease Modeling ◽

Ex Vivo ◽

Cardiac Progenitor Cells ◽

Heart Tube ◽

Whole Embryo Culture ◽

Cardiac Actin ◽

Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

Download Full-text

Abstract P014: Oxidative Stress Mediated Regulation of Antioxidants in Cardiac Progenitor Cells

Circulation Research ◽

10.1161/res.109.suppl_1.ap014 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Gokulakrishnan Iyer ◽

Michael E Davis

Keyword(s):

Oxidative Stress ◽

Xanthine Oxidase ◽

Progenitor Cells ◽

Cardiac Progenitor Cells ◽

Dependent Manner ◽

Akt Inhibitor ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Phosphorylated Akt

Cardiac diseases are the leading causes of death throughout the world and transplantation of endogenous myocardial progenitor population with robust cardiovascular lineage differentiation potential is a promising therapeutic strategy. Therefore, in vitro expansion and transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential treatment for severe cardiac dysfunction. However, poor survival and engraftment of cells is one of the major limitations of cell transplantation therapy. Oxidative stress is increased in the ischemic myocardium and indirect inferences suggest the vulnerability of CPCs to oxidative stress. In this study, we show that in vitro, resident c-kit positive CPCs isolated from rat myocardium are significantly (p<0.05) resistant to superoxide-induced apoptosis compared to cardiomyocytes as analyzed by the number of sub-G1 population following xanthine/xanthine oxidase treatment. Interestingly, CPCs have two to four fold higher basal SOD1 and SOD2 activities (p<0.01) compared to cardiomyocytes and endothelial cells. Superoxide treatment increased expression of SOD1 (p<0.01), SOD2 (p<0.01), and glutathione peroxidase (p<0.05) mRNAs within 6 h of treatment compared to control cells. Recent studies suggest the involvement of AKT in controlling cell death, survival and also expression of SOD enzymes. Therefore, we investigated the involvement of AKT in CPCs subjected to oxidative stress. Western blot analysis revealed that the amount of phosphorylated AKT increased significantly within 10 minutes of xanthine/xanthine oxidase treatment. In addition, treatment with LY294002 - a PI3 kinase/AKT inhibitor, increased apoptosis in CPCs treated with superoxide. Our studies demonstrate a novel finding in which resident progenitor cells are protected from oxidative injury by containing higher basal levels of antioxidants as compared to myocytes. Moreover, under oxidant challenge antioxidant levels are regulated, possibly in an AKT-dependent manner. Further elucidation of this pathway may lead to novel therapeutic opportunities.

Download Full-text