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Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

ABSTRACT: RATIONALE:The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. OBJECTIVE:Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. METHODS AND RESULTS:Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit+ cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit- mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit+ population is further enriched by selection for a CD133+ endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. CONCLUSIONS:Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.

SUBMITTER: Monsanto MM

PROVIDER: S-EPMC5555597 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

Monsanto Megan M MM White Kevin S KS Kim Taeyong T Wang Bingyan J BJ Fisher Kristina K Ilves Kelli K Khalafalla Farid G FG Casillas Alexandria A Broughton Kathleen K Mohsin Sadia S Dembitsky Walter P WP Sussman Mark A MA

Circulation research 20170426 2

<h4>Rationale</h4>The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration.<h4>Objective</h4>Establish a robust cardiac stem cell isolation and culture protocol to consistently generat ...[more]

PMID: 28446444

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Project description:Mesenchymal stem cells (MSCs) are promising cells for cardiovascular regenerative medicine. However, their potential may be limited, because of their restricted cardiovascular differentiation potential and decline in their number and functional characteristics with increasing donor age. We have previously shown that rat fetus heart harbors primitive MSCs and administration of these cells improved left ventricular (LV) function after ischemia/reperfusion injury in rats. To evaluate their potential as a new cell type for clinical cardiovascular cell therapy, we have undertaken this study on the isolation and characterization of human fetal cardiac MSCs (hfC-MSCs).MSCs were isolated from the heart of five 14-16-week-old aborted human fetuses and studied for their growth characteristics, karyotypic stability and senescence over successive passages, expression of mesenchymal and embryonal markers by flow cytometry and immunocytochemistry, constitutive expression of cardiovascular genes by RT-PCR, differentiation into cells of the cardiovascular lineage and their immunomodulatory properties.The hfC-MSCs grew as adherent monolayer with spindle shaped morphology and exhibited rapid proliferation with an average population doubling time of 34 hours and expansion to up to more than 80 population doublings with maintenance of a normal karyotype and without senescence. Immunophenotyping showed that they had similar phenotype as human bone marrow mesenchymal stem cells (hBM-MSCs) expressing CD73, CD90, CD105 and lacking expression of CD31, CD34, CD45, HLA-DR. However, hfC-MSCs expressed significantly higher levels of CD117 and SSEA-4 compared to hBM-MSCs. In addition, hfC-MSCs expressed the embryonal markers Oct-4, Nanog and Sox-2 as compared to hBM-MSCs. Further, hfC-MSCs had significantly higher expression of the cardiovascular genes viz. ISL-1, flk-1, GATA-4, NKX2.5 and MDR-1 as compared to hBM-MSCs, and could be differentiated into major cardiovascular cells (cardiomyocytes, endothelial cells, smooth muscle cells). Interestingly, hfC-MSCs markedly reduced T-lymphocyte proliferation with an increased secretion of TGF-? and IL-10.Our results show that human fetus heart is a novel source of primitive MSCs with cardiovascular commitment which may have a potential therapeutic application in cardiovascular regenerative medicine.

Project description:While the adult human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells, the cellular composition during early development remains largely unknown. Reliable identification of fetal cardiac cell types using protein markers is critical to understand cardiac development and delineate the cellular composition of the developing human heart. This is the first study to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and cardiac troponin I (cTnI), fibroblasts (DDR2, THY1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Two distinct populations of cTnI positive cells were identified through flow cytometry, with MHC positive cardiomyocytes showing high cTnI expression (cTnIHigh) while MHC negative non-myocytes showed lower cTnI expression (cTnILow). cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin (VIM) was expressed in cultured ventricular fibroblast populations and flow cytometry revealed VIMHigh and VIMLow cell populations in the fetal heart. MHC positive cardiomyocytes were VIMLow whilst CD31 positive endothelial cells were VIMHigh. Using markers investigated within this study, we characterised fetal human cardiac populations and estimate that 75-80% of fetal cardiac cells are cardiomyocytes and are MHC+/cTnIHigh/VIMLow, whilst non-myocytes comprise 20-25% of total cells and are MHC-/cTnILow/VIMHigh, with CD31+ endothelial cells comprising ~9% of this population. These findings show distinct differences from those reported for adult heart.

Dataset Information

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

Publications

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

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