Isolation and characterization of mesenchymal stem cells from human fetus heart

<div><p>Background</p><p>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).</p><p>Methods</p><p>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.</p><p>Results</p><p>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-β and IL-10.</p><p>Conclusions</p><p>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.</p></div

Differentiation of hfC-MSCs into cardiovascular cells.

<p>Representative immunocytochemistry images (40X, 20μm) showing differentiation of human fetal cardiac stem cells (hfC-MSCs) into Cardiomyocytes (B: Troponin-T (cTnT); Endothelial cells (D: CD31); Smooth Muscle Cells (F: Smooth Muscle- myosin heavy chain (SM-MHC); (A, C and E, were control cells without induction medium showing only hoechst dye). Data shown are from three independent experiments at passage 3–5.</p

Morphology and Growth Kinetics of hfC-MSCs compared to hBM-MSCs.

<p>Representative photomicrographs (10X, 20μm) of (A) Human fetal cardiac mesenchymal stem cells (hfC-MSCs); (B) Bone marrow mesenchymal stem cells (hBM-MSCs) showing spindle shaped morphology at 5th passage; (C) Growth kinetics of hfC-MSCs and hBM-MSCs seeded at a density of 1,000 cells per cm².</p

hfC-MSCs inhibit PHA-induced proliferation of lymphocytes.

<p><b>(A)</b> PBMCs (1 × 10⁵ cells) stimulated with or without PHA (5 μg/ mL) in the presence or absence of irradiated hfC-MSCs (1 × 104–5 × 10⁴ cells). Data are expressed as the mean ± SE of three independent experiments. *p<0.05 (Control PBMCs vs. PBMCs+ MSCs), NS: Not significant. <b>(B)</b> TGF-β levels were analyzed in the culture supernatants of co-cultured hfC-MSCs and PBMCs stimulated with or without PHA. PBMCs (1 × 10⁵ cells) cultured with PHA (5 μg/mL) in the presence or absence of hfC-MSCs (1 × 104–5× 10⁴ cells). Data are expressed as the mean ± SE of three independent experiments. *p<0.05 (Control PBMC vs. PBMC+ MSC), NS: Not significant. <b>(C)</b> IL-10 levels were analysed in the culture supernatants of co-cultured hfC-MSCs and PBMCs stimulated with or without PHA. PBMCs (1 × 10⁵ cells) cultured with PHA (5 μg/mL) in the presence or absence of hfC-MSCs (1 × 104–5× 10⁴ cells). Data are expressed as mean ± SE of three independent experiments. *p<0.05 (Control PBMCs vs. PBMCs+ MSCs), NS: Not significant.</p

Phenotypic characteristics of hfC-MSCs compared to BM-MSCs.

<p>Representative bar graphs showing a comparison of the expression of CD73, CD90, CD105, SSEA-4, CD117, CD34, CD45, HLA-DR and CD31 on hfC-MSCs and hBM-MSCs as demonstrated by flow cytometry. Values are mean ± SE of three independent experiments of both the cell types at passage-5.</p

Primers used in this study.

<p>Primers used in this study.</p