Effects of fetal kidney cells on morphological structure, proliferation and apoptosis of tubular epithelial cells in rats with IR ARF.

<p>(A) Kidney section of sham operated animal showing normal architecture of tubules and glomeruli. (B) Kidney section of saline treated animal showing dilated distal convoluted tubule (solid arrow), swollen and necrotic epithelial cells with nuclear changes in the most proximal convoluted tubule (open arrow) and epithelial or hyaline cast material in lumen (solid arrow head). (C) Kidney section of fetal kidney cells treated animal showing signs of recovery as revealed by mild tubular dilatation (open arrow), desquamation of few proximal convoluted tubules and preservation of the integrity of the cellular structure (solid arrow) (20 X). (D) Jablonski grading score of tubular necrosis in saline and fetal kidney cells treated kidneys after 72 hours of fetal kidney cells therapy. (E-G) Representative immunofluorescence photomicrographs (40X) of PCNA staining of kidney sections of sham operated (E), saline treated (F) and fetal kidney cells treated (G) animals. (H) Quantification of PCNA positive cells per HPF. (I-K) Representative immunofluorescence photomicrographs (40X) of TUNEL staining of kidney sections of sham operated (I), saline treated (J) and fetal kidney cells treated (K) animals. (L) Quantification of apoptotic cells per HPF. Values expressed Mean±SEM. (n = 6), *p<0.05 vs. sham operated group, #p<0.05 vs. saline treated group.</p

Effects of fetal kidney cells on renal functions in rats with IR ARF.

<p>The difference in (A) BUN, (B) serum creatinine and (C) serum NGAL levels in sham operated, saline treated and fetal kidney cells treated groups at different time points (0, 24, 48, 72 and 96 hours after reperfusion). Values expressed Mean±SEM (n = 6). *p<0.05 vs. sham operated group, #p<0.05 vs. saline treated group.</p

<i>In vivo</i> tracking of PKH26 positive cells in IR induced damaged kidney.

<p>Representative immunoflourescence photomicrographs (40X) of fetal kidney cells treated kidney showing (A) CK 19, green (B) Hoechst, blue (C) PKH26 labeled cells, red, located in the interstitial spaces and peri-tubular areas of the kidney (D) Overlay of images of (A), (B) and (C). (E) Overlay of images of (A) and (B). (F) Overlay of images of (B) and (C).</p

List of primers used for RQ-PCR analysis.

<p>List of primers used for RQ-PCR analysis.</p

Effects of fetal kidney cells therapy on expression of growth factors and pro- and anti-inflammatory cytokines in rat kidney.

<p>(A-I) mRNA expression levels of growth factors viz. bFGF (A), BMP-7 (B), VEGF-A (C) and IGF-1(D), inflammatory cytokines viz. IL-1β (E), TNF-α (F), IFN-γ (G) and IL-6 (H), anti-inflammatory cytokine IL-10 (I). (J) Representative immunoblots showing the expression levels of inflammatory markers viz. NF-kB and ICAM-1 in the kidney tissues of sham operated, saline treated and fetal kidney cells treated groups. (K-L) Bar diagrams showing semi quantitative densitometry of the expression of NFκB and ICAM-1. Comparative gene expression ratio alculated by referring each gene to β-actin as an internal control. Densitometric analysis applied for comparison of relative protein expression and represented in densitometric arbitrary units (a. u.). Values expressed Mean±SEM. *p<0.05 vs. sham operated group. #p<0.05 vs. saline treated group.</p

Morphology, karyotype and phenotypic characterization of fetal kidney cells.

<p>(A) Representative photomicrograph (10X) of fetal kidney cells in culture, showing spindle-shape and polygonal morphology. (B) The fetal kidney cells showing normal karyotype at 3rd passage (10X). (C) Flow cytometric analysis of fetal kidney cells showing expression of surface markers CD29, CD44, CD73, CD90, CD105, CD24, CD133, VEGFR2, EpCAM, CD45 and MHC class II (green or red lines, detected with FITC- or PE- conjugated antibodies, respectively) with isotype controls (black lines).</p

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

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

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