Expression of the G-CSF receptor (G-CSFR) in kidneys.

<p>(<b>A</b>) RT-PCR analysis of G-CSFR mRNA expression in kidney tissue. Hypothalamus tissue was used as a positive control, together with a no-template negative control. (<b>B</b>) G-CSFR immunostained via antibody (green, a) and DAPI (blue, b) in glomeruli of a kidney section (magnification x400). G-CSF receptor, G-CSFR; positive control, P; negative control, N.</p

Inhibition of miRNA-23a and miRNA-92a prevents apoptosis of cardiomyocytes.

<p>(A, B) Transfection efficiency of the miRNA-23a inhibitor (A) and miRNA-92a inhibitor (B) was determined by real-time PCR using TaqMan probes. Quantitative analysis of apoptotic cells in cardiomyocytes transfected either with miRNA-23a inhibitor (C) or miRNA-92a inhibiton (D). Apoptotic cells were measured by annexin V staining. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia without miRNA inhibitor. ^&P < 0.05 vs. normoxia with miRNA inhibitor. †P < 0.05 vs. hypoxia without miRNA inhibitor.</p

Experimental design.

<p>Experiment 1: a rat model of diabetic nephropathy (male OLETF rats), Experiment 2: a rat model of diabetic nephropathy with bone marrow transplantation (BMT) (donors: male OLETF rats, recipients: female OLETF rats).</p

Hypoxia-exposed BM-MSC-conditioned media reduces apoptosis upon exposure of cardiomyocytes to hypoxia for 48 h.

<p>(A) Dot plots display the stages of apoptotic death of cardiomyocytes: Annexin−/PI− (Q3), viable cells; Annexin+/PI− (Q4), cells undergoing apoptosis; Annexin+/PI+ (Q2), cells that are in end-stage apoptosis or are already dead; Annexin−/PI+ (Q1), cells that are in necrosis. MSCs media indicates hypoxia-exposed BM-MSC-conditioned media. (B) Quantitative analysis of apoptotic cells (Q2+Q4). Hypoxia-exposed BM-MSC-conditioned media reduces hypoxia-induced miRNA expression in vitro. MiRNA expression was measured by real-time PCR using TaqMan probes. MiRNA-23a (C) and miRNA-92a (D) expression in response to treatment with hypoxia-exposed BM-MSC-conditioned media in comparison without hypoxia-exposed BM-MSC-conditioned media in hypoxia. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia without hypoxia-exposed BM-MSC-conditioned media. ^&P < 0.05 vs. normoxia with hypoxia-exposed BM-MSC-conditioned media. †P < 0.05 vs. hypoxia without hypoxia-exposed BM-MSC-conditioned media.</p

BM-MSC therapy improves fibrosis and apoptosis in a rat model of MI.

<p>(A) Representative images of Masson's trichrome staining of whole heart tissue at 4 weeks after treatment for each group. (B) Representative photomicrographs showing TUNEL assay in the peri-infarct region at 4 weeks after treatment for each group. Scale bar = 50 μm. (C) Results of quantitative analysis of collagen area as ratio of fibrotic area to whole heart area. (D) Results of quantitative analysis of apoptotic cells. Sham, surgical procedure with no induction of MI; Saline, saline treatment after induction of MI; Cell, cell treatment after induction of MI. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. sham control group. †P < 0.05 vs. saline group.</p

Histological changes in the kidney after treatment.

<p>(<b>A</b>) Stained with periodic acid-Shiff (PAS) (magnification x400). (<b>B</b>) Electron micrograph of a glomerulus (magnification x20.000). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>C</b>) Quantitative analysis of images of PAS-stained kidney sections. (<b>D</b>) Quantitative analysis of images of GBM thickness via electron micrographs. (<b>E</b>) Quantitative analysis of foot process width via electron micrographs. All data are expressed as mean±SE. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 4).</p

FISH imaging and immunostained ED-1 in glomeruli of BMT female rats after treatment.

<p>(<b>A</b>) Stained with hematoxylin and eosin (HE) (magnification x400). (<b>B</b>) Higher magnification views of the boxed regions in (A), stained with FISH using a Cy3-labeled Y-chromosome (red, white arrow) and DAPI-labeled nucleus (blue) (magnification x400). (<b>C</b>) Macrophages immunostained with ED-1 antibody (black arrow). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>D</b>) Quantitative analysis of Y-chromosome-positive cells in glomeruli. (<b>E</b>) Quantitative analysis of ED-1-positive cells in glomeruli. Fluorescence <i>in situ</i> hybridization, FISH; 4′–6-Diamidino-2-phenylindole, DAPI; Bone marrow transplantation, BMT. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 3).</p

BM-MSC-conditioned media contains paracrine factors when cells are exposed to hypoxia.

<p>Paracrine factors were measured in the hypoxia-exposed BM-MSC-conditioned media by Magnetic Luminex assay. VEGF (A), MCP-1 (B), IL-6 (C), and ANG (D) expression in response to hypoxia exposure time in comparison with normoxia. N or H indicate normoxia or hypoxia, respectively. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia for 48 h.</p

Levels of metabolic parameters before treatment with G-CSF or saline.

<p>Long-Evans Tokushima Otsuka rats, LETO; Otsuka Long-Evans Tokushima Fatty rats, OLETF; total cholesterol, TC; triglyceride, TG; urine albumin creatinine ratio, UACR. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rat (LETO, <i>n</i> = 4; OLETF, <i>n</i> = 8).</p

Sequences of primers.

<p>Sequences of primers.</p