Article Figures & Data
Figures
- Figure 1
Adipose-derived stem cells (ADSCs) express Nkx2-5 in response to ISX1. (A) Increased Nkx2-5 gene expression in ADSCs treated for 4 days with ISX1 compared with vehicle (DMSO). Data include n=4–5 in each group and are presented as mean±SEM, *p<0.05. (B) Immunocytochemistry of nuclear Nkx2–5 protein in vehicle-treated and ISX1-treated ADSCs. Nkx2.5 (red) and Hoechst 33342 dye (blue). Bar=40 µm.
- Figure 2
ISX1 augments cardiac gene expression in adipose-derived stem cells (ADSCs) co-cultured with neonatal cardiomyocytes (NRVMs). Increased cardiac gene expression (ANF, BNP, Troponin C) in cells co-cultured with NRVMs for 3 weeks and exposed to ISX1 compared with vehicle (DMSO). Data include n=4–7 in each group and are presented as mean±SEM, *p<0.05.
- Figure 3
Adipose-derived stem cells (ADSCs) manifest diminished capacity to undergo adipogenesis, osteogenesis, and chondrogenesis in response to ISX1. (A) Representative images of Oil Red O staining in ADSCs differentiated (3 weeks) into mature adipocytes and treated with vehicle (DMSO) or ISX1. Bar=20 µm. Black arrows indicate Oil Red O lipid droplets. (B) Quantification of eluted Oil Red O stain. (C) Representative images of Alizarin Red staining in ADSCs differentiated (3 weeks) into mature osteoblasts and treated with vehicle (DMSO) or ISX1. Bar=40 µm. (D) Quantification of eluted Alizarin Red stain. (E) Representative images of von Kossa staining in ADSCs differentiated (3 weeks) into mature osteoblasts and treated with vehicle (DMSO) or ISX1. Bar=40 µm. (F) Quantification of sulfated glyosaminoglycans after 3 weeks in chondrogenesis differentiation medium and exposed to vehicle (DMSO) or ISX1. Data include n=3–4 per group and are presented as mean±SEM (*p<0.05).
- Figure 4
ISX1 increased histone acetyltransferase (HAT) activity but not histone deacetylase (HDAC) activity in adipose-derived stem cells (ADSCs), and HDAC inhibition alone is insufficient to induce Nkx2-5 gene expression. (A) HAT activity of nuclear extracts of ADSCs treated with ISX1 or vehicle (DMSO). Data include n=4 per group and are presented as mean±SEM (p<0.05). (B) HDAC activity in nuclear extracts of ADSCs treated with ISX1 or vehicle (DMSO). Data include n=3 per group (NS, non-significant). (C) Nkx2.5 gene expression after addition of valproic acid (VPA) alone or in combination with ISX1. Data include n=4–8 in each group. (D) Nkx2.5 gene expression after addition of trichostatin A (TSA) alone or in combination with ISX1. Data include n=5–7 in each group.
- Figure 5
ISX1 increased histone 3 and 4 acetylation and histone 4 lysine 8 acetylation. (A) Representative western blot illustrating increased acetylation of histone 3 lysine 9 (H3K9) in response to ISX1. (B) Quantification of acetylated H3K9 compared with total histone 3. Data include n=3 in each group. Data are presented as mean±SEM (*p<0.05). (C) Representative western blot illustrating increased acetylation of histone 4 lysine 8 (H4K8) in response to ISX1. (D) Quantification of acetylated H4K8 compared to total histone 4. Data include n=3 per group.
- Figure 6
Transplantation of adipose-derived stem cells (ADSCs) pretreated with ISX1 in myocardial infarction (MI)-injured hearts leads to improved functional recovery. (A) Representative M-mode echocardiograms. (B) Ventricular systolic function depicted as percent fractional shortening (%FS) in sham-operated mice, in hearts treated with cell diluent (phosphate-buffered saline, PBS), or in hearts transplanted with ADSCs treated with vehicle (DMSO) or ISX1. (C) Left ventricular end-diastolic dimension (LVEDD) in millimeters. (D) Left ventricular end-systolic dimension (LVESD) in millimeters. Data include n=10 in the sham-operated control group, n=12 in diluent of the cells-treated group, n=15 in (PBS), n=15 in the vehicle-treated ADSC-transplanted group, n=13 in the ISX1-treated ADSC-transplanted group. Data are presented as mean±SEM. *p<0.05, Sham versus diluent, vehicle-treated cells, ISX1-treated cells, **p<0.05, ISX1-treated cells versus diluent, vehicle-treated cells.
- Figure 7
Enhanced neovascularization after adipose-derived stem cells (ADSC) transplantation but no difference in the original infarct size. (A) Percent infarct size between all groups (myocardial infarction (MI)+cell diluent (phosphate-buffered saline, PBS), MI+vehicle-treated cells, MI+ISX1-treated cells) as assessed by TTC (2,3,5-triphenyltetrazolium chloride) staining (n=5–6). (B) Representative images of isolectin B4 staining (green) of blood vessels. Bar=200 µm. (C) Quantification of vessel abundance normalized to microscopic field in hearts transplanted with ADSCs treated with cell diluent (PBS), vehicle (DMSO), or ISX1. Data are presented as mean±SEM; (n=7–11); *p<0.05.
- Figure 8
Transplanted GFP+ adipose-derived stem cells (ADSCs) pretreated with ISX1 detected in hearts 3 weeks postmyocardial infarction (MI) and co-localized with cardiac myocytes. (A) Representative images of heart sections immunostained for green fluorescent protein (GFP) (red) and Troponin T (green). Bar=40 µm. (B) Confocal images of hearts transplanted with ISX1-treated ADSCs. GFP (red), Troponin T (green), overlap (yellow). Bar=20 µm.
Tables
- Table 1
Cell marker expression
SVF (%) P0 (%) CD29 79 44 CD34 52 17 Sca1 60 38 CD24 40 7 CD105 30 17 CD117 2 0 CD44 0 2 CD90 9 0 CD73 36 7 CD166 15 0 CD31 2 0 CD45 0 0 CD11b 14 1 CD14 26 0 Ter119 0 0 Cell surface marker expression on freshly isolated stromal vascular fraction (SVF) and cells cultured for 4 days (P0). Both SVF and P0 cells expressed no endothelial cell or inflammatory cell markers but rather expressed markers of adipose progenitor cells, endothelial progenitor cells, and mesenchymal stem cells.
- Table 2
PCR primers and antibodies used in analyses
Real-time PCR TaqMan primers used in gene expression analyses. Antibodies and isotype control antibodies used to detect cell surface markers by flow cytometry TaqMan Nkx2–5 primers Mm00657783_m1 TaqMan Nppa primers (ANF) Mm01255748_g1 TaqMan Nppb primers (BNP) Mm 01255770_g1 TaqMan TnnC primers (Troponin C) Mm00437111_m1 List of antibodies used for flow cytometry Flow cytometry antibody Isotype control PE anti-mouse CD29 PE Armenian Hamster IgG eFluor660 CD34 eFluor660 rat IgG2a, к PE-Cy7 anti-mouse Ly-6A/E (Sca1) PE-Cy7 rat IgG2a, к PE anti-mouse CD24 PE rat IgG2c, к PE anti-mouse CD105 PE rat IgG2a, к PE-Cy7 anti-mouse CD117 (c-Kit) PE-Cy7 rat IgG2a, к PE-Cy7 anti-mouse CD44 PE-Cy7 rat IgG2b, к PE anti-mouse CD90.1 (Thy1.1) PE mouse IgG2a, к PE anti-mouse CD73 PE rat IgG1 PE anti-mouse CD166 (ALCAM) PE rat IgG2a, к PE-Cy7 anti-mouse CD31 PE-Cy7 rat IgG2a, к PerCP-Cy5.5 anti-mouse CD45 PerCP-Cy5.5 rat IgG2b, к PE-Cy7 anti-mouse CD11b PE-Cy7 rat IgG2b, к PE anti-mouse CD14 PE rat IgG2a, к PerCp-Cy5.5 anti-mouseTer119(Ly-76) PerCP-Cy5.5 rat IgG2b, к
