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First published online May 14, 2007
doi: 10.1242/10.1242/jcs.006122

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Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration

¹ Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan
² Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
³ Departments of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
⁴ Molecular Neuroscience Research Center, Shiga University of Medical Science, Shiga 520-2192, Japan

View larger version (60K): [in this window] [in a new window]   Fig. 1. Clonal isolation and characterization of stem cells in the adult heart. (A,B) Single-cell deposition analysis was performed by the limiting dilution technique. At day 1 of the culture period, wells were inspected for the presence of single cells by phase contrast (A) and GFP fluorescence (B). (C) Colony formation from single cells in 96-well plates at 14 days of culture in serum-free medium. Three-independent colonies derived from single cells are shown. (D) FACS analysis of CSCs. Black line, control IgG; red line, corresponding antibody. Data are representative of three independent clonal CSCs. (E) RT-PCR for CSC clones. The numbers on the right indicate the number of individual colonies that expressed the corresponding genes out of the colonies examined. mES, mouse ES cells used as positive control. Data are representative of three independent clonal CSCs. Bars, 20 µm in A,B; 500 µm in C.

View larger version (37K): [in this window] [in a new window]   Fig. 2. TERT-expressing cells in adult heart are associated with Sca-1 expression. (A) Telomerase activity was measured by the TRAP assay in three independent clonal-CSCs. The cells were treated with or without heat and used as templates. Hela cells were used as positive controls. (B) Construction of EGFP transgene under the control of the TERT promoter. (C) PCR of genomic DNA from 2 independent TERT-EGFP transgenic lines and respective NTG littermate controls. (D) The expression of TERT on EGFP-positive and EGFP-negative cells sorted from TERT-EGFP transgenic hearts is shown by RT-PCR. The TERT expression was detectable in all three independent clonal CSCs shown in Fig. 1C. (E) FACS analysis of the primary EGFP-positive cells isolated from TERT-EGFP mice (TG). Expression of Sca-1, KIT, CD45, CD34, and CD31 in EGFP-positive cells was examined. Cells from NTG littermates were used as negative control. Data are representative of six independent experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Generation of Sca-1 KD mice. (A) Construction of the pDECAP-Sca-1 vector. (B,C) Decrease in exogenous Sca-1 expression induced by pDECAP-Sca-1 as shown by RT-PCR (B) and FACS (C) in HEK 293 cells. The total amount (8 µg) of plasmids co-transfected was the same in each experiment (n=3). (D) Decrease in Sca-1 protein levels in the hearts from two independent Sca-1 KD lines. (E) The frequency of CSC colonies from NTG- and Sca-1 KD hearts is shown. Data are expressed as the mean number of colonies formed per 105 single cells deposited ± s.e. (n=4). *P<0.01 vs NTG. (F) RT-PCR for embryonic and mesodermal precursor markers. Cardiac fibroblasts were used as negative control. (G) Decrease in Sca-1 expression from two independent Sca-1 KD CSCs clones. Black line, control IgG; red line, Sca-1. (H) Phase-contrast images of respective CSC clones at 14 days of culture in serum-free medium. Bars, 500 µm. (I) Growth kinetics of two independent clonal CSCs isolated from NTG (black lines, C4 and C6) and Sca-1 KD (red lines, C1 and C2) mice. (J) BrdU incorporation, phosphorylated histone-H3 (p-H3) and p53 expression from five independent experiments are shown. *P<0.01 vs NTG. (K) Loss of telomerase activity in the clonal CSCs (C1 and C2) isolated from two independent lines of Sca-1 KD mice.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Sca-1-mediated signaling activates Akt to support CSC proliferation and survival. (A) Representative photographs of TUNEL assay obtained from NTG- or Sca-1 KD-derived CSCs treated with 200 µM H2O2 for 18 hours. The numbers of apoptotic cells (brown nuclei) in NTG (C6)- or Sca-1 KD-CSCs (C1) are shown (n=8). *P<0.01 versus NTG. (B) Loss of Sca-1 diminished EGF and bFGF-induced Akt activation in CSCs. CSCs treated with 200 µm H2O2 for 15 minutes were used as positive controls. Bars, 50 µm in A.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Loss of Sca-1 transcripts does not affect the differentiation potential of clonal CSCs. (A) Differentiation of NTG-derived or Sca-1 KD-derived clonal CSCs. Cardiac muscle cell (cardiac troponin-T), endothelial cell (CD31) and vascular smooth muscle cell (-SMA) differentiation were 1.24±0.3%, 12.4±1.8% and 31.9±2.5%, respectively, for NTG-CSCs (C2, C3 and C6, respectively), and 1.23±0.3%, 12.1±2.1% and 32.2±4.7%, respectively, for Sca-1 KD-CSCs (C1). Nuclei are stained by DAPI (blue). Bars, 20 µm in A. (B) RT-PCR showed that the differentiation potential into the three different lineages were similar for both types of CSCs (n=3). (C) Representative Ca2+ transient in beating cardiomyocytes. Clone 2, 3 and 6 from NTG and clone 1 from Sca-1 KD mice, all expressed brachyury at baseline, were used for analysis. Intensities were corrected by background amplitude and expressed as arbitrary units (n=3).

View larger version (87K): [in this window] [in a new window]   Fig. 6. Sca-1 KD CSC transplantation fails to prevent cardiac remodeling after myocardial infarction. (A,B) WT mice received transplantation of either NTG- or Sca-1 KD mice-derived CSCs 1 hour after infarction. Cardiac MRI was performed 4 weeks after CSC transplantation (n=8). Arrowheads indicate akinetic regions. White bars, sham-operated. Myocardial infarction with PBS injection (light gray bars), NTG-CSC (C6, dark gray bars) or Sca-1 KD-CSC (C1, black bars) transplantation are shown. *P<0.05 vs PBS; P<0.05 versus NTG-CSC; P<0.01 versus PBS injection. EF, ejection fraction; ESV, end-systolic volume; EDV, end-diastolic volume; IS, infarcted size.

View larger version (71K): [in this window] [in a new window]   Fig. 7. Sca-1 transcripts are required for CSC proliferation and survival in vivo. (A) Immunohistochemistry of transplanted lacZ+ cells 3 days after infarction. Transplanted lacZ+ cells entering cell cycle were detected as Ki67-positive cells (arrowheads) (n=6). Myocardial infarction transplanted with NTG-CSCs (C6) and Sca-1 KD CSCs (C1) are shown. *P<0.01 versus NTG. (B) Apoptotic features (arrowheads, brown nuclei) of lacZ+ engrafted cells are shown at day 3 after NTG- or Sca-1 KD-CSC transplantation. *P<0.01 versus NTG-CSC transplantation (n=6). Bars, 50 µm in A,B.

View larger version (80K): [in this window] [in a new window]   Fig. 8. Loss of Sca-1 transcripts in clonal CSC transplantation shows less donor-cell engraftment, resulting in the decrease in late cardiovascular regeneration. (A) Engrafted lacZ+ cells in NTG (C6)- and Sca-1 KD-CSC (C1)-transplanted hearts at day 7 after infarction. Sections were counterstained using H&E. (B-D) The representative images and frequencies of cardiomyocytes (cardiac troponin-T, red), and endothelial (CD31) and smooth muscle cells (-SMA) in lacZ+ cells at day 28 are shown (n=6). Note that differentiated lacZ+ cardiomyocytes co-express connexin-43 (yellow). Bars, 100 µm in A; 20 µm in B; 50 µm in C,D.

View larger version (70K): [in this window] [in a new window]   Fig. 9. Transplantation of Sca-1 KD CSCs fails to prevent myocardial apoptosis and limits neoangiogenesis after myocardial infarction partially due to the failure of paracrine effector secretion. (A) TUNEL staining revealed the apoptotic cardiomyocytes in the border zone of PBS-treated, NTG (C6)- and Sca-1 KD-CSC (C1)-transplanted hearts at day 3; (n=6). *P<0.01 vs PBS. P<0.01 vs NTG. (B) Capillary density of infarcted border zone of transplanted hearts at day 14 after myocardial infarction. Capillary density was measured by staining of CD31 (brown) and corrected by the area analyzed. (n=8). *P<0.01 vs PBS-treated mice; P<0.01 vs NTG-CSC-transplanted mice. Bars, 50 µm in A,B. (C) Relative mRNA expression levels of VEGF, HGF, and IGF1 normalized by 18S expression in NTG- and Sca-1 KD-CSCs under normoxic and hypoxic conditions (n=7). *P<0.01 vs NTG-CSCs under normoxia; P<0.01 vs NTG-CSCs under hypoxia.