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sc-12-0465_sm_SupplFigure1.pdf23KSupplemental Figure S1. Fluorescence-activated cell sorting (FACS) of human muscle pericytes. Human muscle biopsies were mechanically dissociated and digested with collagenase. After labeling, single cells suspension was subjected to FACS to purify the pericyte population based on their differential expression of cell lineage markers, including robust expression of the pericyte/endothelial cell marker CD146 and absence of the myogenic, endothelial/stem, and hematopoietic cell markers: CD56, CD34 and CD45, respectively. Skeletal muscle-derived pericytes (CD146+/34-/45-/56-) sorted to homogeny were collected and expanded in vitro.
sc-12-0465_sm_SupplFigure2A.pdf62KSupplemental Figure S2. (A) Morphology of purified pericytes in long-term culture. Flow cytometry analysis revealed that cultured pericytes retain original cell surface marker expression, including the robust expression of CD146 and alkaline phosphatase (ALP) with the absence of CD34, CD45, and CD56. (B) Flow cytometry analysis showed that long-term cultured pericytes strongly express classic MSC markers: CD90, CD44, CD73 and CD105.
sc-12-0465_sm_SupplFigure2B.pdf39KSupplemental Figure S2. (A) Morphology of purified pericytes in long-term culture. Flow cytometry analysis revealed that cultured pericytes retain original cell surface marker expression, including the robust expression of CD146 and alkaline phosphatase (ALP) with the absence of CD34, CD45, and CD56. (B) Flow cytometry analysis showed that long-term cultured pericytes strongly express classic MSC markers: CD90, CD44, CD73 and CD105.
sc-12-0465_sm_SupplFigure3.tif2672KSupplemental Figure S3. Representative echocardiographic M-mode images of left ventricle (LV). End-systolic dimension (ESD) and end-diastolic dimension (EDD) were indicated.
sc-12-0465_sm_SupplFigure4.pdf40KSupplemental Figure S4. Echocardiographic measurement of cardiac function in acutely infarcted hearts injected with either APs (CD146) or CD56+ myogenic progenitors (CD56) at 2 weeks post-infarction. APs and CD56+ myogenic progenitors were simultaneously sorted from a single adult muscle biopsy. Pericyte-treated hearts (N=6) exhibited significantly better LV function than CD56+ progenitor-treated ones (N=6) in multiple categories, including LVEDA (p=0.004), LVESA (p=0.002), LVFS (p=0.003), LVFAC (p=0.003), and LVEF (p≤0.001).
sc-12-0465_sm_SupplFigure5.pdf50KSupplemental Figure S5. Detection of host EC proliferation at 2 weeks post-infarction. Representative images of Ki-67 (green) and mouse CD31 (red) co-immunostaining (A) within the infarct region and (B) in the peri-infarct area of hearts injected with pericytes or PBS (scale bars=50μm). Proliferating host ECs were identified as Ki-67/CD31 dual-positive cells (green/red arrows). (C) Pericyte-treated group had a significantly larger number of proliferating ECs both within the infarct region (p=0.034) and in the peri-infarct area (p=0.025) than PBS control group (N=3 per group).
sc-12-0465_sm_SupplFigure6.pdf358KSupplemental Figure S6. (A-D) Donor pericytes (stained in red by anti-GFP) were particularly abundant in the peri-infarct area where host CD31(+) capillaries (mouse CD31 stained in green) remained largely intact (scale bar=50μm).
sc-12-0465_sm_SupplFigure7.tif2320KSupplemental Figure S7. Expression of the pericyte marker NG2 (chondroitin sulphate) was detected in the majority of, but not all, GFP-positive donor pericytes at 2 weeks post-infarction (scale bar=10μm).
sc-12-0465_sm_SupplFigure8-1.pdf273KSupplemental Figure S8. Tracking cardiac cell lineage fates of donor pericytes. Confocal microscopy revealed that in the peri-infarct area (A-C) a minor fraction of GFP-labeled pericytes co-expressed a mature cardiomyocyte marker, cardiac troponin-I (cTn-I), while additional GFP(+) cells remained in the interstitium ([A], main, scale bar=50μm); a few of them appear single-nucleated ([A], inset, scale bar=10μm) (cTn-I in red, Anti-GFP in green). (D-F) Immunofluorescent detection of GFP-cTn-I dual-positive cardiomyocytes integrating within the residual myocardium (scale bar=50μm) with dotted area enlarged in (G-I) showing sarcomeric patterns (scale bar=20μm). (J-L) A very small number of GFP(+) pericytes (<1%) co-expressed human-specific CD31 (hCD31 in red, Anti-GFP in green; scale bar=10μm). (M-O) Few donor pericytes (<0.5%) expressed human-specific SM-MHC (hSM-MHC in red, Anti-GFP in green; scale bar=10μm). (P-R) Negative control images were taken from sections immunostained only with matching fluorescence-conjugated secondary antibodies (no primary antibody) (Cy3 in red, AlexaTM488 in green; scale bar=10μm).
sc-12-0465_sm_SupplFigure8-2.pdf301KSupplemental Figure S8. Tracking cardiac cell lineage fates of donor pericytes. Confocal microscopy revealed that in the peri-infarct area (A-C) a minor fraction of GFP-labeled pericytes co-expressed a mature cardiomyocyte marker, cardiac troponin-I (cTn-I), while additional GFP(+) cells remained in the interstitium ([A], main, scale bar=50μm); a few of them appear single-nucleated ([A], inset, scale bar=10μm) (cTn-I in red, Anti-GFP in green). (D-F) Immunofluorescent detection of GFP-cTn-I dual-positive cardiomyocytes integrating within the residual myocardium (scale bar=50μm) with dotted area enlarged in (G-I) showing sarcomeric patterns (scale bar=20μm). (J-L) A very small number of GFP(+) pericytes (<1%) co-expressed human-specific CD31 (hCD31 in red, Anti-GFP in green; scale bar=10μm). (M-O) Few donor pericytes (<0.5%) expressed human-specific SM-MHC (hSM-MHC in red, Anti-GFP in green; scale bar=10μm). (P-R) Negative control images were taken from sections immunostained only with matching fluorescence-conjugated secondary antibodies (no primary antibody) (Cy3 in red, AlexaTM488 in green; scale bar=10μm).
sc-12-0465_sm_SupplFigure9.pdf19KSupplemental Figure S9. Comparison of total and proliferating cell density within the infarct area at 2 weeks post-infarction. (A) Pericyte-injected hearts had a significantly less number of cells within the infarct area than the PBS-injected controls (p<0.05, N=5 per group). (B) A cell proliferation marker, Ki-67, was used to detect proliferating cells within the infarct area. No statistical difference in Ki-67(+) proliferating cell density was observed between pericyte- and PBS-injected hearts (p=0.808, N=3 per group).
sc-12-0465_sm_SupplFigure10.pdf27KSupplemental Figure S10. (A) Terminal dUPT nick end-labeling (TUNEL) staining revealed the apoptotic cells within the infarct region of pericyte- or PBS-injected hearts (scale bars=100μm). (B) Quantification of the apoptotic cell number (N=5 per group) showed no statistical difference between pericyte treatment (12.55±1.87 cells/mm2) and PBS injection (9.01±2.55 cells/mm2) at 2 weeks post-infarction (p=0.296).
sc-12-0465_sm_SupplTable1.pdf49KSupplementary Table 1
sc-12-0465_sm_SupplTable2.pdf56KSupplementary Table 2
sc-12-0465_sm_SupplTable3.pdf9KSupplementary Table 3
sc-12-0465_sm_Supplinfor.pdf172KSupplementary Data

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