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Additional Supporting Information may be found in the online version of this article.

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stem1736-sup-0001-suppfig1.tif356K

Supplemental figure 1. Representative images of cycling NRVMs in different cell cycle phases in CDC co-culture system. Prophase (A, C, white arrow), anaphase (B, D, yellow arrow), telophase (A, B, turquoise arrow), and cytokinesis (D, red arrow).

stem1736-sup-0002-suppfig2.tif166K

Supplemental figure 2. Gating strategy for neonatal BrdU+ cycling cardiomyocytes by flow cytometry.

stem1736-sup-0003-suppfig3.tif343K

Supplemental figure 3. A, Cardiac myocytes were isolated enzymatically by Langendorff perfusion. B, Representative flow cytometry plots for gating strategy of adult cycling cardiomyocytes. CDCs were labeled with far red-fluorescent dye, DiD, before transplantation. DiD-/TnT+ cardiomyocytes were further gated for Ki67 positivity.

stem1736-sup-0004-suppfig4.tif107K

Supplemental figure 4. Anti-β1 integrin antibody blocks the effect of CDCs on NRVM proliferation in admixed direct contact co-culture, but not in conditioned media, by immunocytochemistry. A and B, NRVMs co-cultured with CDCs in admixed direct contact co-culture and in conditioned media in the presence of an anti-β1 integrin antibody or an isotype control (n=4 for each group). Representative images (A) and pooled data (B) of Ki67+ NRVMs are shown (n=4 per group). Arrows point to Ki67+ cardiomyocytes. * indicates p<0.05 when compared to other three groups. Scale bar indicates 50 μm.

stem1736-sup-0005-suppfig5.tif89K

Supplemental figure 5. Anti-β1 integrin antibody blocks the effect of CDCs on NRVM proliferation in admixed direct contact co-culture, not in conditioned media by flow cytometry. A and B, NRVMs co-cultured with CDCs in admixed direct contact co-culture and in conditioned media in the presence of an anti-β1 integrin antibody or an isotype control (n=4 per group). Representative flow cytometry plots (A) and pooled data (B) of BrdU+ NRVMs are shown (n=4 per group). * indicates p<0.05 when compared to other three groups.

stem1736-sup-0006-suppfig6.tif238K

Supplemental figure 6. Beta1 integrin on cardiomyocytes can be effectively blocked by anti-β1 integrin antibody. To block β1 integrin in vivo, mice were pretreated with anti-β1 integrin antibody by intraperitoneal injection every 5 days at a dose of 2 mg/kg. The day after the second injection, LAD ligation was performed. One week after LAD ligation, the heart was harvested for immunohistochemistry. A: Animals were pretreated with isotype control. The heart section was immunostained with no primary antibody, but with a secondary antibody conjugated with Alexa488. The data indicate no non-specific signal from the secondary antibody. B: The same histology sections as in (A) were immunostained with primary antibody against β1 integrin and the Alexa488 secondary antibody. The data confirm the effectiveness of the primary antibody in detecting β1 integrin on the surface of cardiomyocytes. C: Animals were pretreated with anti-β1 integrin antibody prior to CDC injection. Heart sections were immunostained with no primary antibody, but with a secondary antibody conjugated with Alexa488 as in (A). The image illustrates that the pre-injected β1 integrin antibody is effectively bound to the β1 integrin on the surface of cardiomyocytes. Scale bar indicates 50 μm.

stem1736-sup-0007-suppfig7.tif88K

Supplemental figure 7. Prior blockade of β1 integrin in mice decreases pro-proliferative effects of transplanted CDCs in vivo. Representative flow cytometry plots (A) and quantification (B) of Ki67+ cardiomyocytes from sham-operated, vehicle control, mice pretreated with isotype control or anti-β1 integrin antibody at day 7 after MI (n=4 per group). * indicates P<0.05.

stem1736-sup-0008-suppfig8.tif91K

Supplemental figure 8. Ki67+ adult cardiomyocytes are smaller than Ki67- adult cardiomyocytes from SCID mice. A, Representative images of single enzymatically-isolated cardiomyocytes stained for Ki67 (red), α-sarcomeric actinin (green) and DAPI (blue). B, Quantification of cell area of Ki67+ and Ki67- adult cardiomyocytes from SCID mice. * indicates p<0.05. Scale bars indicate 20 μm.

stem1736-sup-0009-suppmovie1.avi35469K

Supplemental movie 1. Representative time-lapse images of human CDCs (red) and NRVMs (green). In top left, top right, and low right corner, NRVMs could be visualized in NRVM-CDC contact and cell-cell contact free dividing patterns. Numbers in top right corner show running hours. Scale bar indicates 100 μm.

stem1736-sup-0010-suppmovie2.avi41500K

Supplemental movie 2. Representative time-lapse images of solitary NRVMs (green). No dividing NRVMs were identified for recording >20 hours. Numbers in top right corner show running hours. Scale bar indicates 100 μm.

stem1736-sup-00011-suppinfo.docx21KSupporting Information

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