Importance of Cell-Cell Contact in the Therapeutic Benefits of Cardiosphere-Derived Cells
Version of Record online: 18 AUG 2014
© 2014 AlphaMed Press
Volume 32, Issue 9, pages 2397–2406, September 2014
How to Cite
Xie, Y., Ibrahim, A., Cheng, K., Wu, Z., Liang, W., Malliaras, K., Sun, B., Liu, W., Shen, D., Cheol Cho, H., Li, T., Lu, L., Lu, G. and Marbán, E. (2014), Importance of Cell-Cell Contact in the Therapeutic Benefits of Cardiosphere-Derived Cells. STEM CELLS, 32: 2397–2406. doi: 10.1002/stem.1736
- Issue online: 18 AUG 2014
- Version of Record online: 18 AUG 2014
- Accepted manuscript online: 6 MAY 2014 01:01PM EST
- Manuscript Accepted: 4 APR 2014
- Manuscript Revised: 19 MAR 2014
- Manuscript Received: 21 JUN 2013
- National Institutes of Health . Grant Number: R01HL083109
- National Natural Science Foundation of China . Grant Number: 81170122
- American Heart Association Beginning . Grant Number: 12BGIA12040477
Additional Supporting Information may be found in the online version of this article.
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).
Supplemental figure 2. Gating strategy for neonatal BrdU+ cycling cardiomyocytes by flow cytometry.
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.
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.
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.
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.
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.
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.
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.
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.
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