Abstract 16688: Human Cardiosphere-Derived Cells Stimulate Cardiomyocyte Proliferation in vivo and in Co-Culture

Abstract

Background: Cardiosphere-derived cells (CDCs) are effective in regeneration after myocardial infarction (MI); however, direct differentiation of transplanted cells only accounts for a minuscule fraction of the benefit. Methods and Results: SCID mice underwent LAD ligation and were injected with 10 5 human CDCs or vehicle. Two weeks later, hearts were explanted for myocyte isolation by Langendorff perfusion, or for tissue analysis. Immunohistochemistry revealed decreased numbers of apoptotic cells in the MI border zone of CDC-treated animals vs. controls (14.2 ± 2.9% vs. 32.8 ± 5.2% TUNEL+ cells; n=3; P<0.01). Myocytes isolated from CDC-treated hearts demonstrated increased numbers of cycling cardiomyocytes (1.3 ± 0.1% vs. 0.6 ± 0.4% α-sarcomericactinin/Ki67 double positive cells; n=3; P<0.05). To probe the mechanism of the increase in cycling (Ki67+) myocytes, we co-cultured human CDCs or dermal fibroblasts (DFs) with neonatal rat cardiomyocytes (NRCMs). After 3 days, Ki67+ NRCMs were increased in the CDC co-cultures (22.8 ± 0.8% vs. 9.5 ± 4.7% in DF cocultures; n=3; P=0.004). Cycling NRCMs were smaller than non-cycling NRCMs (cross-sectional area 2.3 ± 1.3E+09 vs. 3.4 ± 1.5E+09 nm 2 ; n=11; P<0.05), and the number of beating NRCM clusters was higher in CDC co-cultures (56.5 ± 15.7% vs. 28.0 ± 13.0%; n=3; P<0.05). To investigate paracrine effects, we cultured CDCs (or DFs) and NRCMs in a transwell contact-free system. After 3 days of contact-free co-culture, there were more Ki67+ NRCMs in the CDC group (15.6 ± 2.6% vs. 11.5 ± 1.5% in DF controls; n=3; P<0.05), while cycling NRCMs were again smaller (P<0.01) than non-cycling NRCMs. However, the percentage of cycling NRCMs was less than that in mixed co-cultures (15.6 ± 2.6% vs. 22.8 ± 0.8%; n=3; P<0.01). Conclusions: CDCs decrease apoptosis and increase the number of cycling myocytes after MI in vivo . CDCs stimulate cardiomyocyte proliferation in vitro ; the enhancement of proliferation is greater in mixed co-culture than with conditioned media. Although soluble paracrine factors are involved, contact-dependent cell-cell interactions may also play an important role.