Letter to the Editor

Dear Editor,

Zhao et. al.¹ cast into doubt the conclusions of our published work² showing that Cardiosphere-Derived Cells (CDCs) induce cardiac and systemic rejuvenation in senescent rats. However, Zhao et al.’s conclusions are questionable given that the cells they isolated and studied are not CDCs. Indeed, they differ fundamentally from CDCs in various important ways, enumerated below.

First, the method used to prepare the cells is inconsistent with established protocols for CDCs. Zhao et al. harvested only phase-bright explant-derived cells to prepare cardiospheres (their Figure 1B, C). This is in contrast with the widely-accepted method of preparing CDCs (whether human or murine) using all cells growing around the explant^3–5.

Second, the culture period used to prepare “cardiospheres” from explant-derived cells was unconventionally prolonged. Zhao et al. describe a seven-day culture period to form their spheroids, which contrasts with the standard 72-hour period used to prepare cardiospheres. Genuine cardiosphere formation happens quite rapidly, and time periods longer than 72 hours result in the formation of apoptotic cores ^{2–4, 6}. Consistent with the unconventional culture conditions, the “cardiospheres” shown by Zhao et al. (their Figure 1D, E) resemble non-specifically-aggregated multicellular fragments of cardiac tissue. Another troubling conclusion is the observation that their spheres are beating, indicating they are enriched in functional, differentiated cardiomyocytes. Genuine cardiospheres and CDCs do not contain cardiomyocytes⁷.

Third, marker expression data presented by the authors fail to establish Zhao et al.’s cells as CDCs (their Figure 1G-J). Data on established CDC identity markers, including CD105 and CD45 (consistently high and absent respectively), are conspicuously absent⁸. Furthermore, the selective data provided are inconsistent with previous characterizations of CDCs. For example, Sca-1 is expressed in only 13% of the cell population whereas we and others have shown Sca-1 to be expressed in >50% of CDCs⁹. CD34, which is a classic negative marker in CDCs (less than 1%), is quite high in the Zhao et al. cells (16%).

The considerations above give reason to question the identity of the cells isolated and used by Zhao et al. In addition, several concerns with the aging C57BL/6 mouse model raise further issues. While this is an established model for age-related neurodegeneration, it is unclear from the literature if these mice develop cardiac dysfunction. For this reason, it is difficult to rationalize why Zhao et al. did not include young controls to validate the presence of cardiac dysfunction in this model (their Figure 2 and 3). Based on the Masson trichrome staining (their Figure 4A), there is no age-related fibrosis; indeed, the only fibrosis seen is quite focal, consistent with iatrogenic needle track injury. Finally, the authors claim that telomere length in cardiomyocytes of these aged mice is shorter than in younger animals (their Figure 5). However, previous, more thorough descriptions conclude that telomere length is unchanged in cardiomyocytes from this model ¹⁰.

In conclusion, the title and conclusions of the study by Zhao et al. are misleading. Given substantive technical concerns, namely the questionable identity of the cells and major issues with the animal model, the study falls significantly short of evaluating the potential of CDCs to ameliorate age-related dysfunction.