A New Method to Stabilize C-Kit Expression in Reparative Cardiac Mesenchymal Cells

Abstract

Cell therapy improves cardiac function. Few cells have been investigated more extensively or consistently shown to be more effective than c-kit sorted cells; however, c-kit expression is easily lost during passage. Here, our primary goal was to develop an improved method to isolate c-kitpos cells and maintain c-kit expression after passaging. Cardiac mesenchymal cells (CMCs) from wild-type mice were selected by polystyrene adherence properties. CMCs adhering within the first hours are referred to as rapidly adherent (RA); CMCs adhering subsequently are dubbed slowly adherent (SA). Both RA and SA CMCs were c-kit sorted. SA CMCs maintained significantly higher c-kit expression than RA cells; SA CMCs also had higher expression endothelial markers. We subsequently tested the relative efficacy of SA vs. RA CMCs in the setting of post-infarct adoptive transfer. Two days after coronary occlusion, vehicle, RA CMCs, or SA CMCs were delivered percutaneously with echocardiographic guidance. SA CMCs, but not RA CMCs, significantly improved cardiac function compared to vehicle treatment. Although the mechanism remains to be elucidated, the more pronounced endothelial phenotype of the SA CMCs coupled with the finding of increased vascular density suggest a potential pro-vasculogenic action. This new method of isolating CMCs better preserves c-kit expression during passage. SA CMCs, but not RA CMCs, were effective in reducing cardiac dysfunction. Although c-kit expression was maintained, it is unclear whether maintenance of c-kit expression per se was responsible for improved function, or whether the differential adherence property itself confers a reparative phenotype independently of c-kit.

Myocardial Reparative Properties of Cardiac Mesenchymal Cells Isolated on the Basis of Adherence

“…As is the case for virtually all other cell types, the vast majority of transplanted c-kit POS CPCs disappeared soon after transplantation (9–12,14,17). Interestingly, several studies suggested that c-kit POS CPCs possess mesenchymal stromal characteristics and express classic mesenchymal stromal markers, including CD90, CD105, CD29, and CD73 (13,18–22), intimating that c-kit POS CPCs might, in fact, be a subset of mesenchymal stromal cells. If this is the case, their reparative actions after MI might relate to the general properties of mesenchymal cells rather than to the expression of c-kit per se.…”

“…Although c-kit POS CPCs are effective in repairing the infarcted heart, c-kit–based selection is prone to errors and controversy because of variability in c-kit expression levels and reagents (3,13). Furthermore, the expression of c-kit in culture appears to be labile.…”

“…Furthermore, the expression of c-kit in culture appears to be labile. For example, we have found that in murine sorted c-kit POS CPCs, the expression of c-kit decreased from 78% to <30% after 4 or 5 passages (13). Because c-kit expression can be lost easily during expansion in culture, we posit that many investigators are observing beneficial effects with cells that are no longer c-kit POS at the time of transplantation despite selection for this marker.…”

“…Because c-kit expression can be lost easily during expansion in culture, we posit that many investigators are observing beneficial effects with cells that are no longer c-kit POS at the time of transplantation despite selection for this marker. Recently, we discovered that the ability of myocardial cells to adhere to plastic enabled separation of a population of cells that maintained prominent c-kit positivity in vitro (13). Specifically, after cardiac digestion, we identified 2 cell populations with distinct properties: those that require more than 24 h to adhere to plastic, which we termed slowly adhering (SA), and those that adhere in < 24 h, which we termed rapidly adhering (RA).…”

Dose-dependency and reversibility of radiation-induced injury in cardiac explant-derived cells of mice

“…It will be also interesting to know the radiation effect specially on the small fraction of c-kit-positive cells in heart2526. However, it is technically difficult for us to get enough number of c-kit-positive cells for experiments because the expression of c-kit is extremely low in the heart tissue of mouse and may loss during ex vivo expansion27. Second, radiation exposure was done at a high dose rate (>12 Gy per hour) in our study.…”