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-kit(pos) 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.

Keywords: c-kit; cell therapy; heart failure; mesenchymal cell; myocardial repair.

Figure 1

Isolation and characterization of mouse c-kit^pos CMCs. Enzymatically dissociated mouse hearts were left in culture medium for 24 h. When reached 70% confluence (4–6 days of expansion), c-kit positive CMCs were positively selected using magnetic beads. (A) Immediately after sorting, qPCR was performed to compare gene expression for c-kit, pluripotency markers, cardiac markers, and chemokine receptors (n = 5). C-kit (B) and Sca-1 (C) were monitored by flow cytometry up to 11 passages after sort (n = 13). Gene expression comparison in c-kit^pos CMCs between passage 1 and 5 (n = 4) (D,E). Values are mean ± SD ^*p < 0.05 vs. vehicle.

Figure 2

Expression of c-kit, pluripotency and cardiac markers in CMCs isolated from RA and SA fractions. Schematics of CMCs RA and SA isolation (A) expression of c-kit in CMCs from RA and SA fractions 4–6 days after isolation and expansion at mRNA level by qPCR (n = 4) (B) and protein level by flow cytometry (n = 4) (C). Real-time PCR on cells from RA and SA cells without sorting (n = 3) (D). Values are mean ± SD ^*p < 0.05 vs. SA.

Figure 3

Characterization of RA and SA c-kit^pos CMCs. Flow cytometric analysis of c-kit expression in RA and SA c-kit^pos CMCs (n = 3) (A). Clonogenicity performed using Terasaki plates (n = 4) (B). Population doubling times (n = 7) (C). Expression of pluripotency markers by qPCR at mRNA level (n = 3) (D). Mesenchymal and endothelial marker analysis by flow cytometry (n = 2) (E,F). Expression of endothelial (G), cardiac (H), smooth muscle (I) and mesenchymal (J) markers evaluated by qPCR (n = 3). Values are mean ± SD ^*p < 0.05 vs. RA.

Figure 4

Cardiac function 37 days after MI. Two days following myocardial infarction, vehicle, RA c-kit^pos CMCs, or SA c-kit^pos cMSCs, were percutaneously injected with echocardiographic guidance into the lumen of the left ventricular cavity (A). Echocardiography was used to determine diastolic and systolic volumes; the resulting ejection fractions are shown in (B). Representative frames from B-mode imaging (labeled PSLAX) and M-mode imaging are shown for the three groups in (C). Values are mean ± SD. ^*p < 0.05 vs. vehicle.

Figure 5

Morphometric analysis. Representative Masson's trichrome stained heart short axis sections from mice 35 days after injection of vehicle, RA, or SA c-kit^pos CMCs (A). Non-infarcted (i.e., Remote) and infarct wall thickness (B), scar as percent of LV (C) and expansion index (D). Values are mean ± SD. ^*p < 0.05 vs. vehicle.

Figure 6

Pro-angiogenic activity of RA and SA c-kit^pos CMCs in vivo. Capillary density in the ischemic zone (A) and border zone (B) of hearts 37 days after MI was evaluated by isolectin B4 staining. Values are mean ± SD. ^*p < 0.05 vs vehicle (Veh).