Cardiac Explant-Derived Cells Are Regulated by Notch-Modulated Mesenchymal Transition

Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT).Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of β-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in β-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells.Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells

Notch signaling regulates mesenchymal transition of c-Kit+ and c-Kit- cells.

<p>Notch signaling was up-regulated by over-expressing NICD-GFP adenovirus or suppressed with γ-secretase inhibitor XXI (GSI). (<b>A</b>) NICD transfection efficiency was monitored by GFP expression. (<b>B</b>) Notch modulation was confirmed by a Western blot analysis with anti-NICD antibody and (<b>C</b>) by qRT-PCR measuring expression level of Hey1. (<b>D</b>) qRT-PCR expression analysis after Notch signaling modulation. Expression levels were normalized to the level of β-actin. Fold changes were calculated as a ratio of expression in experimental (NICD- or GSI-treated) group to the expression in control group, * p<0.05. (<b>E, F</b>) FACS analysis of c-Kit+ (<b>E</b>) and c-Kit- (<b>F</b>) cells after Notch signaling modulation. N = 5 per group, *, p<0.05 compared to control.</p

Expression of cell-specific markers in cardiac cell outgrowths is time-dependent.

<p>(<b>A</b>) Cell outgrowths were cultured for various times and analyzed by immunofluorescence for the expression of cell lineage markers: stem cell marker, c-Kit; cardiac structural proteins, α-myosin heavy chain (MHC); mesenchymal cell marker, α-smooth muscle actin (SMA), epithelial cell marker, E-cadherin (E-cad) and epicardial progenitor marker Willms tumor 1 (Wt1). Nuclei were counterstained with DAPI (blue). (<b>B</b>) Percentage of cells expressing cell lineage markers. * p<0.05, compared to day 4. (<b>C</b>) Temporal changes of Snail gene expression were measured by qRT-PCR; *, p<0.05 compared to day 4. (<b>D</b>) Proliferation of outgrowth sub-populations. Proliferating (BrdU-positive) marker-specific outgrowth sub-populations were detected by flow cytometry as double positive events. The percentage of marker-specific proliferating cells was calculated as number of BrdU+/marker+ cells normalized to the total number of marker+ cells. (<b>E</b>) Apoptosis of cell sub-populations. Apoptotic (cleaved caspase 3-positive) marker-specific outgrowth sub-populations were detected by flow cytometry as double positive events. The percentage of marker-specific apoptotic cells was calculated as a number of caspase-3+/marker+ cells normalized to the total number of marker+ cells.</p

Expression of Notch1 receptor and Jagged1 in c-Kit+ and c-Kit- cells.

<p>(<b>A</b>) c-Kit+ cells displayed positive staining for Notch1 (green) co-localized with MHC (red, top row) or FSP1 (red, bottom row). (<b>B</b>) C-Kit- cells displayed positive staining for Jagged (green) co-localized with FSP1 (red, top row) or SMA (red, bottom row) as indicated. (<b>C</b>) Low number of c-Kit+ cells also express Jagged1 (green) co-localized with FSP1 (red). (<b>A–C</b>) Scale bar, 20 µm.</p

Suppression of Notch reversed mesenchymal phenotype of explant-derived cells.

<p>C-Kit+ and c-Kit- cells were cultured in presence of GSI. Representative images of c-Kit- cells are shown. (<b>A</b>) Transmitted light images demonstrate changes in cell morphology upon treatment with NICD or GSI. (<b>B</b>) Cells were labeled with antibodies to E-cadherin (white), N-cadherin (red) or β-catenin (green) as indicated. Nuclei were counterstained with DAPI (blue). Scale bars, 100 µm (A) or 20 µm (B).</p

Characterization of c-Kit+ cells isolated from total outgrowths.

<p>(<b>A</b>) Flow cytometry analysis of outgrowths cultured for 21 days shows percent of Wt1+ cells. (<b>B</b>) FACS analysis of double c-Kit and Wt1 labeling. Percentage of c-Kit+/Wt1+ cells indicated in upper right quadrant of histogram was calculated as a ratio of c-Kit+/Wt1+ (UR) to the total number of c-Kit+ cells (UR + LR). (<b>C–D</b>) Heart coronal sections are labeled with anti c-Kit (red) or anti Wt1 (green) antibodies. Nuclei were stained with DAPI (blue). Right column represents merged images of consecutive sections labeled with c-Kit and Wt1. (<b>C</b>) Representative images of left atrium are shown. Scale bar, 100 µm. (<b>D</b>) Higher magnification of the area selected in the box. Scale bar, 20 µm. (<b>E</b>) Purity of c-Kit+ and c-Kit- cell populations were confirmed by FACS analysis. C-Kit+ (black) and c-Kit- (blue) cells were labeled with anti-c-Kit antibody followed by secondary antibody conjugated with Alexa 488. For negative control, isotype IgG was used instead of primary antibody (grey). Representative histogram is shown. (<b>F</b>) qRT-PCR analysis of c-Kit+ and c-Kit- cell populations. Expression levels were normalized to the level of β-actin, fold changes were calculated as a ratio of expression in c-Kit- group to expression in c-Kit+ group, * p<0.05.</p

Notch modulation affects state of Wnt signaling.

<p>(<b>A</b>) Comparative qRT-PCR analysis of selected Wnt signaling-related genes in c-Kit+ and c-Kit- cells. (<b>B</b>–<b>E</b>) Notch signaling was up-regulated by over-expressing NICD-GFP adenovirus or suppressed with GSI. (<b>B</b>–<b>C</b>) Western blot of selected Wnt cascade components. β-Actin was used as a loading control. Representative images are shown. (<b>D</b>) Density of specific bands were quantified and normalized to the density of beta-actin. Level of inactive GSK3β was determined as ratio between pGSK3β (Ser 9) and total GSK3β. (<b>E</b>) qRT-PCR analysis of selected Wnt target genes in NICD-treated or GSI-treated cells. N = 5 per treatment condition. *, p<0.05.</p