+ + A. Standard Isolation Method: B. New Isolation Method: c-kit SORT

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+ + A. Standard Isolation Method: B. New Isolation Method: c-kit SORT Enzymatically dissociated myocardial cells Removal of floating cells and debris 70% confluence 4-6 days 24 h c-kit SORT Expansion of adherent cells Tissue Culture Labeled Cells Anti- c-kit conjugated with magnetic beads Magnetic Column + Positive selection c-kit cells Cell Labeling (one step method) Flow through c-kit depleted cells B. New Isolation Method: Enzymatically dissociated myocardial cells Re-plating of non-adherent cells into new culture flasks 1’ 2’ 3’ 4’ 5’ 2 h 2 h 24 h 24 h RA – rapidly adherent SA – slowly adherent Labeled Cells Anti-FITC conjugated with magnetic beads Magnetic Column Anti-c-kit conjugated with FITC + Positive selection c-kit cells Cell Labeling (two step method) Flow through c-kit depleted cells Supplementary Figure 1. Workflow schematics for isolation of c-kitpos CMCs. Comparison of c-kitpos CMCs isolation with standard (A) and new improved isolation method (B). Detailed description in material and methods.

A. B. Supplementary Figure 2. Expression of c-kit RA (1’ and 2’) and SA (3’, 4’ and 5’) fractions. 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) (A) and protein level by flow cytometry (n=4) (B).

RA SA Empty CD105 CD29 CD73 0.8% 1% 98.6% 97.9% 99.1% 99.7% 62.1% 54.6% Supplementary Figure 3. Flow Cytometric analysis of RA and SA CMCs. Mesenchymal marker analysis by flow cytometry (n=2).