Multi-Potent Sca-1+ CPCs Figure 3. Multiple differentiation potential of isolated colony-derived Sca-1 high expressing population. A, Differentiation assay of Sca- 1 high expressing populations into mesenchymal cell lineage. Upper panel shows osteogenic differentiation of Sca-1 high expressing populations with von Kossa staining. Magnification =6320. Lower panel shows chondrogenic differentiation with Toluidine Blue O staining. Magnification =6200. Inserts show magnified area from same sample. Magnification =61000. B, Gene expression profiles of Sca-1 high expressing cell populations analyzed by RT-PCR. doi:10.1371/journal.pone.0025265.g003 cells possessed multiple stem cell-related features and exhibited great potential for myocardial regeneration in a mouse model of acute MI. Gene profiling studies suggested that CSH2 cells originated from precursor cells of secondary heart field and showed functional superiority in terms of proliferation, clonogeni- city, self-renewal, and Sca-1 expression besides multiple stem cell- related genes as compared to MSH. Contrarily, MSH cells, isolation of which was based on Sca-1 expression alone, were heterogeneous and contained cell populations from different sources including primary heart field, secondary heart field, and neural crest-derived cells. Purification of a cardiac stem cell population from the heart is challenging, especially due to lack of specific surface markers. We therefore exploited clonogenic potential of the stem cells, which is considered as one of the functional feature of stem/progenitor cells [25], in combination with Sca-1 expression for their purification. We cultured the heart-derived cells in serum-free culture conditions at a very low density which alleviated their cross- contamination from the presence of mature cells in the culture besides recovery of sub-clonal cell populations with strong survivability and proliferation. Interestingly, some of these cell populations thus obtained showed spontaneous high Sca-1 positivity with very low c-kit expression stem/progenitor cell marker [1,8]. This may be attributed to the difference in the culture conditions and different expression period of these two surface markers during maturation of heart [26]. CSCs in their undifferentiated status express stem-cell antigens c-kit, MDR1, and Sca-1 either alone or in combination, however only a small percentage of cells is positive for anyone of the three antigens alone [27]. Of these three populations, ckit+ CSCs are the most extensively studied and well characterized CSCs for stemness characteristics including clonogenicity, self renewal and PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25265