Absence of Cardiomyocyte Differentiation Following Transplantation of Adult Cardiac-Resident Sca-1+ Cells Into Infarcted Mouse Hearts.

Although several lines of evidence suggest that the glycosyl phosphatidylinositol–anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT–enhanced green fluorescent protein (EGFP); MHC-nLAC double-transgenic mice ([C57/Bl6J × DBA/2J]F1 genetic background; all procedures followed were in accordance with institutional guidelines). The ACTEGFP transgene targets ubiquitous expression of an EGFP reporter, and the MHCnLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, whereas cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-d-galactoside (XGAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme, and the resulting cells reacted with an allophycocyanin-conjugated anti–Sca-1 antibody and a phycoerythrin-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage– cells were then isolated via fluorescence-activated cell sorting (characterization of the donor cells is provided in Figure [A]), and 100 000 cells were injected into the infarct border zone of nontransgenic [C57/Bl6J × DBA/2J]F1 mice immediately following permanent coronary artery occlusion. To screen for cardiomyogenic activity, hearts were harvested at 11 (n=3), 12 (n=3), 14 (n=2), or 21 (n=2) days postengraftment, fixed, and sectioned on a vibratome at 200 μm; sections were then reacted with X-GAL and visualized on a dissecting microscope. No nuclear β-galactosidase activity was detected (Figure [B]), despite the presence of Sca-1+ donor cells as evidenced by the presence of EGFP fluorescence (inset). To confirm the absence of cardiomyogenic differentiation, all vibratome sections exhibiting EGFP fluorescence were cryosectioned at 10 μm and analyzed (example shown in Figure [C]). No nuclear β-galactosidase activity was observed (8577 EGFP+ cells analyzed; the engraftment efficiency was 0.87%). Immune cytology confirmed that the green fluorescent signal colocalized with EGFP protein (not shown). As a positive control for the reporter system, cardiomyocytes prepared from embryonic day 15.5 ACT-EGFP; MHC-nLAC double-transgenic mice were engrafted into the hearts of syngeneic [C57/Bl6J × DBA/2J]F1 recipients; the hearts were harvested at 14 days postengraftment and processed exactly as described above. Donor cells with nuclear β-galactosidase activity and EGFP fluorescence were readily observed in the engrafted hearts (Figure [D]). Immune histology revealed that at least a portion of the Sca-1+ donor cells express vimentin (Figure Mark H. Soonpaa, PhD Pascal J. Lafontant, PhD Sean Reuter, BA John A. Scherschel, MD Edward F. Srour, PhD Marc-Michael Zaruba, MD Michael Rubart-von der Lohe, MD Loren J. Field, PhD