Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction

Abstract

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5'-azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (alphaMHC-Cre/R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells.

Figures

Fig. 4.

Homing, differentiation, and fusion of donor cardiac Sca-1+ cells in host myocardium. Diagram of the dye-labeling (A) and Cre/Lox (B) donor/recipient strategies. (B) RT-PCR analysis showing lack of Cre expression in newly isolated cardiac Sca-1+ cells from αMHC-Cre mice. (C) Western blot showing expression of neo in R26R mice. (D) Homing of dye-labeled cardiac Sca-1+ cells at 24 h (Left) and engraftment at 2 wk (Right). (E) Neo (FITC; yellow in merged image) was ubiquitously expressed in R26R adult ventricular myocardium. (D and E) Identical fields are shown, Upper and Lower. Cardiomyocytes were identified by sarcomeric α-actin (Texas red) and nuclei with 4′,6-diamidino-2-phenylindole. (F–I) Animals were analyzed by confocal microscopy 2 wk after ischemia-reperfusion injury and infusion of ScaI+ cells. (F) Neo (blue) was ubiquitously expressed in adult ventricular myocardium of R26R mice. Grafted Sca-1+ cells from αMHC-Cre mice activate cardiomyocyte-specific Cre, and recombination of R26R. Cre protein (green) was localized to nuclei. Three phenotypes resulted. Unfused donor-derived cells express neither neo nor LacZ (circled in column 2). Fusion with host R26R myocardium typically results in LacZ+ muscle cells (red; circled in column 3). Fused cells without recombination were detected very rarely (circled in column 4). (G) Mean ± SE for donor-derived myocytes with (Cre+ LacZ+ neo–; Cre+ LacZ– neo+) and without (Cre+ LacZ– neo–) fusion after grafting. BZ, infarct border zone in anterolateral (A, L) myocardium; NI, noninfarcted control regions; P, posterior wall; R, right ventricle, interventricular septum. (H) Delineation of Cre+ cells by laminin. (I) All Cre+ cells coexpressed sarcomeric α-actin, cardiac troponin I, and Cx43. Mitotic phosphorylation of histone H3 2 wk after grafting was seen almost exclusively in donor-derived Cre+ myocytes. Arrow, Cre+ phospho-H3+ cardiomyocyte nuclei (blue-green in merged image). (Bar = 20 μm.) ntg, nontransgenic.

Fig. 1.

Isolation of Sca-1+ cells from adult mouse myocardium. (A) Sca-1 was analyzed by flow cytometry (MoFlo, Cytomation, Ft. Collins, CO), by using IgG2a + 2b-FITC as the control. (B) Immunostaining of adult mouse myocardium for Sca-1, laminin, and CD31. Yellow-orange in the merged images denotes colocalization. Representative cells are highlighted in white and shown at higher magnification (Insets). (Bar = 15 μm.) (C) Cells were labeled with Sca-1 plus the indicated markers (EPICS XL-MCL, Beckman Coulter). Values in the bar graph denote prevalence in the myocyte-depleted population, e.g., 12% are Sca-1+CD31+. Bone marrow cells ± collagenase are shown (Right). (D) Cardiac SP cells were identified with Hoechst 33342 (MoFlo, Cytomation). Labeling with Sca-1 vs. c-kit and CD45 is shown as contour plots. (E) Enrichment for SP cells in the cardiac Sca-1+ population.

Fig. 2.

Purification and culture of cardiac Sca-1+ cells. Purity of cardiac Sca-1+ and -1– cells after magnetic enrichment. (A Left) Flow cytometry. FSC-H, forward-angle light scatter. (Right) Immunostaining. (B) Telomerase activity (30) was detected in cardiac Sca-1+ cells but not Sca-1– cells. Numbers above each lane indicate the amount of adult lysate, relative to neonatal mouse heart (“neonate”). (C) RT-PCR analysis of cardiac Sca-1+ and -1– cells vs. adult mouse heart. (Bar in A = 5 μm).

Fig. 3.

In vitro differentiation of cardiac Sca-1+ cells is induced by 5-aza and depends on a BMP receptor. (A) Phase-contrast microscopy. (B) Induction of sarcomeric α-actin and cardiac troponin I by using 5-aza, shown by immunostaining (4 wk). Differentiated cells (red) are found in the monolayer with 4′,6-diamidino-2-phenylindole (blue). (C) Induction of Nkx-2.5, Bmpr1a, and cardiac MHC genes using 5-aza, shown by RT-PCR. (D) Cartoon of the floxed and null Bmpr1a alleles at exon 2. (Bottom Right) Excision of exon 2 after viral delivery of Cre, shown by PCR. (E) Cardiac Sca-1+ cells from Bmpr1aF/– mice were subjected to viral gene transfer, differentiated with 5-aza, and analyzed by quantitative real-time-PCR. White, β-gal; black, Cre. *, P < 0.05; †, P < 0.01; n = 6. [Bars = 20 μm (A) and 10 μm (B).]