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. 2009 Oct 6;106(40):17169-74.
doi: 10.1073/pnas.0903089106. Epub 2009 Sep 17.

Clonality of mouse and human cardiomyogenesis in vivo

Toru Hosoda  1 , Domenico D'Amario, Mauricio Castro Cabral-Da-Silva, Hanqiao Zheng, M Elena Padin-Iruegas, Barbara Ogorek, João Ferreira-Martins, Saori Yasuzawa-Amano, Katsuya Amano, Noriko Ide-Iwata, Wei Cheng, Marcello Rota, Konrad Urbanek, Jan Kajstura, Piero Anversa, Annarosa Leri
Affiliations

Clonality of mouse and human cardiomyogenesis in vivo

Toru Hosoda et al. Proc Natl Acad Sci U S A. .

Abstract

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genetic tagging of CPCs in situ. (A) Cardiac niche containing seven CPCs (c-kit, white). Two CPCs are labeled by EGFP (green, arrows). One EGFP-positive myocyte (α-sarcomeric actin, α-SA, red) is visible (asterisk). (B) Scatterplots of cells co-expressing EGFP and c-kit (CPCs), EGFP and CD31 (ECs), and EGFP and Thy1.2/CD90 (fibroblasts). Forward scatter (FSC) and side scatter (SSC) are also shown. Double positive cells were back-gated and are depicted by colored dots in FSC/SSC plots.
Fig. 2.
Fig. 2.
Clonal marking of mouse CPCs in vivo. (A) Transcripts for α-MHC (Myh6), CD31 and procollagen (Col3a1) in myocytes (Myo), ECs and fibroblasts (Fbl). Transcript for c-kit were detected by two or three rounds of nested PCR. MC, mouse myocardium; Actb, β-actin. (B–E) FACS-sorted cardiac cells express c-kit (B, green), α-SA (C, red), CD31 (D, yellow), or procollagen (E, magenta). (F) Four distinct clones were identified in CPCs, ECs, Fbl, and Myo isolated from one mouse heart 4 months after EGFP-lentivirus injection. Bands of the same molecular weight correspond to identical sites of integration of the proviral sequence in the host genome. (see Fig. S4 in SI Appendix for clonal mapping in the mouse genome and sequences).
Fig. 3.
Fig. 3.
CPCs and cardiomyogenesis. (A) FISH for EGFP viral integrants (white dot in the nucleus) found in EGFP-positive (green) myocytes (α-SA, red). Area in the rectangle is shown at higher magnification in the insets. (B) At 6 months, EGFP-labeled myocytes in the mid-portion of the LV are also BrdU-positive (white, arrowheads). One EGFP-positive BrdU-negative myocyte (asterisk) is visible. *, P < 0.05 vs. BrdU-positive myocytes. (C) Most BrdU positive (white) LV myocytes do not express EGFP (arrowheads). A few BrdU-positive EGFP-positive myocytes are present (double arrowheads). CPCs express c-kit (magenta) and are labeled by BrdU (arrows). Areas in the rectangles are shown at higher magnification in the lower panels. ***, P < 0.05 vs. Atrioventricular groove (AVG) and mid-region (Mid), respectively.
Fig. 4.
Fig. 4.
Validation of BrdU labeling. (A and B) Spectral analysis of BrdU-labeled nuclei. See text for detail. (C) Fraction of BrdU- and EdU-labeled myocytes. (D–F) BrdU (white) and EdU (green) co-localize in myocyte nuclei (arrows) at the border zone of the infarct.
Fig. 5.
Fig. 5.
Viral tagging of cultured hCPCs and myocardial regeneration. (A) Genomic DNA extracted from EGFP-positive-hCPCs was subjected to three rounds of PCR. The first (1st) round of PCR resulted in a DNA smear, and the second (2nd) led to the identification of seven bands. Arrows of the same color point to corresponding bands in different lanes. After the third (3rd) round, the size of the amplicons decreased by 112 bp for the 5′-side products and 138 bp for the 3′-side products. The seven bands reflect different sites of integration of the EGFP lentivirus in the human genome. PCR products were reamplified (Re-amp) and sequenced. In this example, the reamplified products of the two bands indicated by the yellow and pink arrows are shown (see Fig. S10A in SI Appendix for sequences). (B) Multiple clones were identified in cultured hCPCs (see Fig. S10 B and C in SI Appendix for chromosome mapping and sequences). (C) Band of regenerated myocardium (arrowheads) within the infarcted region of the LV. Newly formed myocytes express α-SA (upper panel, red) and EGFP (central panel, green). Lower panel, merge of upper and central panel. Spared myocytes (*).
Fig. 6.
Fig. 6.
Clonal tracking of hCPCs in the regenerated myocardium. (A) Scatterplots of EGFP-positive cells isolated from the regenerated myocardium expressing c-kit, CD31, and CD146. (B) Various clones were detected in distinct cardiac cell populations from the regenerated myocardium of one treated rat (see Fig. S13 in SI Appendix for chromosome mapping and sequences). (C) Sites of integration in isolated cell populations. Some clones were common to different cell classes (same color arrowheads). Key DNA sequences are ≈80 bp shorter than the corresponding clonal bands.
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