Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle

Abstract

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD(-/-) myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD(-/-) myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD(-/-) myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD(-/-) myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD(-/-) myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD(-/-) myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD(-/-) myoblasts. Therefore, MyoD(-/-) myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.

Fig. 1.

Increased engraftment of MyoD^−/− myoblasts after i.m. transplantation. (A) More than 90% of stable transformants of wild-type and MyoD^−/− myoblasts expressed nuclear lacZ after cotransfection and puromycin selection (Left). Twenty-four hours after CTX injection, 1 × 10⁶ wild-type and MyoD^−/− myoblasts with nls-lacZ were i.m. injected into regenerating TA muscle. By 1 week, X-Gal staining indicated that MyoD^−/− myoblasts were more greatly engrafted in regenerating muscle than wild-type myoblasts (Left Center). Engraftment of MyoD^−/− myoblasts (blue) was higher than wild-type myoblasts in muscle sections by 2 weeks after cell injection (Right Center, arrowheads). Luciferase expression in TA muscle injected with MyoD^−/− myoblasts was much higher than that of wild-type myoblasts by 1 week (Right, arrows). (B) Engraftment of MyoD^−/− myoblasts (blue) was much higher than wild-type myoblasts in muscle sections by 2 weeks after cell injection, and both wild-type and MyoD^−/− myoblast-derived, centrally located nuclei were integrated into myosin heavy chain-positive regenerating muscle fibers (arrowheads). (Insets) Magnified views. (C) A larger number of MyoD^−/− myoblasts can engraft in the TA muscle at days 1–14 after transplantation, compared with wild-type myoblasts (*, P < 0.05; **, P < 0.01). y axis indicates survival rates of engrafted cells after injection.

Fig. 2.

Satellite cell differentiation of MyoD^−/− myoblasts in TA muscle. (A–C) EM analysis indicates that satellite cells on muscle fibers contain X-Gal precipitation (A), in the perinuclear region (B, arrows), and in the nucleus (C, arrows), suggesting satellite cell differentiation of MyoD^−/− myoblasts. (D–I) By 1 month, TA muscles were used for immunostaining experiments to detect MyoD^−/− myoblast-derived satellite cells. The lacZ-expressing satellite cell (arrowheads) was positive for Pax7 (D–F) in the nucleus (E). The lacZ-expressing satellite cell (G, arrowheads) also was detected in between the sarcolemma (dystrophin⁺: green, arrowheads in H) and the basal lamina (laminin⁺: red, arrowheads in I). DAPI staining indicates nuclei (blue).

Fig. 3.

Gene expression profiles in wild-type vs. MyoD^−/− myoblasts. (A) GeneChip microarray data for myogenic, hematopoietic markers, and apoptosis-related genes. *, Mean fold change for pairwise comparisons of wild-type (WT)/MyoD^−/− (MD^−/−) myoblasts. **, Negative value indicates mean fold change for pairwise comparisons of MyoD^−/−/wild-type myoblasts. (B) Expression of skeletal muscle-specific, HSC, and apoptosis-related genes in wild-type and MyoD^−/− myoblasts were confirmed by semiquantitative RT-PCR. RNA was isolated from growth (G), differentiation days 1–5 (–5) of wild-type and MyoD^−/− myoblasts, and skeletal muscle (S).

Fig. 4.

MyoD^−/− myoblasts express stem cell markers. (A) MyoD^−/− myoblasts expressed stem cell markers Sca-1 and CD34 to a higher degree and in a larger proportion compared with wild-type myoblasts. (B) FACS analysis for Hoechst dye exclusion indicated that MyoD^−/− myoblasts possess a higher proportion of the SP fraction compared with wild-type myoblasts. Both SP fractions decreased after treatment with verapamil.

Fig. 5.

MyoD^−/− cells are resistant to apoptosis. (A) Under proliferation and differentiation (day 2) conditions, apoptosis detected by annexin-V staining was much higher in wild-type (WT) myoblasts compared with MyoD^−/− (MD^−/−) myoblasts. (B) After UV exposure at day 1, annexin-V-positive apoptotic cells were significantly increased in wild-type myoblasts vs. MyoD^−/− myoblasts. (C) MyoD^−/− myoblasts had increased survival over wild-type myoblasts after UV exposure. Ectopic expression of MyoD by infection with lentiviral MyoD expression vector (CS2-EF-MyoD) expression vector (+) in MyoD^−/− myoblasts increased cell death after UV exposure at days 1 and 2, compared with MyoD^−/− myoblasts infected with control lentiviral empty vector (−). (D) Ectopic expression of MyoD decreased engraftment rate in MyoD^−/− myoblasts after 1 × 10⁶ cell injections into TA muscle by day 7, compared with control MyoD^−/− myoblasts (*, P < 0.05; **, P < 0.01). y axis indicates survival rates of engrafted cells after injection.

Fig. 6.

Effect of cell survival of wild-type and MyoD^−/− myoblasts by pro- and antiapoptotic proteins. (A) Ectopic expression of human Bcl-2 [CMV-Bcl-2 (+)] in wild-type (WT) and MyoD^−/− (MD^−/−) myoblasts decreased cell death after UV exposure at days 1 and 2, compared with control myoblasts (−). (B) Ectopic expression of human Puma [CMV-Puma (+)] in wild-type and MyoD^−/− myoblasts increased cell death in growth medium, compared with control myoblasts (−). (C) Ectopic expression of human Bcl-2 [CMV-Bcl-2 (+)] increased engraftment rate in wild-type myoblasts after 1 × 10⁶ cell injections into TA muscle by day 7, compared with control wild-type myoblasts (−). By contrast, ectopic expression of human Puma [CMV-Puma (+)] decreased engraftment rate in MyoD^−/− myoblasts after 1 × 10⁶ cell injections into TA muscle by day 7, compared with control MyoD^−/− myoblasts (−) (*, P < 0.05; **, P < 0.01). y axis indicates survival rates of engrafted cells after cell injection.