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.

Duchenne muscular dystrophy (DMD) is a progressive disorder in which the absence of the protein dystrophin results in a loss of the dystrophin-associated protein complex linked to the extracellular matrix (1). Definitive treatment for DMD likely requires that the dystrophin-associated protein complex is restored in all affected muscle groups to improve muscle function. Possible approaches to restoring muscle fibers include cell therapy, gene therapy, or a combination of the two.

Muscle satellite cells are a small population of myogenic stem cells found in skeletal muscle, characterized by the expression of desmin, Pax7, MyoD, Myf5, and M-cadherin (2–5). Satellite cells are responsible for muscle repair as skeletal muscle stem cells. Evidence indicates that there are at least two populations of satellite cells resident in skeletal muscle. One population rapidly contributes to muscle repair, whereas the other population is more stem cell-like and remains longer in an undifferentiated state in the recipient muscle (6, 7). However, it remains unknown which population is more efficient in repairing continuously degenerating muscle.

Myoblasts derived from satellite cells isolated from adult skeletal muscle can be easily expanded ex vivo. The capacity for myoblasts to become muscle fibers in regenerating muscle and to form ectopic muscle fibers in nonmuscle tissues is exploited by myoblast transplantation, a potential therapeutic approach for DMD (8), urological dysfunction (9), and heart failure (10). Ex vivo expanded myoblasts have been successfully transplanted in the muscle of both mdx mice (model animals for DMD) and DMD patients (11). The normal, unaffected myoblasts fuse with host muscle fibers, produce dystrophin, and improve the strength of the injected muscle. Although positive effects are seen with myoblast transplantation, severe limitations exist, including immune rejection of allogeneic donor cells, poor cellular survival, and limited spread of the injected cells, hindering the practical application to patients (8). In addition, it is clear that transient muscle fiber repair as well as a continuous supply of myoblasts for damaged muscle fibers are essential for long-term therapy.

The myogenic regulatory factors, a group of skeletal muscle-specific basic helix–loop–helix transcription factors consisting of MyoD, Myf5, myogenin, and MRF4, play an essential role in satellite cell activation, proliferation, and differentiation (1, 2, 12). The adult skeletal muscle in mice lacking MyoD (MyoD^−/−) displays a deficiency in regeneration and strongly supports the assertion that MyoD plays an essential role in regulating the satellite cell myogenic program (13). In addition, we and other groups have demonstrated that satellite cell-derived primary myoblasts isolated from adult MyoD^−/− mice display an accelerated growth rate and delayed terminal differentiation (2, 14–19). Therefore, MyoD^−/− myoblasts have been suggested to display characteristics that are more primitive than wild-type myoblasts and may represent an intermediate stage between stem and myogenic precursor cells. We examined whether MyoD^−/− myoblasts represent more primitive stem cells and display efficient engraftment and contribution to regenerating muscle fibers, as well as to satellite cells, after transplantation.

Results

Enhanced Survival of MyoD^−/− Myoblasts After i.m. Injection.

MyoD is a skeletal muscle-specific transcription factor that plays an essential role in muscle stem cell differentiation and muscle regeneration. To further investigate the function of MyoD in muscle stem cells, we examined myoblast transplantation into regenerating muscle. Wild-type and MyoD^−/− satellite cell-derived primary myoblasts (1 × 10⁶ cells) transfected with ubiquitous promoter-driving nls-lacZ (Fig. 1A) were i.m. injected into cardiotoxin (CTX)-induced regenerating tibialis anterior (TA) muscle of adult Scid/beige mice (Fig. 1A). By 1 and 2 weeks after cell injections, engraftment and survival of MyoD^−/− myoblasts were significantly increased compared with wild-type myoblasts (Fig. 1A). Both wild-type and MyoD^−/− myoblast-derived, lacZ-positive nuclei were integrated into myosin heavy chain-positive regenerating muscle fibers (Fig. 1B). These lacZ-positive nuclei were centrally located in the newly formed muscle fibers. The time course for the engraftment rate of myoblasts after 1 × 10⁶ cell injections was analyzed by counting nuclear lacZ expression in every 10 serial sections of whole TA muscle at days 1, 3, 7, and 14 after cell injections (Fig. 1C). The results showed that significantly more MyoD^−/− myoblasts than wild-type myoblasts were engrafted each day (day 1: wild type, 9.2 ± 3.1%; MyoD^−/− myoblasts, 21.6 ± 6.3% of total injected cells; day 3: wild type, 3.0 ± 0.5%; MyoD^−/− myoblasts, 13.0 ± 6.0%; day 7: wild type, 1.5 ± 0.5%; MyoD^−/− myoblasts, 18.5 ± 2.6%; day 14: wild type, 1.3 ± 0.5%; MyoD^−/− myoblasts, 6.0 ± 3.0%). In addition, transplanted MyoD^−/− myoblasts not only differentiated into multinucleated muscle fibers, but also gave rise to the satellite cell compartment 1 month after transplantation (Fig. 2). By 1 month after injection of MyoD^−/− myoblasts, EM observation detected satellite cells with X-Gal precipitation in the nucleus (Fig. 2 A and C) and perinuclear region (Fig. 2 A and B). Furthermore, a few lacZ/Pax7 double-positive nuclei were detected in the peripheral region of the muscle fibers (Fig. 2 D–F). Some lacZ-positive nuclei also were found between the basal lamina (laminin⁺) and sarcolemma (dystrophin⁺) of muscle fibers (Fig. 2 G–I), indicating that the lacZ-positive nuclei were within a satellite cell. By contrast, we could not find any wild-type myoblast-derived satellite cells in the recipient muscle. These data support the hypothesis that MyoD^−/− myoblasts represent progenies derived from satellite cells maintaining stem cell activity.

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).

GeneChip Microarray Analysis of the Effects of MyoD Deficiency.

Myoblast transplantation experiments demonstrate that injected MyoD^−/− myoblasts can survive more efficiently in regenerating muscle, compared with wild-type myoblasts. Previous work has suggested that MyoD^−/− myoblasts display characteristics that are more primitive than wild-type myoblasts and may represent an intermediate stage between a stem cell and a myogenic precursor cell (2, 14). However, molecular pathways regulated by MyoD during satellite cell differentiation and survival remain to be elucidated. To reveal the molecular pathways downstream of MyoD, the transcriptional profiles of proliferating MyoD^−/− and wild-type myoblasts were examined by Affymetrix GeneChip microarray (Santa Clara, CA) to assess the effects of MyoD deficiency on gene expression as previously reported (20, 21). The findings revealed that ≈350 of 13,000 genes demonstrated at least a 2-fold average change in their expression between wild-type and MyoD^−/− cells [supporting information (SI) Table 1]. To classify altered gene expression between wild-type and MyoD^−/− cells, down- and up-regulated genes were placed into broad functional categories (SI Table 1 and SI Fig. 7). Interestingly, many different categories of genes (including genes for cell-cycle regulators, apoptosis-related signaling pathways, RNA processing, secreted molecules and their receptors, cell adhesion molecules and extracellular matrix, hematopoietic cell markers, mitochondrial function and redox regulation, and cytoskeletons) had a higher frequency of up-regulation in MyoD^−/− myoblasts. As anticipated, genes for muscle-specific proteins, such as muscle structural proteins and channels involved in muscle contraction, were significantly less prevalent in MyoD^−/− than in wild-type myoblasts. In addition, genes for RNA processing were more abundant in wild-type myoblasts. Among the genes involved in signaling pathways, genes involved in Notch and TGFβ cascades were placed into a separate category because these signaling cascades are well known for regulating satellite cell proliferation and differentiation (22, 23). Genes involved in the Notch cascade were more abundant in wild-type cells. The TGFβ family and their downstream genes were more abundant in MyoD^−/− cells. Taken together, these results suggest that MyoD^−/− myoblasts possess broad activity for many signaling cascades and high cell motility activities, whereas wild-type myoblasts partially undergo terminal differentiation.

We also confirmed the GeneChip microarray data for myogenic, hematopoietic stem cell (HSC) markers, and apoptosis-related genes by semiquantitative RT-PCR (Fig. 3 A and B and SI Fig. 8). Many muscle-specific genes were highly up-regulated in wild-type, but not in MyoD^−/−, cells. These genes included MyoD (11.2-fold), desmin (55.0-fold), and M-cadherin (5.4-fold), consistent with previous data (14). Earlier work showed that many of the muscle-specific genes listed in Fig. 3A contain E-boxes that are essential for MyoD-mediated transcriptional activation, indicating that these muscle-specific genes are directly activated by MyoD in myoblasts (24). In this category, only Myf5 (4.1-fold) was found to be up-regulated in MyoD^−/− cells, consistent with reported results (14, 25).

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 (1–5) of wild-type and MyoD^−/− myoblasts, and skeletal muscle (S).

Interestingly, the loss of MyoD resulted in the up-regulation of several cell-surface markers expressed in HSC (Fig. 3 A and B and SI Fig. 8). In particular, the up-regulation of Sca-1 (10.8-fold) and CD34 (5.5-fold) is quite interesting because both are markers for HSCs and hematopoietic progenitor cells, as well as muscle-derived stem cells (5, 26–28). Immunostaining clearly confirmed this expression profile: Both expression levels and numbers of cells positive for Sca-1 or CD34 were significantly increased in MyoD^−/− cells compared with wild-type cells (Fig. 4A). In addition, the MyoD deficiency also up-regulated Meis1 (5.7-fold), which belongs to the TALE family of homeodomain-containing proteins and is specifically expressed in HSCs, myeloid leukemia, and neural stem cells (Fig. 3A).

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.

Recent work demonstrated that many tissue-specific stem cells, including HSCs and muscle-derived stem cells, can be isolated by FACS of side population (SP) cells on the basis of Hoechst dye exclusion (26, 28, 29). Therefore, SP cells have been suggested to possess characteristics common to stem cells. Therefore, we used the FACS/Hoechst method to examine whether wild-type and MyoD^−/− myoblasts contained SP cells. Strikingly, compared with wild-type SP cells, significantly increased numbers of SP cells were detected in MyoD^−/− cells (2.4% vs. 32.6% of total cells in Fig. 4B). Most of the wild-type and MyoD^−/− SP cells stained with Hoechst dye were sensitive to verapamil, indicating involvement of ABC transporter proteins for Hoechst dye exclusion (30). Taken together, increased up-regulation of stem cell markers and expansion of MyoD^−/− SP cells further support the hypothesis that MyoD^−/− myoblasts display characteristics that are more primitive than wild-type cells and may represent an intermediate stage between stem cell and myogenic precursor cells.

MyoD Deficiency Alters Gene Expression Involved in Mitochondrial Function, Redox, and Apoptosis.

Interestingly, many genes that mediate apoptotic cell death, including mitochondrial function and redox, differed in expression between wild-type and MyoD^−/− myoblasts (Fig. 3 A and B and SI Fig. 8). For example, antiapoptotic proteins such as PEA15 (a death-effecter, domain-containing protein), TNFα-induced protein 2, Bcl-2, and caspase-8 (FLICE) inhibitory protein were 4.6-, 4.5-, 2.8-, and 2.6-fold up-regulated in MyoD^−/− cells, respectively. By contrast, wild-type cells up-regulated PEG3/Pw1 (17.2-fold), a zinc finger protein that is downstream of the TNFα pathway and positively regulates p53-dependent apoptosis. In addition, MyD116 and GADD45α genes regulating growth arrest after stress induction, such as UV exposure, are highly up-regulated (>100- and 58.4-fold, respectively) in wild-type cells compared with MyoD^−/− cells. Furthermore, we noticed that genes controlling intracellular redox potential, such as CD53 and microsomal GST (mGST3), and genes involved in mitochondrial function, such as cytochrome C oxidase VIB (COX6B), differed between wild-type and MyoD^−/− cells. The loss of MyoD resulted in up-regulation of CD53 (4.1-fold) and mGST3 (13.0-fold). Expression of CD53 and mGST3 can lead to an increase in the total cellular level of glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. By contrast, wild-type myoblasts up-regulated gene expression of COX6B (>100-fold), a protein known to be induced by oxidative stress, inducing apoptosis. Release of oxidative cytochrome C from mitochondria, which causes caspase-3 activation, is one of the most critical steps for inducing many forms of apoptotic cascades. RT-PCR clearly showed data consistent with the expression profiles (Fig. 3B and SI Fig. 8). These results indicate that, compared with wild-type myoblasts, MyoD^−/− myoblasts have a greater survival rate after transplantation because of altered apoptotic cascades in MyoD^−/− myoblasts.

MyoD^−/− Cells Are Resistant to Apoptosis.

Gene expression profiles and results from RT-PCR strongly suggest that MyoD may regulate apoptosis pathways in satellite cells. Previous work showed that a subset of myoblasts undergoes apoptosis during proliferation and apoptosis is increased during differentiation (31). Therefore, we examined whether there is any difference in apoptosis between wild-type and MyoD^−/− myoblasts under both proliferation and differentiation conditions. Annexin-V staining revealed that, under high-serum conditions, almost no apoptotic cells were detected in MyoD^−/− myoblasts, whereas a small number of wild-type myoblasts underwent apoptosis (Fig. 5A and SI Fig. 9). Under low-serum (differentiation) conditions by day 2 after UV exposure-induced DNA damage by day 1, the number of annexin-V-positive apoptotic cells increased in wild-type myoblasts (Fig. 5 A and B and SI Fig. 9). By contrast, annexin-V-positive apoptotic cells were barely detected in MyoD^−/− myoblasts, suggesting that MyoD is required for proper apoptosis during proliferation and differentiation phases and after DNA damage.

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.

We previously showed that ectopic expression of MyoD rescues differentiation deficiency in MyoD^−/− myoblasts (14). Therefore, we examined whether ectopic expression of MyoD in MyoD^−/− myoblasts can increased apoptosis. As a result, UV-induced cell death was significantly increased in MyoD^−/− myoblasts expressing ectopic MyoD by days 1 and 2 after UV exposure (Fig. 5C and SI Fig. 10). Ectopic expression of MyoD in MyoD^−/− myoblasts also reduced the engraftment rate after transplantation into CTX-induced TA muscle in syngenic BALB/c mice by day 7 (Fig. 5D).

To show that wild-type myoblasts display decreased engraftment after transplantation, compared with MyoD^−/− myoblasts because of apoptosis, we examined whether overexpression of proapoptotic proteins could induce cell death in MyoD^−/− myoblasts independent of MyoD expression. Ectopic expression of Puma, a proapoptotic BH3-only protein that binds to the antiapoptotic protein Bcl-2 (32) in MyoD^−/− myoblasts, induced a similar level of cell death in comparison to Puma-expressing wild-type myoblasts in vitro (Fig. 6B). In addition, ectopic expression of Puma decreased the number of MyoD^−/− myoblasts that engrafted after transplantation into CTX-induced TA muscle in BALB/c mice by day 7 (Fig. 6C). These results suggest that the overexpression of proapoptotic proteins can induce apoptosis in myoblasts in a MyoD-null environment. Next, we examined whether overexpression of the antiapoptotic protein Bcl-2 (32) could convert wild-type myoblasts to a MyoD^−/− myoblast phenotype by inhibiting apoptosis. Ectopic expression of Bcl-2 clearly protected wild-type myoblasts from UV-induced cell death in vitro (Fig. 6A) and increased engraftment rate of wild-type cells after transplantation into CTX-induced TA muscle in BALB/c mice by day 7 (Fig. 6C). The results presented here clearly indicate that MyoD has a pivotal role in the induction of apoptotic cascades induced by various signals. The MyoD deficiency results in increased resistance to many forms of apoptosis cascades, some of which may be at the mitochondrial level presumably by up-regulation of antiapoptotic proteins such as Bcl-2. Taken together, MyoD-deficient, myoblast-based stem cell therapy may be used to effectively treat DMD to improve skeletal muscle. In addition, MyoD may positively regulate apoptotic cascades in myoblasts, which may be a reason for the limited success of current myoblast transplantations.

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.

Discussion

Skeletal myoblasts have been used for many stem cell-based therapies, both experimentally and clinically, especially for DMD and heart failure. There are some advantages to the use of skeletal myoblasts for patients with DMD and heart failure. First, skeletal myoblasts can be isolated from patient or donor skeletal muscle by biopsy and can be expanded ex vivo before autologous or allogenic cell transplantation. Second, after transplantation, engrafted myoblasts can form skeletal muscle fibers in skeletal muscle or the heart and, thus, can improve muscle or heart contractile function. However, severe limitations exist, including immune rejection of allogenic donor cells, poor cellular survival, and limited spread of the injected cells, hindering practical application (8). Previously, Grounds and colleagues (16, 17) performed transplantation experiments by using sliced and whole muscle derived from MyoD^−/− mice, demonstrating increased migratory activity and delayed myotube formation of MyoD^−/− myoblasts in the host muscle. In this study, we demonstrate that myoblasts derived from muscle satellite cells lacking the MyoD gene are a potentially useful source for repair of damaged muscle in DMD patients. MyoD, a skeletal muscle-specific master transcription factor, plays an essential role in myogenic determination. MyoD^−/− myoblasts have been suggested to represent an intermediate stage between quiescent satellite and myogenic precursor cells (14). Therefore, myoblasts lacking MyoD activity may prove useful for cell therapy in damaged skeletal muscle.

The first important finding in this study is that a larger number of MyoD^−/− myoblasts can survive in regenerating muscle after transplantation, compared with wild-type myoblasts (Fig. 1). An increased cell proliferation rate for MyoD^−/− myoblasts may be the cause of enhanced survival in recipient muscle. However, this is unlikely because 24 h after cell injection the number of surviving MyoD^−/− myoblasts is already three times higher than that of wild-type myoblasts. Because high levels of apoptotic cell death were detected during terminal differentiation of wild-type myoblasts (Fig. 5) (31), the slower terminal differentiation of MyoD^−/− myoblasts may protect them from apoptosis. Alternatively, MyoD^−/− myoblasts are partially deficient for apoptotic cascades through direct or indirect gene activation or repression by MyoD because many genes involved in apoptosis are altered in MyoD^−/− myoblasts. Supporting this idea, the overexpression of proapoptotic protein Puma (32) can induce apoptosis in myoblasts in MyoD-null environments (Fig. 6 B and C). By contrast, overexpression of antiapoptotic protein Bcl-2 (32) can convert wild-type myoblasts to the MyoD^−/− phenotype by inhibiting apoptosis (Fig. 6 A and C). Interestingly, Bcl-2 gene expression is up-regulated in MyoD^−/− myoblasts, compared with wild-type myoblasts, in growth conditions (Fig. 3), suggesting that MyoD may be negatively regulating Bcl-2 gene expression. Among other apoptosis-related genes, Myd116 and GADD45α, which regulate growth arrest after stress induction such as UV exposure (33), are highly up-regulated in wild-type myoblasts compared with MyoD^−/− myoblasts. This finding suggests a defect in the cell-cycle arrest after DNA damage occurs in MyoD^−/− myoblasts. In addition, genes involved in mitochondrial function and redox potential also are markedly altered in MyoD^−/− myoblasts. MyoD deficiency may protect myoblasts from generating reactive oxygen species and stimulating mitochondria, which can trigger release of cytochrome C, a mitochondrial initiator of major apoptosis cascades (34, 35). In any case, the evidence for substantially increased survival of engrafted MyoD^−/− myoblasts after transplantation into damaged muscle provides reason to believe that they may effectively improve muscle function compared with wild-type myoblasts.

We also found up-regulation of stem cell markers such as Sca-1, CD34, and Meis1 in MyoD^−/− myoblasts (Figs. 3 and 4). Sca-1, a cell-surface protein, was detected in many adult stem cells, including HSCs (26), muscle-derived stem cells (27), muscle SP cells (26, 28), and some proliferating satellite cells (36). Sca-1 negatively regulates myoblast proliferation and muscle differentiation (36) or positively regulates cell-cycle withdrawal (37). CD34 also is detected in muscle-derived stem cells (27) and quiescent satellite cells, but its expression is down-regulated and becomes more heterogeneous after satellite cell activation (5). Interestingly, CD34-positive myoblasts are negative for MyoD in proliferating myoblasts, indicating that MyoD negatively regulates CD34 expression. Overexpression of FGF6 expands SP cells in the C2C12 myoblast cell line and suppresses terminal differentiation through up-regulation of multidrug-resistant genes and down-regulation of MyoD expression (38), supporting the idea that MyoD deficiency expands the SP phenotype in myoblasts. Taken together, these data indicate that MyoD negatively regulates Sca-1, CD34, and multidrug-resistant genes causing the SP phenotype. Therefore, MyoD deficiency may maintain a more primitive stem cell phenotype.

Recent studies demonstrate that freshly isolated satellite cells contribute remarkably to myofiber regeneration after transplantation (39, 40). In addition, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. After injury, these satellite cells expand extensively and regenerate a large number of new myofibers (39). Expansion of these satellite cells in culture before transplantation reduced their regenerative capacity. Because freshly isolated satellite cells do not express MyoD until activation, they may possess more resistance to the apoptotic cell death than normally happens after transplantation.

For clinical purposes, MyoD^−/− myogenic progenitor cells, including myoblasts and mesoangioblasts (41), are not available for humans. However, isolated myogenic progenitor cells, in which MyoD expression or function is chemically, transcriptionally, or posttranslationally suppressed, could be generated to mimic MyoD^−/− cell effects in transplantation. For example, a possible method to suppress MyoD is gene silencing for myogenic progenitor cells by an RNA interference technique such as siRNA or the lentivirus shRNA expression vector for the MyoD gene. Therefore, myogenic progenitor cells with genetically or chemically suppressed MyoD gene expression could be created and tested for their potential to efficiently engraft in damaged muscle, their contribution to muscle fiber regeneration, and their improvement of muscle function in DMD patients. Therefore, suppression of MyoD in myogenic progenitor cells, including myoblasts and mesoangioblasts (41), would be beneficial to therapy by providing a selective advantage for expansion of the stem cells.

Materials and Methods

Extended details are published in SI Materials and Methods. Briefly, MyoD^−/− mice and wild-type BALB/c mice were used for isolation of primary myoblasts. For cell transplantation, myoblasts were transfected with PGK-lacZ-MAR, and PGK-Puro plasmids by Lipofectamine (Invitrogen, Carlsbad, CA). Stable transformants were pooled after selection in puromycin. Myoblasts also were infected with adenovirus CMV-luciferase, lentiviral pCS2-EF-MyoD, adenovirus CMV-human Puma (Ad-PUMA), CMV-human Bcl-2 (Ad-Bcl2A1), or adenovirus Ad5 CMV ntlacZ. For cell transplantation, Ad5 CMV ntlacZ-infected myoblasts also were used at day 1 before cell injection. Scid/beige-immunodeficient or syngenic BALB/c mice were used as recipient mice for cell transplantation. Muscle regeneration was induced in TA muscle by injection of CTX. Twenty-four hours later, 1 × 10⁶ cells myoblasts were injected into regenerating TA muscle. The engrafted cell number was analyzed by X-Gal and DAPI double-positive nuclei in every 10 serial sections of whole TA muscle. A Xenogen IVIS imaging system also was used to quantify the efficiency of engraftment after transplantation of cells infected with adenovirus CMV-luciferase vector. After X-Gal staining, sections were used for immunofluorescence, and satellite cell fields in TA muscle were viewed with a Jeol 1200EX biosystem transmitted electron microscopy after fixation in 2% glutaraldehyde and 0.1 M cacodylate (pH 7.4). Affymetrix microarrays done by using the Mu11K chip arrays contained ≈13,000 mouse genes and analyzed by GeneSpring 3.2.2 software. Total RNA was isolated by TRIzol, and semiquantitative RT-PCR was performed by using primer pairs described in SI Table 2. Hoechst staining and FACS analysis were performed for the detection of SP cells. For detection of apoptotic cell death, annexin-V staining was performed, and dead cell number was assessed after Trypan blue staining.

Abbreviations

CTX

cardiotoxin

DMD

Duchenne muscular dystrophy

HSC

hematopoietic stem cell

SP

side population

TA

tibialis anterior.