Baohong Cao

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells

Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle. Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34-positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo.

Antioxidant levels represent a major determinant in the regenerative capacity of muscle stem cells.

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.

The Use of Adeno-Associated Virus to Circumvent the Maturation-Dependent Viral Transduction of Muscle Fibers

Muscle-based gene therapy using adenovirus, retrovirus, and herpes simplex virus has been hindered by viral cytotoxicity, host immune response, and the maturation-dependent viral transduction of muscle fibers. The development of new mutant vectors has greatly reduced the toxicity and the immune rejection problems, but the inability of viral vectors to penetrate and transduce mature myofibers remains an important issue. Research has been focused on the characterization of barriers to viral transduction in mature myofibers to develop strategies to circumvent the maturation-dependent viral transduction of myofibers. Here, we report that adeno-associated virus (AAV) can be used to overcome the maturation-dependent viral transduction of myofibers. We have investigated by which mechanism AAV can penetrate and efficiently transduce mature muscle fibers, and have shown that this viral vector is not blocked by the basal lamina and that AAV transduction of myofibers is independent of myoblast mediation. Although AAV can efficiently transduce mature myofibers, a differential transduction is still observed among the different types of myofibers that correlates with the expression of the heparan sulfate proteoglycan receptors, the muscle maturity, the number of viral particles used, and the time postinjection. The identification of the mechanisms by which AAV transduces mature myofibers will help in the development of strategies to achieve an efficient muscle-based gene therapy for inherited and acquired diseases.

Modeling Stem Cell Population Growth: Incorporating Terms for Proliferative Heterogeneity

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle‐derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Muscle-derived cell transplantation and differentiation into lower urinary tract smooth muscle.

OBJECTIVES To explore the feasibility of primary skeletal muscle-derived cell (MDC)-based tissue engineering and gene transfer into the lower urinary tract and to explore whether the injected primary skeletal MDCs can persist and differentiate into myotubes and myofibers in the bladder wall. METHODS Primary MDCs isolated from normal mice were first transduced with adenovirus encoding the expression of the beta-galactosidase reporter gene. Adult severe combined immunodeficiency mice (n = 12) were used in this study. The MDCs were injected into the right and left lateral bladder walls with a 10-microL Hamilton microsyringe. The amount of injected MDCs ranged from 1 to 1.5 x 10(6) cells. The tissue was harvested after 5, 35, and 70 days, sectioned, stained for fast myosin heavy chain, and assayed for beta-galactosidase expression. RESULTS We observed a large number of cells expressing beta-galactosidase in the bladder wall at each time point. Many myotubes and myofibers expressing beta-galactosidase and positively stained for fast myosin heavy chain were also seen in the bladder wall at 35 and 70 days after injection. Additionally, the size of the injected MDCs significantly increased during the course of the study (P <0.05). CONCLUSIONS We have demonstrated the long-term survival and beta-galactosidase expression of MDCs injected into the bladder wall. Moreover, our results suggest that some injected MDCs can differentiate into myofibers. These results suggest that MDCs can be a desirable substance for tissue engineering and an ex vivo method for gene transfer into the lower urinary tract.

Muscle-derived stem cells.

No abstract yet available.

Dystrophin Delivery in Dystrophin-Deficient DMDmdx Skeletal Muscle by Isogenic Muscle-Derived Stem Cell Transplantation

Duchennes muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

Muscle stem cells can act as antigen-presenting cells: implication for gene therapy

Research has shown that the use of a muscle-specific promoter can reduce immune response and improve gene transfer to muscle fibers. We investigated the efficiency of direct and ex vivo gene transfer to the skeletal muscles of 6- to 8-week-old mdx mice by using two adenoviral vectors: adenovirus (AD) encoding the luciferase gene under the cytomegalovirus (CMV) promoter (ADCMV) and AD encoding the same gene under the muscle creatine kinase (MCK) promoter (ADMCK). Direct intramuscular injection of ADMCK triggered a lower immune response that enabled more efficient delivery and more persistent expression of the transgene than did ADCMV injection. Similarly, ex vivo gene transfer using ADCMV-transduced muscle-derived stem cells (MDSCs) induced a stronger immune response and led to shorter transgene expression than did ex vivo gene transfer using ADMCK-transduced MDSCs. This immune response was due to the release of the antigen after MDSC death or to the ADCMV-transduced MDSCs acting as antigen-presenting cells (APCs) by expressing the transgene and rapidly initiating an immune response against subsequent viral inoculation. The use of a muscle-specific promoter that restricts transgene expression to differentiated muscle cells could prevent MDSCs from becoming APCs, and thereby could improve the efficiency of ex vivo gene transfer to skeletal muscle.

The role of receptors in the maturation-dependent adenoviral transduction of myofibers

One of the major hurdles facing the application of adenoviral gene transfer to skeletal muscle is the maturation-dependent transduction of muscle myofibers. It was recently proposed that the viral receptors (Coxsackie and adenovirus receptor (CAR) and the integrins αvβ3/β5) play a major role in the poor adenoviral transduction of mature myofibers. Here we report the findings of morphological studies designed to determine experimentally the role of receptors in the adenoviral transduction of mature myofibers. First, we observed that the expression of both attachment and internalization receptors did not change significantly during muscle development. Second, when an extended tropism adenoviral vector (AdPK) that attaches to heparan sulfate proteoglycan (HSP) is used, a significant reduction of adenoviral transduction still occurs in mature myofibers despite HSPs high expression in mature skeletal muscle fibers. Third, when the adeno-associated virus (AAV) is used, which also utilizes HSP as a viral receptor, muscle fibers at different maturities can be highly transduced. Fourth, the pre-irradiation of the skeletal muscle of newborn mice to inactivate myoblasts dramatically decreased the transduction level of Ad and AdPK, but had no effect on AAV-mediated viral transduction of immature myofibers. These results taken together suggest that the viral receptor(s) is not a major determinant in maturation-dependent adenoviral transduction of myofibers.