Skeletal Muscle Repair and Muscular Dystrophy
Lab Personnel
Lab Projects
The isolation of adult stem cells from a variety of tissues and organs has invigorated the stem cell research community. Like stem cells obtained from other tissue types, the stem cells isolated from skeletal muscle can give rise to cells of other lineages. At least 2 types of stem cells exist in skeletal muscle. Muscle satellite cell populations have long been recognized as a source of stem cells that generate daughter myogenic precursor cells in adult skeletal muscle. Recent reports indicate that skeletal muscle also harbors multipotent stem cells (MPSCs). Various researchers have identified different MPSCs that all exhibit the ability to differentiate into a variety of cell types. Gussoni et al. demonstrated the existence of side population (SP) cells that can be isolated by fluorescence-activated cell sorting (FACS) of Hoechst-stained muscle cells. These SP cells possess stem cell characteristics. After the systemic delivery of SP cells into lethally irradiated mdx mice, an analysis of both dystrophin expression and donor nuclei demonstrated the participation, of the SP cells in muscle regeneration. The SP cells also displayed hematopoietic potential by reconstituting the ablated bone marrow of the lethally irradiated mice.
Muscle-derived Stem Cells:
Researchers in our group have isolated muscle-derived stem cells (MDSCs) by modifying
a method called the preplate technique. The preplate technique, which is based on the varied propensity of cells to adhere to collagen-coated flasks, previously was used to purify myogenic cells.1-5 We purified the cells that required 5 to 7 days to attach to these flasks, and obtained clones (MDSCs) from this highly-enriched myogenic cell population. MDSC clones expressed both myogenic (desmin [-/+] and MyoD) and stem cell markers (CD34 [+/-], Sca-1+, Bcl-2+); however, they were negative for the hematopoietic stem cell marker c-Kit and CD45.4,5 They also were negative for other blood lineage markers (i.e., B220, Mac-1, Gr-1, CD3, CD4, CD5, and CD8). When cultivated in proliferation medium at low confluence (‹30%), the MDSC clones continue to proliferate. When cultivated in fusion medium (low serum–supplemented medium), they spontaneously differentiate into myotubes.
One of the most common techniques used is the fluorescent in situ hybridization (FISH). In this method, DNA sequences from the mouse X or Y chromosome are amplified by degenerate oligonucleotide primer PCR (DOP-PCR), tagged with a fluorophore like FITC or an antigen like digoxigenin or biotin. This sequence (probe or paint) is hybridized overnight at 37 °C to the target cells. On day 2, the slides are washed and the hybridization signal is detected by antibodies conjugated to fluorescent molecules. Typically, we engraft male stem cells into female mice. Engraftment site is checked for donor cells by searching for the Y chromosomal paint signal that appears in one color and the female recipient tissue which shows two X chromosome signals in another color.
Other Projects:
Other research projects include the use of G-banding and C-banding techniques to examine the effect of culture conditions on stem cells. Chromosomal numerical and structural abnormalities are recorded and metaphases are photographed and analyzed.
- Characterization of muscle stem cells.
- To establish efficient and fast method to isolate muscle stem cells.
- Systemic delivery of muscle stem cells to deliver dystrophin gene to mdx mice,animal model of Duchenne Muscular Dystrophy (DMD).
- Gene therapy for DMD using viral vectors. Explore different viral vectors, i.e. adenovirus, adeno-associate virus, and retrovirus.
- Ex vivo gene transfer using viral vector and muscle stem cells to skeletal muscle.
- Investigate the multipotency of muscle stem cells, i.e., hematopoietic differentiation.
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