The pig is an excellent biological model for the human supported by the anatomic similarities and the more comparable lifespan than, for instance, rodents. We explain protocols how to isolate muscle satellite cells and progenitor cells from porcine muscle tissue and to purify the side population (SP). Isolation of muscle satellite cells is based on stimulation of cell migration from muscle fibers. We describe how to purify for a specific cell population from this heterogeneous population based on Hoechst dye exclusion ability by the ABC-G2 transporter present on the SP. Furthermore, we discuss the benefits and drawbacks of each isolation method and provide lists of antibodies cross-reactive to porcine antigen epitopes for muscle stem cell characterization. Stem cells can be obtained from the pig muscle within 1 week.
A hierarchy of several progenitor cell populations resides in muscle tissue. The skeletal muscle stem and progenitor cells contribute to muscle growth and maintenance, and are important for repair and regeneration of damaged tissue caused by injury or disease1-5. For several stem cell populations the in vivo localization of the cells remains rather elusive. Some cells are associated with the vascular network, others are in more close contact with the muscle niche6-8. Upon muscle development or maintenance, stem cells are triggered to form myoblasts were they will fuse with the muscle fibers for growth or restoration.
The various stem and progenitor cells in muscle tissue differ in self-renewal capacity, differentiation potential and show diversity in biomarker expression9-11. The myogenic stem cell populations are widely used to study tissue regeneration for the treatment of muscle diseases such as muscular dystrophy or cardiac dysfunction5, 12-14. In addition, muscle tissue can serve as a potential source of myogenic cells to use in toxicology screening, drugs discovery and tissue engineering.
Pigs can be used as animal models to define strategies for treatment of human diseases. Their similarities in life-span and in organ size make these animals excellent models for humans15.
Muscle satellite cells are the most prominent muscle stem cells which were discovered by Mauro in 196116. These cells are located beneath the basal lamina and the sarcolemma of muscle fibers. They are indicated as the muscle stem cells with a high contribution to muscle development and regeneration based on their anatomic position towards the muscle fiber. To isolate these cells, single fibers are first obtained from total muscle biopsies by enzymatic dissociation. Satellite cells are then stimulated to migrate from the fiber and adhere onto tissue culture plastic. The cell isolation technique generates a rather pure population of satellite cells with low numbers of non-myogenic cells1, 17-19. Limitations of this isolation are the low cell yield achievable and the time-consuming technique. Transplanted satellite cells show poor migration and poor survival rates. Furthermore, cell expansion reduces their myogenic differentiation capacity20.
To attain larger amounts of skeletal muscle progenitor cells a more robust isolation method can be performed, in particular by enzymatic digestion of muscle parts of the hind leg of a pig21, 22. Subsequently, these cells can be purified for myogenic stem cell populations such as the side population (SP).
The SP contains stem cells that have a more hematopoietic character. SP cells reside in bone marrow, but also have been found in muscle tissue23-25. It has been shown that muscle-derived SP cells exhibit both hematopoietic and myogenic potentials in vivo. Although, SP cells do not express muscle cell markers, they contribute to the muscle lineage upon transplantation24, 26-28. The sensitivity to verapamil, an inhibitor of multidrug resistance transporter like ABG2, allows the discrimination of SP cells from the main population (MP). Using flow cytometry, we can observe the secretion of toxic Hoechst by the ATP binding cassette ABG2-transporter present on the SP cells. The population of cells with the ability to exclude the Hoechst dye can be selected from the MP and sorted for in vitro expansion. Although SP cells have a poor myogenic differentiation capacity in vitro, they are capable to migrate through the circulation system and contribute to muscle regeneration in vivo. This makes the SP cells excellent candidates for new therapeutic strategies in regenerative medicine29, 30.
To date, several methods have been used to isolate muscle stem cells including muscle-derived stem cells (MDSC), distinct from satellite cells and other progenitor cells6. MDSCs have a high survival capacity, efficient tissue engraftment and their self-renewal and multilineage character make them potentially important for regenerative medicine31-33. This technique has been extensively described by the group of Huard et al. and can be applied to porcine muscle33, 34.
Numerous isolation methods have been described in the last decades to derive high potential muscle stem cell populations. We have adapted protocols from previous studies to improve isolations of cells from larger animals such as the pig 35-39. In addition we report cross-reactive antibodies applicable to characterize and identify in situ porcine stem and progenitor cells.
Comparison with other methods
To obtain large amounts of stem cells, the isolation of muscle progenitor cells method is recommended. The drawback here is the heterogeneous character of the cell population which comprises satellite cells, muscle progenitor cells and non-myogenic cells. The population of cells can be purified using Percoll gradient centrifugation39, 40. Furthermore, membrane-associated biomarkers, such as NCAM (CD56), CD34, M-Cadherin and integrins can be used to select specifically for a stem cell population using flow cytometric cell sorting1, 5, 22, 36, 39, 40.
The isolation of muscle stem cells can be applied to all muscle tissues. Commonly used muscle groups are the semimembrinosus and semitendinosus muscle from the hamstring. However, muscles from the lower leg can also be used. Interestingly, the single fiber satellite cell isolation method allows us to derive cells from a special fiber type (slow or fast) preserving putative intrinsic specializations of the myogenic cell.
The muscle niche is important to control muscle stem cells and direct the behavior in order to maintain their function8, 21. The knowledge concerning important extracellular matrix (ECM) proteins can be applied to stimulate in vitro stem cell growth and myogenic cell differentiation. The correct surface coating of cell culture plastic is crucial to sustain the stem cell character21 (Table 1). For porcine muscle stem cells, it has been shown that fibronectin surface coating supports satellite cell adhesion to the culture plastic. However, improved myogenic differentiation was observed when cells were cultured onto Matrigel and laminin coated areas.
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