Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting.
Fluorescent-activated cell sorting (FACS) has enabled the direct isolation of highly enriched skeletal muscle stem cell, or satellite cell, populations from postnatal tissue. Several distinct surface marker panels containing different positively selecting surface antigens have been used to distinguish muscle satellite cells from other non-myogenic cell types. Because functional and transcriptional heterogeneity is known to exist within the satellite cell population, a direct comparison of results obtained in different laboratories has been complicated by a lack of clarity as to whether commonly utilized surface marker combinations select for distinct or overlapping subsets of the satellite cell pool. This study therefore sought to evaluate phenotypic and functional overlap among popular satellite cell sorting paradigms.



Utilizing a transgenicPax7-zsGreen reporter mouse, we compared the overlap between the fluorescent signal of canonical paired homeobox protein 7 (Pax7) expressing satellite cells to cells identified by combinations of surface markers previously published for satellite cells isolation. We designed two panels for mouse skeletal muscle analysis, each composed of markers that exclude hematopoietic and stromal cells (CD45, CD11b, Ter119, CD31, and Sca1), combined with previously published antibody clones recognizing surface markers present on satellite cells (β1-integrin/CXCR4, α7-integrin/CD34, and Vcam1). Cell populations were comparatively analyzed by flow cytometry and FACS sorted for functional assessment of myogenic activity.



Consistent with prior reports, each of the commonly used surface marker schemes evaluated here identified a highly enriched satellite cell population, with 89-90 % positivity for Pax7 expression based on zsGreen fluorescence. Distinct surface marker panels were also equivalent in their ability to identify the majority of the satellite cell pool, with 90-93 % of all Pax7-zsGreen positive cells marked by each of the surface marker schemes. The direct comparison among surface marker schemes validated their selection for highly overlapping subsets of cells. Functional analysis in vitro showed no differences in the abilities of cells sorted by these different methods to grow in culture and differentiate.



This study demonstrates the equivalency of several previously published and widely utilized surface marker schemes for isolating a highly purified and myogenically active population of satellite cells from the mouse skeletal muscle, which should facilitate cross-comparison of data across laboratories.
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138 USA.
Claire C
CC
Maesner
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138 USA.
Albert E
AE
Almada
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138 USA.|Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA 02115 USA.
Amy J
AJ
Wagers
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D039001:Integrin alpha Chains / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D051763:PAX7 Transcription Factor / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000378:metabolism; D032496:Satellite Cells, Skeletal Muscle / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D015703:Antigens, CD; D018952:Antigens, CD34; C514936:CXCR4 protein, mouse; D039001:Integrin alpha Chains; D019012:Integrin beta1; D008565:Membrane Proteins; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; D019718:Receptors, CXCR4; D019010:Vascular Cell Adhesion Molecule-1; C085725:integrin alpha7
Fluorescence-activated cell sorting; Satellite cells; Surface marker