RESUMEN
Investigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.
Asunto(s)
Células Satélite del Músculo Esquelético/citología , Animales , Separación Celular/métodos , Células Cultivadas , Distrofina/metabolismo , Humanos , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Regeneración/fisiología , Células Satélite del Músculo Esquelético/metabolismo , Trasplante de Células Madre/métodos , Células Madre/citología , Células Madre/metabolismo , Trasplante Heterólogo/métodosRESUMEN
Coreceptor CD4 and CD8αß double-negative (DN) TCRαß(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαß(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαß(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αß(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαß(+) T cells form a third lineage of TCRαß T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.