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Satellite cells, the canonical muscle stem cells, are responsible for postnatal muscle growth and regeneration. They exist in a quiescent state, located in a niche between the basal lamina and muscle sarcolemma1,2,3,4,5. During muscle regeneration, these cells activate, divide asymmetrically to self-renew or form a proliferative population of committed progenitors called myoblasts. These myoblasts differentiate and fuse to form myotubes, the functional component of skeletal muscle6,7,8. Without satellite cells, skeletal muscle regeneration is completely abolished2,9,10. After muscle development or regeneration, some satellite cells return to quiescence and repopulate the niche in preparation for future rounds of regeneration8.
Many have hypothesized that transplanted satellite cells could treat muscle diseases and traumatic muscle injuries8,11. However, satellite cells are rare in skeletal muscle, so large quantities cannot be obtained from postnatal muscle tissue. Furthermore, although several decades of research have provided a deep understanding of the functional properties of satellite cells, it is not known yet how to effectively increase their numbers in culture while maintaining their stem cell characteristics, particularly preserving their ability to repopulate the niche12,13. In conventional tissue culture conditions, satellite cells enter the cell cycle and become myoblasts8. However, satellite cells and myoblasts have key differences. Although both can produce muscle fibers, after transplant, satellite cells are up to 1,000 times more efficient at this process7,12,14. Only satellite cells can repopulate the stem cell niche, self-renew and sustain muscle regeneration over the lifetime of an organism6,7,12,14. These critical differences between satellite cells and myoblasts underscore the need to generate therapeutically relevant quantities of satellite cells in vitro.
Culturing cells in three dimensions can recreate stem cell niche conditions for several tissues, enabling adult stem cell expansion while retaining their capacity to differentiate and mature15,16,17,18. Evidence shows that adult progenitor cells in the tooth, liver, gut and pancreas dedifferentiate into a stem cell-like state in vivo and, in some cases, in three-dimensional (3D) cultures19,20,21. We studied the effect of 3D culture on proliferating adult myoblasts. Myoblasts can generate skeletal muscle organoids (SkMOs) containing cells resembling satellite cells, termed in vitro-derived satellite cells (idSCs). Upon transplantation into damaged or diseased muscle, idSCs regenerate muscle similarly to satellite cells. Although idSCs share key pathways known to regulate quiescence and myogenic commitment, they are transcriptionally and epiginomically distinct, raising several interesting questions regarding the relationship between in vitro and in vivo cellular identities.
ResultsSkMOs contain satellite-like cells
Satellite cells express Pax7, an essential transcription factor (TF) for satellite cell maintenance and commitment2,22. During skeletal muscle regeneration, myoblasts maintain Pax7 expression but activate MyoD, a TF involved in myogenic commitment and differentiation23,24. MyoD expression, coupled with the downregulation of Pax7, commits cells to differentiation, resulting in the loss of stem cell potential12. To investigate whether myoblasts (Pax7+MyoD+) can reduce the expression of MyoD and dedifferentiate back to a satellite cell-like state (Pax7+MyoD−), we seeded myoblasts into spinner flasks in proliferation medium17, where they self-assembled in three dimensions into SkMOs25. After 20 d, we reduced the serum concentration and allowed the cells to differentiate. After an additional 10 d, the SkMOs were predominantly composed of cells that expressed myosin heavy chain (MyHC), a marker for terminally differentiated myotubes26, and cells that expressed Pax7, many of which were present near myotubes (Fig. 1a,b). Although MyoD was expressed in the vast majority (98% ± 0.70%) of the Pax7+ myoblasts used to generate SkMOs, it was expressed in only a small percentage (9% ± 4.28%) of the Pax7+ cells present in late-stage SkMOs (Fig. 1c,d).
Fig. 1: Mouse myoblasts can form SkMOs that share in vitro characteristics with satellite cells.
a, Schematic of the isolation and expansion of mouse myoblasts in vitro followed by the formation, growth and maturation of an SkMO. b, Immunofluorescent images of a late-stage SkMO stained with Pax7 (green), MyHC (red) and nuclear counterstain Hoechst (blue). Scale bars, 100 µm. c, Immunostaining of myoblasts and late-stage SkMOs for MyoD (red), Pax7 (green) and Hoechst (blue). Scale bars, 20 µm. d, Percentage of Pax7+MyoD+ cells in myoblasts and late-stage SkMOs. Box bounds represent s.e.m.; red line represents mean (n = 3, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P value: Myo versus SkMO 3.37 × 10−5; ***P < 0.001. e, Immunofluorescent images from EdU pulse chase experiments over 48 h comparing myoblasts, quiescent satellite cells and late-stage SkMO cells. Images show Pax7 (green), EdU (red) and Hoechst (blue). Scale bars, 10 µm. f, Percentage of Pax7+EdU+ cells in each cell condition. Box bounds represent s.e.m.; red line represents mean (satellite cell n = 4, myoblast n = 3, SkMO n = 3, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: SC versus SkMO 0.205, SC versus Myo 2.0 × 10−6; Myo versus idSC 3.5 × 10−5; ***P < 0.001. g, Representative images of myoblasts, SkMO-derived GFP+ cells and quiescent satellite cells immediately after FACS isolation. Scale bars, 10 µm. h, Comparisons of cell diameter in proliferative myoblasts, SkMO-derived GFP+ cells and quiescent satellite cells. Box bounds represent s.e.m.; red line represents mean (n = 3, biological replicates with ≥100 cells per replicate). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: Myo versus SC 2.22 × 10−4; Myo versus SkMO 8.98 × 10−4; SkMO versus SC 1.7 × 10−4; ***P < 0.001. NS, not significant; Myo, myoblast; SC, satellite cell.
To validate our findings, we used a transgenic Pax7nGFP reporter mouse strain (Tg:Pax7nGFP)27 to FACS purify satellite cells (>95% GFP+) and derive, in standard two-dimensional (2D) growth conditions, several primary myoblast lines subsequently used to generate SkMOs (Supplementary Fig. 1a,b). After 30 d of organoid culture, GFP+ cells accounted for 18% ± 2% of the mononuclear cells within each organoid (Supplementary Fig. 1c). This provided approximately 3 ± 1.5 million GFP+ cells per spinner flask (Supplementary Fig. 1d). Finally, we compared the presence of canonical myogenic cell surface markers among myoblasts, SkMO GFP+ cells and freshly isolated satellite cells (Supplementary Fig. 1e) and found that SkMO GFP+ cells closely resembled the profile of satellite cells (Cxcr4+, CD104− and CD200+)28.
In intact muscle, satellite cells are normally quiescent. Although the parental myoblasts used to form SkMOs incorporated the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) (93% ± 4.20% EdU+), indicative of cycling cells, very few Pax7+ cells (3% ± 1.5% EdU+) present in late-stage SkMOs were EdU+ (Fig. 1e,f). Additionally, a quiescent satellite cell can be distinguished from an activated satellite cell by cell size alone29,30. Consistent with those findings, FACS-sorted late-stage SkMO-derived GFP+ cells were significantly smaller (9.15 ± 0.17 µm in diameter) than FACS-sorted GFP+ myoblasts (15.39 ± 0.68 µm), more closely resembling freshly isolated GFP+ primary satellite cells (6.76 ± 0.05 µm) (Fig. 1g,h). To further confirm that SkMO GFP+ cells have acquired properties of satellite cells, we set up a clonal growth assay modified from those used frequently to ascertain stem cell function in the neural and hematopoietic fields31,32,33,34. Using FACS to sort one GFP+ cell per well of a 96-well spheroid plate, we quantified the ability of freshly isolated Pax7nGFP satellite cells, myoblasts (derived from the satellite cells in two dimensions) and SkMO-derived GFP+ cells to form colonies in suspension (Supplementary Fig. 2a). After the confirmation that a single cell resided per well after 24 h in culture (Supplementary Fig. 2b), we found that freshly isolated satellite cells and SkMO-derived GFP+ cells, but not myoblasts, could form distinct clones (≥3 cells in a colony) (Supplementary Fig. 2c,d). Quantification of the approximate number of cells per clone revealed that satellite cells and SkMO-derived GFP+ cells gave rise to an average of 12.5 ± 0.9 cells per well and 6.9 ± 1.5 cells per well, respectively, whereas myoblasts rarely proliferated under these conditions (1.18 ± 0.16 cells per well) (Supplementary Fig. 2d). The regenerative properties of SkMO GFP+ cells were further illustrated by their ability to expand the number of SkMOs when passaged in spin culture (Supplementary Fig. 2e). Finally, we found that purified SkMO-derived GFP+ cells, as would be expected, retained their capacity to give rise to proliferative myoblasts capable of fusing to form multinucleated myotubes when cultured under standard differentiation conditions in vitro (Supplementary Fig. 2f).
Thus, using multiple criteria, including loss of MyoD expression, quiescence, cell size and clonal growth, combined with the ability to form differentiated muscle, it appears as if a population of cells resembling satellite cells arises when myoblasts are cultured in organoid-like conditions. For the remainder of this manuscript, we refer to SkMO-derived GFP+ cells as idSCs.
Satellite cells and idSCs share transcriptional similarities
To study transcriptional changes that define idSCs as they arise in myoblast-seeded organoids, we conducted multiplexed RNA sequencing (RNA-seq) analyses (3′ DGE) of freshly isolated satellite cells, myoblasts and idSCs, all derived from the Tg:Pax7nGFP mouse. We further obtained Pax7+ reserve cells, which are present in small numbers after traditional 2D differentiation in vitro of Tg:Pax7nGFP myoblasts into myotubes, as they represent the closest in vitro analogue to satellite cells35,36 (Supplementary Fig. 3a). A focused analysis of genes known to be involved in satellite cell maintenance/activation11 clustered idSCs with endogenous satellite cells while clearly distinguishing them from both myoblasts and reserve cells (Fig. 2a). Among the genes separating idSCs and satellite cells from myoblasts were genes associated with the Notch signaling pathway (Notch3, HeyL and Jag1), early activation/stress response (Fos, Egr1 and Socs3), quiescence (Spry1, Gas1 and Timp3), cell surface (Cdh15, Cxcr4 and Fgfr4), extracellular matrix (ECM) (Dcn, Dag1 and Col15a1) and secreted growth factors (Igf1, Bmp4 and Wnt4). Quantitative polymerase chain reaction (qPCR) measurements and transcripts per million (TPM) analysis from our bulk RNA-seq dataset confirmed many of these findings (Supplementary Fig.
