A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during culture. Here we describe an artificial niche for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. Using a machine learning method, we identified a molecular signature of quiescence and used it to screen for factors that could maintain mouse MuSC quiescence, thus defining a quiescence medium (QM). We also engineered muscle fibers that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in QM on engineered fibers showed enhanced potential for engraftment, tissue regeneration and self-renewal after transplantation in mice. An artificial niche adapted to human cells similarly extended the quiescence of human MuSCs in vitro and enhanced their potency in vivo. Our approach for maintaining quiescence may be applicable to stem cells isolated from other tissues.
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Montarras, D. et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 2064–2067 (2005).
Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. & Blau, H.M. Self-renewal and expansion of single transplanted muscle stem cells. Nature 456, 502–506 (2008).
Cerletti, M. et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell 134, 37–47 (2008).
Li, L. & Clevers, H. Coexistence of quiescent and active adult stem cells in mammals. Science 327, 542–545 (2010).
Fuchs, E. The tortoise and the hair: slow-cycling cells in the stem cell race. Cell 137, 811–819 (2009).
Pietras, E.M., Warr, M.R. & Passegué, E. Cell cycle regulation in hematopoietic stem cells. J. Cell Biol. 195, 709–720 (2011).
Ottone, C. et al. Direct cell-cell contact with the vascular niche maintains quiescent neural stem cells. Nat. Cell Biol. 16, 1045–1056 (2014).
Cheung, T.H. & Rando, T.A. Molecular regulation of stem cell quiescence. Nat. Rev. Mol. Cell Biol. 14, 329–340 (2013).
Schepers, K., Campbell, T.B. & Passegué, E. Normal and leukemic stem cell niches: insights and therapeutic opportunities. Cell Stem Cell 16, 254–267 (2015).
Gupta, N. et al. Neural stem cell engraftment and myelination in the human brain. Sci. Transl. Med. 4, 155ra137 (2012).
Gilbert, P.M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).
Chakkalakal, J.V., Jones, K.M., Basson, M.A. & Brack, A.S. The aged niche disrupts muscle stem cell quiescence. Nature 490, 355–360 (2012).
Fu, J. et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7, 733–736 (2010).
Nishikawa, S.-I., Osawa, M., Yonetani, S., Torikai-Nishikawa, S. & Freter, R. Niche required for inducing quiescent stem cells. Cold Spring Harb. Symp. Quant. Biol. 73, 67–71 (2008).
Cheung, T.H. et al. Maintenance of muscle stem-cell quiescence by microRNA-489. Nature 482, 524–528 (2012).
Collins, C.A. et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 (2005).
Dellatore, S.M., Garcia, A.S. & Miller, W.M. Mimicking stem cell niches to increase stem cell expansion. Curr. Opin. Biotechnol. 19, 534–540 (2008).
Lutolf, M.P., Gilbert, P.M. & Blau, H.M. Designing materials to direct stem-cell fate. Nature 462, 433–441 (2009).
Fu, X. et al. Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion. Cell Res. 25, 655–673 (2015).
Choi, J.S., Mahadik, B.P. & Harley, B.A.C. Engineering the hematopoietic stem cell niche: frontiers in biomaterial science. Biotechnol. J. 10, 1529–1545 (2015).
Lutolf, M.P., Doyonnas, R., Havenstrite, K., Koleckar, K. & Blau, H.M. Perturbation of single hematopoietic stem cell fates in artificial niches. Integr. Biol. (Camb) 1, 59–69 (2009).
Lugert, S. et al. Quiescent and active hippocampal neural stem cells with distinct morphologies respond selectively to physiological and pathological stimuli and aging. Cell Stem Cell 6, 445–456 (2010).
Csaszar, E. et al. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell 10, 218–229 (2012).
Schultz, E., Gibson, M.C. & Champion, T. Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J. Exp. Zool. 206, 451–456 (1978).
MAURO. A. Satellite cell of skeletal muscle fibers 9, 493–495 (1961).
Fukada, S.-I. et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25, 2448–2459 (2007).
Xu, C. et al. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell 155, 909–921 (2013).
Cosgrove, B.D. et al. Rejuvenation of the muscle stem cell population restores strength to injured aged muscles. Nat. Med. 20, 255–264 (2014).
Bernet, J.D. et al. p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice. Nat. Med. 20, 265–271 (2014).
Rathbone, C.R. et al. Effects of transforming growth factor-beta (TGF-β1) on satellite cell activation and survival during oxidative stress. J. Muscle Res. Cell Motil. 32, 99–109 (2011).
Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).
Discher, D.E., Janmey, P. & Wang, Y. Tissue cells feel and respond to the stiffness of their substrate. Science 18, 1139–1143 (2005).
Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
Defranchi, E. et al. Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres. Microsc. Res. Tech. 67, 27–35 (2005).
Trensz, F. et al. Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation. Skelet. Muscle 5, 5 (2015).
Brightman, A.O. et al. Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro. Biopolymers 54, 222–234 (2000).
Badylak, S.F., Freytes, D.O. & Gilbert, T.W. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 5, 1–13 (2009).
Kirkwood, J.E. & Fuller, G.G. Liquid crystalline collagen: a self-assembled morphology for the orientation of mammalian cells. Langmuir 25, 3200–3206 (2009).
Urciuolo, A. et al. Collagen VI regulates satellite cell self-renewal and muscle regeneration. Nat. Commun. 4, 1964 (2013).
Bjornson, C.R.R. et al. Notch signaling is necessary to maintain quiescence in adult muscle stem cells. Stem Cells 30, 232–242 (2012).
Dean, D.C., Iademarco, M.F., Rosen, G.D. & Sheppard, A.M. The integrin alpha 4 beta 1 and its counter receptor VCAM-1 in development and immune function. Am. Rev. Respir. Dis. 148, S43–S46 (1993).
Jülich, D., Geisler, R. & Holley, S.A. Tübingen 2000 Screen Consortium. Integrinalpha5 and delta/notch signaling have complementary spatiotemporal requirements during zebrafish somitogenesis. Dev. Cell 8, 575–586 (2005).
McDonald, K.A., Lakonishok, M. & Horwitz, A.F. Alpha v and alpha 3 integrin subunits are associated with myofibrils during myofibrillogenesis. J. Cell Sci. 108, 975–983 (1995).
Hirsch, E. et al. Alpha v integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development. Dev. Dyn. 201, 108–120 (1994).
Sastry, S.K., Lakonishok, M., Thomas, D.A., Muschler, J. & Horwitz, A.F. Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J. Cell Biol. 133, 169–184 (1996).
Yang, J.T. et al. Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle. J. Cell Biol. 135, 829–835 (1996).
Yin, H., Price, F. & Rudnicki, M.A. Satellite cells and the muscle stem cell niche. Physiol. Rev. 93, 23–67 (2013).
Sanes, J.R. The basement membrane/basal lamina of skeletal muscle. J. Biol. Chem. 278, 12601–12604 (2003).
Gulberg, D., Tiger, C.F. & Velling, T. Laminins during muscle development and in muscular dystrophies. Cell. Mol. Life Sci. 56, 442–460 (1999).
Breiman, L. Random forests. Mach. Learn. 45, 5–32 (2001).
Rando, T.A. The adult muscle stem cell comes of age. Nat. Med. 11, 829–831 (2005).
Hall, J.K., Banks, G.B., Chamberlain, J.S. & Olwin, B.B. Prevention of muscle aging by myofiber-associated satellite cell transplantation. Sci. Transl. Med. 2, 57ra83 (2010).
Bareja, A. et al. Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis. PLoS One 9, e90398 (2014).
Charville, G.W. et al. Ex vivo expansion and in vivo self-renewal of human muscle stem cells. Stem Cell Rep. 5, 621–632 (2015).
Konieczny, P., Swiderski, K. & Chamberlain, J.S. Gene and cell-mediated therapies for muscular dystrophy. Muscle Nerve 47, 649–663 (2013).
Partridge, T.A. Impending therapies for Duchenne muscular dystrophy. Curr. Opin. Neurol. 24, 415–422 (2011).
Busch, K. et al. Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518, 542–546 (2015).
Shandalov, Y. et al. An engineered muscle flap for reconstruction of large soft tissue defects. Proc. Natl. Acad. Sci. USA 111, 6010–6015 (2014).
Nishijo, K. et al. Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J. 23, 2681–2690 (2009).
Johnson, W.E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).
Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 1540–1542 (2006).
Singh, G., Mémoli, F. & Carlsson, G.E. Topological Methods for the Analysis of High Dimensional Data Sets and 3D Object Recognition. in Symposium on Point Based Graphics 07 (European Association for Computer Graphics) 91–100. http://dx.doi.org/10.2312/SPBG/SPBG07/091-100 (2007).
Rosenblatt, J.D., Lunt, A.I., Parry, D.J. & Partridge, T.A. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell. Dev. Biol. Anim. 31, 773–779 (1995).
Roure du, O. et al. Force mapping in epithelial cell migration. Proc. Natl. Acad. Sci. USA 102, 2390–2395 (2005).
Hansen, C.L., Sommer, M.O.A. & Quake, S.R. Systematic investigation of protein phase behavior with a microfluidic formulator. Proc. Natl. Acad. Sci. USA 101, 14431–14436 (2004).
We thank members of the Rando laboratory for comments and discussions, in particular M. Hamer and I. Akimenko for helping with some of the experiments performed with human MuSCs; L.-M. Joubert for technical support with scanning electron microscopy; E. Araci for technical support at the Stanford Microfluidic Foundry; C. Horst of Cellscale Biomaterials Testing for support in the tensile measurements of the EMF; M. Adorno from Stanford Stem Cell Institute for providing the lentivirus construct expressing luciferase and GFP; E. Cadag for the technical support with topological data analysis; M. Lynch for technical support with the micropost array fabrication; and A. Wang for technical support with the AFM measurements. This work was supported by the Glenn Foundation for Medical Research and by grants from the National Institutes of Health (NIH) (P01 AG036695, R01 AG23806 (R37 MERIT award), R01 AR062185, and R01 AG047820) the California Institute of Regenerative Medicine, and the Department of Veterans Affairs (BLR&D and RR&D Merit Reviews) to T.A.R.; by NIH P41 program: RESBIO The Technology Resource for Polymeric Biomaterials (EB001046); and by the CalPoly funding award TB1-01175.
The authors declare no competing financial interests.
Integrated supplementary information
a. Analysis of differential gene regulation between quiescent and activated single MuSCs. PCA loadings were generated for genes after combinatorial Q-RT-PCR of single MuSCs FACS isolated at 0, 1.5, and 3.5 DPI. Shown are standard deviational ellipses (radius = 1 SD) after k-medians clustering with k = 2. b. Topological data analysis (TDA) of single MuSCs freshly isolated by FACS. TDA was used to identify the quiescent signature (in grey) in an unsupervised fashion (see Methods) within single cell populations. Nodes in the network correspond to collections of samples, and edges connect two nodes where samples exist in both nodes; edge distance does not carry any meaning in this representation. Node size is correlated with the number of samples in each node, and the coloring corresponds to Pax7 expression levels, with a blue-red gradient signifying low-to-high.
a. Heat map analysis of gene expression profiles obtained from MuSCs cultured in different concentrations of candidate molecules. In 96 well plates, 500 MuSCs were directly isolated by FACS and cultured in the presence of the candidate molecules titrated at different concentrations. After 2.5 days, cells were collected for combinatorial Q-RT-PCR to screen for the conditions that result mostly closely resemble freshly isolated MuSCs based on the gene expression profiles. A heat map for absolute deviations from expression levels of freshly isolated quiescent MuSCs (QSC) is shown. On the X axis, the molecules tested and the different dilutions employed from the working concentrations are indicated as in Table S2. On the Y axis the names of the genes that were tested are listed. The gene names were organized in general functional groups. Each functional group is based on the main function and role in MuSCs.
a. Heat map analysis of gene expression profiles obtained from MuSCs cultured in different combinations of candidate molecules. Based on the panel of candidate molecules previously screened as in Supplementary Figure 2, a combinatorial screening for the top 8 molecules was performed on 500 FACS freshly isolated MuSCs cultured for 60 hours. Results are represented in a heat map clustering, based on Manhattan distance after UV-scaling and mean-centering genes Ward linkage. A line at the bottom of the heat map separates the profile of freshly isolated MuSCs (indicated on the right in dark blue as FI) and MuSCs cultured in the combination of the 8 molecules that resulted in the gene expression prolife that was closest to that of freshly isolated MuSCs (indicated in light blue as QM). The scale bar (lower right) indicates the relative gene expression (Log 2).
a. Representative AFM surface scanning of a single myofiber explant. (Top panel) The topography of the surface (scale bar in microns) is shown. (Bottom panel) Stiffness measurements obtained with nanoindentation scanning are shown (scale bar in kPa). Freshly isolated single fibers were incubated overnight on a Laminin/Collagen-coated glass coverslip to allow them to adhere to the substrate. The following day the fibers were mounted onto the AFM stage for analysis. b. Representative immunofluorescence images of FACS isolated MuSCs cultured for 72 hours on microposts of 2 kPa or 12 kPa. Staining for EdU is in green; nuclei are stained blue. c. Percentages of quiescent (i.e. EdU-ve) MuSCs cultured on microposts of different elasticity. Freshly FACS isolated MuSCs were cultured for 72 hours. A pulse of EdU was administered to the cells for the last 12 hours. Twenty-two fields per condition in three technical replicates were counted (error bars are s.e.m.). d. Representative immunofluorescence images of FACS isolated MuSCs cultured for 72 hours on microposts. Two differently labeled MuSCs (one is Pax7+ve/MyoD-ve and the other is Pax7-ve/MyoD+ve) are shown. e. Percentages of Pax7+ve or MyoD+ve cells cultured for 72 hours on microposts of different elasticity as shown in “d”. In this experiment, 50 fields per condition in three technical replicates were counted (error bars are s.e.m.).
a. AFM nanoindentation measurements of the stiffness of Collagen-based hydrogels generated using different formulations. b. Percentages of EdU+ve MuSCs cultured on Collagen-based hydrogels generated using different formulations, as in panel “a”. MuSCs were freshly isolated by FACS and cultured for 36 hours in the presence of EdU (error bars are s.e.m., n = 3). c. Percentages of Caspase+ve MuSCs on Collagen-based hydrogels generated using different formulations, as in panel “a”. MuSCs were freshly isolated by FACS and cultured for 36 hours (error bars are s.e.m., n = 3).
a. AFM nanoindentation measurements of the stiffness of Collagen-based hydrogel or EMFs generated with the same formulation (error bars are s.e.m., n = 3). b. Percentages of proliferating MuSCs cultured alone or on EMFs. Cells were cultured in the presence of EdU for 48 hours (error bars are s.e.m., n = 3). c. Micro-tensile mechanical stress capacity measurements of EMFs. (Left panel) Representative micrographs of the experimental set up, showing the cantilever (above) used to clip and pull an EMF (visible in the middle and indicated by the green arrow in the lower right panel) and the manipulator used to hold the EMF below the cantilever. (Right panel) Measurements of EMF tensile mechanical stress capacity expressed in Pa are shown (Young’s Modulus is Et = 26.9 kPa).
a. Representative images of IF-IHC staining of an uninjured TA muscle showing in cross section myofibers that are Integrin α4β1+ve. b. Representative images of immunofluorescence staining of single myofiber explants showing Integrin α4β1+ve staining in proximity to a MuSC. Individual myofibers were dissociated and immediately fixed. Percentages of EdU+ve (c) or Caspase+ve (d) MuSCs plated on different Integrins at different concentrations. FACS-isolated MuSCs were cultured for 36 hours on substrates coated with different concentrations of Integrin heterodimers (error bars are s.e.m., n = 3).
a. Percentages of MuSCs that were EdU+ve on differently functionalized EMFs. MuSCs were FACS-isolated and cultured for 36 hours in the presence of EdU. The percentage of EdU+ve MuSCs grown in the absence of EMFs (“No Fiber”) is shown for comparison (error bars are s.e.m., n = 3). b. ATP measurements of FACS-isolated MuSCs cultured on differently functionalized EMFs for 36 hours (10,000 cells were analyzed per condition in three biological replicates, error bars are s.e.m.). c. Myogenic progression of FACS-isolated MuSCs cultured on EMFs with different functionalization. MuSCs were stained for Pax7 and MyoD to assess their state in the myogenic progression from quiescence after 72 hours in culture (error bars are s.e.m., n = 3). d. Percentages of EdU+ve MuSCs cultured on Collagen-based soft thick hydrogels or on rigid substrates with a thin Collagen coating, generated at different Collagen formulations and similarly functionalized with Integrin and Laminin proteins. MuSCs were freshly isolated by FACS and cultured for 36 hours in the presence of EdU (error bars are s.e.m., n = 3).
Supplementary Figures 1–8 and Supplementary Note (PDF 3924 kb)
Gene expression array. (XLSX 453 kb)
Small molecules list. (XLSX 14 kb)
Quiescence media formulation. (XLSX 12 kb)
Primers sequence. (XLSX 19 kb)
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Quarta, M., Brett, J., DiMarco, R. et al. An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy. Nat Biotechnol 34, 752–759 (2016). https://doi.org/10.1038/nbt.3576
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