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An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy

Nature Biotechnology volume 34, pages 752–759 (2016)Cite this article

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Abstract

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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Figure 1: Formulation and functional characterization of QM.
Figure 2: Artificial muscle fibers support functional quiescence of MuSCs in vitro.
Figure 3: Mouse MuSCs cultured in QM on EMFs are transcriptionally similar to quiescent MuSCs in vivo.
Figure 4: Transplant potency enhancement via the artificial niche.
Figure 5: EMF maintains MuSC self-renewal capacity after in vitro manipulations.
Figure 6: Artificial niche preserves the potency of human MuSCs.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA

    Marco Quarta, Jamie O Brett, Antoine De Morree, Stephane C Boutet, Robert Chacon, Michael C Gibbons, Victor A Garcia & Thomas A Rando

  2. Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, California, USA

    Marco Quarta, Jamie O Brett, Antoine De Morree, Stephane C Boutet, Robert Chacon, Michael C Gibbons, Victor A Garcia & Thomas A Rando

  3. Center for Tissue Regeneration, Repair and Restoration, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, USA

    Marco Quarta, Robert Chacon, Michael C Gibbons, Victor A Garcia & Thomas A Rando

  4. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA

    Jamie O Brett

  5. Department of Bioengineering, Stanford University, Stanford, California, USA

    Rebecca DiMarco

  6. Department of Materials Science and Engineering, Stanford University, Stanford, California, USA

    James Su & Sarah Heilshorn

  7. Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California, USA

    Joseph B Shrager

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  1. Marco Quarta
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Contributions

M.Q. conceived and designed most of the experiments reported. T.A.R. provided guidance throughout. M.Q., J.O.B., A.D.M. and S.C.B. performed experiments and collected data. S.H. provided guidance throughout. M.Q., R.D. and J.S. designed EMF fabrication and performed EMF experiments. J.O.B. analyzed single cell gene expression data and developed the random forests model. M.Q. and R.D. designed and performed AFM experiments and analysis. M.Q., A.D.M. and S.C.B. designed, performed and analyzed single cell gene expression experiments and the screening for factors promoting quiescence of MuSCs. M.Q. and R.C. performed transplant experiments, in vivo imaging and data analysis. M.Q. and M.C.G. designed fabrication and experiments with microfluidic chips for EMFs. M.Q. and J.S. designed the EMF and performed EMFs fabrication experiments and imaging. V.A.G. performed the experiments with the human MuSCs. J.B.S. collected operative samples during surgeries. M.Q. and T.A.R. analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Thomas A Rando.

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Integrated supplementary information

Supplementary Figure 1 Combinatorial Q-RT-PCR reveals genes predictive of quiescence and activation

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.

Supplementary Figure 2 Screening of molecules titrated at different concentrations

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.

Supplementary Figure 3 Combinatorial screening of molecules

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).

Supplementary Figure 4 Micropost-array study of the MuSC niche stiffness

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.).

Supplementary Figure 5 Mechanical properties of Collagen-based substrates regulate MuSC properties

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).

Supplementary Figure 6 Mechanical characterization of EMFs

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).

Supplementary Figure 7 Integrin α4β1 is a component of the niche that promotes quiescence of MuSCs

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).

Supplementary Figure 8 Effect of functionalization of EMFs

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 information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Note (PDF 3924 kb)

Supplementary Table 1

Gene expression array. (XLSX 453 kb)

Supplementary Table 2

Small molecules list. (XLSX 14 kb)

Supplementary Table 3

Quiescence media formulation. (XLSX 12 kb)

Supplementary Table 4

Primers sequence. (XLSX 19 kb)

Confocal movie of artificial niche with MuSCs on EMF. (MOV 261 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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