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Cited by 218Muscle Physiology and DisordersTissue Engineering and Regenerative MedicineCell Adhesion Molecules Research

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Basal lamina remodeling at the skeletal muscle stem cell niche mediates stem cell self-renewal

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Rayagiri, Shantisree Sandeepani, Ranaldi, Daniele, Raven, Alexander, Mohamad Azhar, Nur Izzah Farhana, Lefebvre, Olivier, Zammit, Peter S, Borycki, Anne-Gaëlle·2018·Nature Communications
DOI 10.1038/s41467-018-03425-3PMC PMC5852002
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Abstract

A central question in stem cell biology is the relationship between stem cells and their niche. Although previous reports have uncovered how signaling molecules released by niche cells support stem cell function, the role of the extra-cellular matrix (ECM) within the niche is unclear. Here, we show that upon activation, skeletal muscle stem cells (satellite cells) induce local remodeling of the ECM and the deposition of laminin-α1 and laminin-α5 into the basal lamina of the satellite cell niche. Genetic ablation of laminin-α1, disruption of integrin-α6 signaling or blocking matrix metalloproteinase activity impairs satellite cell expansion and self-renewal. Collectively, our findings establish that remodeling of the ECM is an integral process of stem cell activity to support propagation and self-renewal, and may explain the effect laminin-α1-containing supports have on embryonic and adult stem cells, as well as the regenerative activity of exogenous laminin-111 therapy.

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Figures

Fig. 1 Laminin-α1 and laminin-α5 deposition in the niche of activated satellite cells. a Quantitative PCR analysis of Lama1-5, Lamb1-3, and Lamc1-3 genes together with Pax7 and MyoD in non-injured (dark gray; n = 3) and 4 dpi injured (light gray; n = 3) TA muscles. b Schematic representation of the myofiber culture system showing the sequential activation (blue), proliferation (yellow), self-renewal (green), and differentiation (purple) of satellite cells. c Representative immunofluorescence images of cultured myofibers analyzed for laminin-α1 (green), Pax7 (red), Myf5 (yellow at 24 h), and MyoD (yellow at 72 h). Panels on the right are high magnification images of the area boxed. White arrows indicate satellite cells associated with laminin-α1. Scale bar, 50 μm. d Immunofluorescence analysis of laminin-α5 (green) and Pax7 (red) distribution in cultured myofibers. Arrows indicate Laminin-α5 deposition in Pax7−cells at 72 h. Scale bar, 50 μm. e Quantification of the number of satellite cells expressing Pax7, MyoD, and laminin-α1 after myofiber culture. The color code relates to the cell populations described in (b). n = 3 experiments with 20–32 fibers analyzed per time point. Graph shows mean + sem; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test)
Fig. 1 Laminin-α1 and laminin-α5 deposition in the niche of activated satellite cells. a Quantitative PCR analysis of Lama1-5, Lamb1-3, and Lamc1-3 genes together with Pax7 and MyoD in non-injured (dark gray; n = 3) and 4 dpi injured (light gray; n = 3) TA muscles. b Schematic representation of the myofiber culture system showing the sequential activation (blue), proliferation (yellow), self-renewal (green), and differentiation (purple) of satellite cells. c Representative immunofluorescence images of cultured myofibers analyzed for laminin-α1 (green), Pax7 (red), Myf5 (yellow at 24 h), and MyoD (yellow at 72 h). Panels on the right are high magnification images of the area boxed. White arrows indicate satellite cells associated with laminin-α1. Scale bar, 50 μm. d Immunofluorescence analysis of laminin-α5 (green) and Pax7 (red) distribution in cultured myofibers. Arrows indicate Laminin-α5 deposition in Pax7−cells at 72 h. Scale bar, 50 μm. e Quantification of the number of satellite cells expressing Pax7, MyoD, and laminin-α1 after myofiber culture. The color code relates to the cell populations described in (b). n = 3 experiments with 20–32 fibers analyzed per time point. Graph shows mean + sem; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test)CC BY · PubMed Central
Fig. 2 Spatio-temporal distribution of laminin-α1 and laminin-α5 in regenerating muscles. a Immunofluorescence analysis of laminin-α2 (green, top panels), laminin-α1 (green, middle panels), and laminin-α5 (gray, bottom panels) in regenerating TA muscle following cardiotoxin-mediated injury. Panels on the right are high magnification images of the 2 dpi images (indicated by a white rectangle, with small inserts showing the same image with the laminin channel only). The insert shown at 4 dpi in the laminin-α5 panels illustrates increased laminin α5 (white) deposition at the surface of satellite cells. White arrows indicate satellite cells. Yellow arrows indicate MyoD− or Pax7−(GFP−) cells positive for laminin-α1 or α5. Scale bar, 50 μm. b Quantification of the number of laminin-α1+ myogenic cells in regenerating muscles. c qPCR analysis of Lama1 and Lama5 gene expression in myofibers cultured for 0–72 h. n = 6 experiments. Graphs show mean + sem
Fig. 2 Spatio-temporal distribution of laminin-α1 and laminin-α5 in regenerating muscles. a Immunofluorescence analysis of laminin-α2 (green, top panels), laminin-α1 (green, middle panels), and laminin-α5 (gray, bottom panels) in regenerating TA muscle following cardiotoxin-mediated injury. Panels on the right are high magnification images of the 2 dpi images (indicated by a white rectangle, with small inserts showing the same image with the laminin channel only). The insert shown at 4 dpi in the laminin-α5 panels illustrates increased laminin α5 (white) deposition at the surface of satellite cells. White arrows indicate satellite cells. Yellow arrows indicate MyoD− or Pax7−(GFP−) cells positive for laminin-α1 or α5. Scale bar, 50 μm. b Quantification of the number of laminin-α1+ myogenic cells in regenerating muscles. c qPCR analysis of Lama1 and Lama5 gene expression in myofibers cultured for 0–72 h. n = 6 experiments. Graphs show mean + semCC BY · PubMed Central
Fig. 3 MMP2 and MMP9 catalyze SC basal lamina remodeling. a Immuno-localization of MMP2 and MMP9 (red) in EDL myofibers cultured for 72 h. Inserts are high magnification of satellite cells indicated by red arrows. Green arrows indicate Pax7+ satellite cells that are likely to be self-renewing cells and are negative for MMP2 and MMP9. Scale bar, 50 μm. b Quantification of the number of SCs expressing MMP2 and MMP9 in cultured EDL myofibers. The color code used relates to the cell populations described in Fig. 1b. n = 5 experiments with 50 myofibers per time point. c Representative images of control (DMSO) or treated myofibers cultured in the presence of the MMP inhibitors ARP-100 and Marimastat. Arrows indicate SCs shown at high magnification in inserts. Note the reduced deposition of laminin α1 after 24 h and 48 h in the presence of ARP-100 and the complete absence of laminin α1 after 24 h and 48 h in the presence of Marimastat. Scale bar, 50 μm. d Quantification of the effect of MMP inhibition by ARP-100 and Marismastat on the progression of SCs through the myogenic program in the ex-vivo myofiber culture system. n = 3 experiments with 25–40 fibers per time point. Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test)
Fig. 3 MMP2 and MMP9 catalyze SC basal lamina remodeling. a Immuno-localization of MMP2 and MMP9 (red) in EDL myofibers cultured for 72 h. Inserts are high magnification of satellite cells indicated by red arrows. Green arrows indicate Pax7+ satellite cells that are likely to be self-renewing cells and are negative for MMP2 and MMP9. Scale bar, 50 μm. b Quantification of the number of SCs expressing MMP2 and MMP9 in cultured EDL myofibers. The color code used relates to the cell populations described in Fig. 1b. n = 5 experiments with 50 myofibers per time point. c Representative images of control (DMSO) or treated myofibers cultured in the presence of the MMP inhibitors ARP-100 and Marimastat. Arrows indicate SCs shown at high magnification in inserts. Note the reduced deposition of laminin α1 after 24 h and 48 h in the presence of ARP-100 and the complete absence of laminin α1 after 24 h and 48 h in the presence of Marimastat. Scale bar, 50 μm. d Quantification of the effect of MMP inhibition by ARP-100 and Marismastat on the progression of SCs through the myogenic program in the ex-vivo myofiber culture system. n = 3 experiments with 25–40 fibers per time point. Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test)CC BY · PubMed Central
Fig. 4 Laminin-α1 is essential for SC proliferation. a Control and Lama1cko Tibialis anterior (TA) muscle analyzed at 14 days post injury by Haematoxylin and Eosin staining, and immunofluorescence for laminin-α2 (green), MyoD (red), and collagen I (red). Black and white arrowheads indicate the presence of smaller regenerated myofibers in Lama1cko mice. White arrows indicate sites of fibrosis. The graph shows the minimal Feret diameter analysis of control and Lama1cko TA muscles at 14 dpi. Scale bar, 50 μm. n = 3 per genotype. b Number of MyoD+ cells in injured TA muscles at 2, 4, and 7 dpi in control and Lama1cko mice. n = 3 per genotype. c Number of Ki67+ cells in injured TA muscles at 2 and 4 dpi in control and Lama1cko mice. d Quantification of the number of satellite cells activated (blue), proliferating (yellow), self-renewing (green), and differentiating (purple) in a 72-hour ex-vivo culture of control, heterozygous, and Lama1cko myofibers. n = 3 per genotype with 31–79 myofibers per time point. Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test for b, c, and d, and one-way ANOVA for a)
Fig. 4 Laminin-α1 is essential for SC proliferation. a Control and Lama1cko Tibialis anterior (TA) muscle analyzed at 14 days post injury by Haematoxylin and Eosin staining, and immunofluorescence for laminin-α2 (green), MyoD (red), and collagen I (red). Black and white arrowheads indicate the presence of smaller regenerated myofibers in Lama1cko mice. White arrows indicate sites of fibrosis. The graph shows the minimal Feret diameter analysis of control and Lama1cko TA muscles at 14 dpi. Scale bar, 50 μm. n = 3 per genotype. b Number of MyoD+ cells in injured TA muscles at 2, 4, and 7 dpi in control and Lama1cko mice. n = 3 per genotype. c Number of Ki67+ cells in injured TA muscles at 2 and 4 dpi in control and Lama1cko mice. d Quantification of the number of satellite cells activated (blue), proliferating (yellow), self-renewing (green), and differentiating (purple) in a 72-hour ex-vivo culture of control, heterozygous, and Lama1cko myofibers. n = 3 per genotype with 31–79 myofibers per time point. Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test for b, c, and d, and one-way ANOVA for a)CC BY · PubMed Central
Fig. 5 Impaired long-term regenerative capacity in Lama1-deficient mice. a Number of satellite cells returning to a sublaminal position in regenerated myofibers after one round (n = 3), two rounds (n = 3), and three rounds (n = 3) of injury. b Control and Lama1cko mice analyzed at 14 dpi following three rounds of repeated injuries at 21-day intervals analyzed by Haematoxylin and Eosin staining and immunofluorescence for MyoD and collagen I (red). Black arrows indicate the presence of infiltrating inflammatory cells in Lama1cko mice. White arrows indicate the site of fibrosis. The graph shows the minimal Feret diameter analysis of control and Lama1cko mice after 3 rounds of injury. Scale bar, 50 μm. n = 6 (Lama1cko) and n = 3 (control). Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test for a and one-way ANOVA for b)
Fig. 5 Impaired long-term regenerative capacity in Lama1-deficient mice. a Number of satellite cells returning to a sublaminal position in regenerated myofibers after one round (n = 3), two rounds (n = 3), and three rounds (n = 3) of injury. b Control and Lama1cko mice analyzed at 14 dpi following three rounds of repeated injuries at 21-day intervals analyzed by Haematoxylin and Eosin staining and immunofluorescence for MyoD and collagen I (red). Black arrows indicate the presence of infiltrating inflammatory cells in Lama1cko mice. White arrows indicate the site of fibrosis. The graph shows the minimal Feret diameter analysis of control and Lama1cko mice after 3 rounds of injury. Scale bar, 50 μm. n = 6 (Lama1cko) and n = 3 (control). Graphs show mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test for a and one-way ANOVA for b)CC BY · PubMed Central
Fig. 6 Integrin-α6 mediates laminin-α1 control of SC expansion. a Immunofluorescence analysis of cultured EDL myofibers showing integrin-α6 upregulation in activated SCs. Inserts show high magnification images of satellite cells indicated by the white arrows. Top panels show images in the green channel only, bottom panels show the merge images, inserts are higher magnification of merge image. Scale bar, 50 μm. b Quantification of integrin-α6 expression in satellite cells from cultured myofibers. n = 3 with 20–30 fibers per time point. c Integrin-α6 (red) distribution in 2 dpi and 4 dpi injured TA muscle of Tg(Pax7-GFP) mice. Images of the blue/red channels and green/red channels show colocalization of integrin-α6 with satellite cells (white arrows) and with predicted endothelial cells (white arrowheads). Scale bar, 20 μm. d Immuno-colocalization of integrin-α6 (red) with either MyoD (green, activated satellite cell marker), F4/80 (green, pan macrophage marker), CD206 (green, M2 macrophage marker), or CD31 (green, endothelial cell marker) following cardiotoxin-mediated injury of TA muscles. Inserts are high magnification images of areas delineated by a dotted white line. Note that integrin-α6 is primarily associated with endothelial cells at 2 dpi, whereas it is primarily detected at the surface of macrophages (M1 and M2) and satellite cells at 4 dpi. By 7 dpi, integrin-α6 returns to being restricted to endothelial cells. Scale bar, 50 μm. Graph shows mean + sem
Fig. 6 Integrin-α6 mediates laminin-α1 control of SC expansion. a Immunofluorescence analysis of cultured EDL myofibers showing integrin-α6 upregulation in activated SCs. Inserts show high magnification images of satellite cells indicated by the white arrows. Top panels show images in the green channel only, bottom panels show the merge images, inserts are higher magnification of merge image. Scale bar, 50 μm. b Quantification of integrin-α6 expression in satellite cells from cultured myofibers. n = 3 with 20–30 fibers per time point. c Integrin-α6 (red) distribution in 2 dpi and 4 dpi injured TA muscle of Tg(Pax7-GFP) mice. Images of the blue/red channels and green/red channels show colocalization of integrin-α6 with satellite cells (white arrows) and with predicted endothelial cells (white arrowheads). Scale bar, 20 μm. d Immuno-colocalization of integrin-α6 (red) with either MyoD (green, activated satellite cell marker), F4/80 (green, pan macrophage marker), CD206 (green, M2 macrophage marker), or CD31 (green, endothelial cell marker) following cardiotoxin-mediated injury of TA muscles. Inserts are high magnification images of areas delineated by a dotted white line. Note that integrin-α6 is primarily associated with endothelial cells at 2 dpi, whereas it is primarily detected at the surface of macrophages (M1 and M2) and satellite cells at 4 dpi. By 7 dpi, integrin-α6 returns to being restricted to endothelial cells. Scale bar, 50 μm. Graph shows mean + semCC BY · PubMed Central
Fig. 7 Laminin-111 treatment increases SC proliferation and self-renewal. a Representative images of myofibers cultured in control conditions or in the presence of laminin-111 or Cd49f, and analyzed by immunofluorescence for Pax7 (red), Myf5 (green), MyoD (green), Caveolin-1 (green), and Myogenin (red). Scale bar: 50 μm. b Quantification of satellite cell populations in myofiber cultures supplemented with Laminin-111, Cd49f, or both. Graph shows mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001 (t-test)
Fig. 7 Laminin-111 treatment increases SC proliferation and self-renewal. a Representative images of myofibers cultured in control conditions or in the presence of laminin-111 or Cd49f, and analyzed by immunofluorescence for Pax7 (red), Myf5 (green), MyoD (green), Caveolin-1 (green), and Myogenin (red). Scale bar: 50 μm. b Quantification of satellite cell populations in myofiber cultures supplemented with Laminin-111, Cd49f, or both. Graph shows mean + sem. *P < 0.05, **P < 0.01, ***P < 0.001 (t-test)CC BY · PubMed Central
Fig. 8 Laminin-111 treatment alters SC cell polarity and stimulates planar cell division. a Quantification of planar and apico-basal cell divisions (symmetric and asymmetric) based on the expression of Pax7 and MyoD in T46 myofibers treated with PBS (control; dark gray) or laminin-111 (light gray). n = 3 with 50–82 doublets analyzed per culture. *P < 0.05, **P < 0.01, ***P < 0.001 (t-test). b Representative immunofluorescence images of planar and apico-basal cell divisions in T46 myofibers analyzed using antibodies against Pax7 (red) and MyoD (green). White arrows indicate cell doublets. Individual color channels are shown. Scale bar: 50 μm c Representative immunofluorescence of Par3 (green) and Pax7 (red) in myofibers cultured for 46 h in in the presence of PBS (control) or laminin-111. Arrows indicate Par3 asymmetric distribution in control, but not in laminin-111 treated fibers. The white star indicates background staining. Scale bar: 10 μm. d–g Proposed model for laminin-111 control of SC self-renewal: d Upon activation, SCs upregulate MMP2 and MMP9 expression, leading to a local digestion of the laminin-α2-containing basal lamina (double orange line) at the SC niche. e Simultaneously, SCs re-express laminin-α1, which is secreted and deposited into the SC basal lamina (double red line), and the laminin-α1 receptor, integrin-α6 (blue line). f Laminin-α1-mediated signaling initiates or maintains SC polarity through the asymmetric distribution of the basal determinant Par1b (brown) and the apical determinant Par3 (purple), leading to apico-basal cell polarity. g Apico-basal asymmetric cell division yields two distinct daughter cells, including a self-renewing SC associated with the basal lamina (Pax7+ in green) and a differentiating SC (MyoD+ in purple)
Fig. 8 Laminin-111 treatment alters SC cell polarity and stimulates planar cell division. a Quantification of planar and apico-basal cell divisions (symmetric and asymmetric) based on the expression of Pax7 and MyoD in T46 myofibers treated with PBS (control; dark gray) or laminin-111 (light gray). n = 3 with 50–82 doublets analyzed per culture. *P < 0.05, **P < 0.01, ***P < 0.001 (t-test). b Representative immunofluorescence images of planar and apico-basal cell divisions in T46 myofibers analyzed using antibodies against Pax7 (red) and MyoD (green). White arrows indicate cell doublets. Individual color channels are shown. Scale bar: 50 μm c Representative immunofluorescence of Par3 (green) and Pax7 (red) in myofibers cultured for 46 h in in the presence of PBS (control) or laminin-111. Arrows indicate Par3 asymmetric distribution in control, but not in laminin-111 treated fibers. The white star indicates background staining. Scale bar: 10 μm. d–g Proposed model for laminin-111 control of SC self-renewal: d Upon activation, SCs upregulate MMP2 and MMP9 expression, leading to a local digestion of the laminin-α2-containing basal lamina (double orange line) at the SC niche. e Simultaneously, SCs re-express laminin-α1, which is secreted and deposited into the SC basal lamina (double red line), and the laminin-α1 receptor, integrin-α6 (blue line). f Laminin-α1-mediated signaling initiates or maintains SC polarity through the asymmetric distribution of the basal determinant Par1b (brown) and the apical determinant Par3 (purple), leading to apico-basal cell polarity. g Apico-basal asymmetric cell division yields two distinct daughter cells, including a self-renewing SC associated with the basal lamina (Pax7+ in green) and a differentiating SC (MyoD+ in purple)CC BY · PubMed Central

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Metadata

Authors
Rayagiri, S. S., Ranaldi, D., Raven, A., Mohamad Azhar, N. I. F., Lefebvre, O., Zammit, P. S., & Borycki, A.-G.
Affiliations
  • University of Sheffield (GB)
  • MRC Centre for Regenerative Medicine (GB)
  • King's College London (GB)
Corresponding author
Borycki, Anne-Gaëlle
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-03-14
Volume
9
Issue
1
Pages
1075-1075
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-03425-3
PMC
PMC5852002
OpenAlex
W2791349152
Cited by
218
References
62
Citations by year
2026: 16 · 2025: 26 · 2024: 25 · 2023: 35 · 2022: 25 · 2021: 31 · 2020: 33 · 2019: 20 · 2018: 6
FWCI
8.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Research Portal (King's College London) (submittedVersion, cc-by)
Funders
  • FSH Society
  • Agence Nationale de la Recherche (ANR-10-LABX-0034)
  • Majlis Amanah Rakyat
  • Directorate for Biological Sciences
  • Medical Research Council
  • Biotechnology and Biological Sciences Research Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
Keywords
skeletal muscle stem cells, satellite cells, extracellular matrix, stem cell niche, stem cell self-renewal, laminin alpha1, laminin alpha5, matrix metalloproteinases, muscle regeneration
MeSH terms
Cell Self Renewal, Animals, Basement Membrane, Cell Differentiation, Extracellular Matrix, Humans, Mice, Inbred C57BL, Signal Transduction, Muscle, Skeletal, Muscle Fibers, Skeletal, Satellite Cells, Skeletal Muscle, Stem Cell Niche

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