1. Developmental Biology
  2. Stem Cells and Regenerative Medicine
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The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair

  1. Yulong Wei
  2. Hao Sun
  3. Tao Gui
  4. Lutian Yao
  5. Leilei Zhong
  6. Wei Yu
  7. Su-Jin Heo
  8. Lin Han
  9. Nathaniel A Dyment
  10. Xiaowei Sherry Liu
  11. Yejia Zhang
  12. Eiki Koyama
  13. Fanxin Long
  14. Miltiadis H Zgonis
  15. Robert L Mauck
  16. Jaimo Ahn
  17. Ling Qin  Is a corresponding author
  1. Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, United States
  2. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China
  3. Department of Orthopedics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, China
  4. Department of Bone and Joint Surgery, Institute of Orthopedic Diseases, The First Affiliated Hospital, Jinan University, China
  5. Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz VA Medical Center, United States
  6. School of Biomedical Engineering, Science and Health Systems, Drexel University, United States
  7. Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, United States
  8. Translational Research Program in Pediatric Orthopaedics, The Children's Hospital of Philadelphia, United States
  9. Department of Orthopaedic Surgery, University of Michigan Medical School, United States
Research Article
Cite this article as: eLife 2021;10:e62917 doi: 10.7554/eLife.62917
9 figures, 1 table and 2 additional files

Figures

Figure 1 with 4 supplements
Gli1 labels mesenchymal progenitors in mouse meniscus during development.

(A) Schematic graph of the study protocol. Male Gli1ER/Td mice were treated with Tam at 1, 2, 4, 8, and 12 weeks of age and analyzed at 24 hr (pulse) or 6 weeks (tracing) after the last Tam dosing. (B) Schematic cartoon of meniscus shows sectioning sites. M: meniscus; ACL: anterior cruciate ligament; PCL: posterior cruciate ligament. (C) Representative fluorescence images of meniscus sections at indicated ages and sectioning sites. n = 3 mice/age/sectioning site. Scale bars, 200 μm. F: femur; T: tibia; A: anterior; P: posterior; M: medial meniscus; L: lateral meniscus; Med: medial; Ant: anterior; Post: posterior. Red: Td; Blue: DAPI. (D) Representative fluorescence images of meniscus body at coronal (a) and sagittal (b) planes from 12-week-old Gli1ER/Td mice. Meniscus were harvested at 24 hr after the last Tam injection. n = 3 mice/sectioning site. Scale bars, 200 μm. Boxed areas in a and b are shown at high magnification as c and d, respectively. Dashed lines outline meniscus. (E) qRT-PCR analysis of Hh signaling component genes in mouse meniscus tissues at 1, 4, 8 weeks of age. n = 4 independent experiments. (F) Top panel is a schematic representation of the study protocol. Gli1ER/Td mice were injected with EdU at P3-6 and Tam at P25-29. Joints were harvested 24 hr later. Representative confocal images of coronal sections of mouse knee joints are presented at the bottom panel. Boxed area in a (Scale bars, 200 μm) is shown at high magnification in b (Scale bars, 50 μm). Green: EdU. (G) The percentage of EdU+ cells within Gli1+ or Gli1- meniscus cells was quantified. n = 6 mice/group. (H) Gli1ER/Td mice were treated with Tam at 24 or 48 weeks of age and analyzed 24 hr later. Representative fluorescence images of sagittal (a, b) and coronal (c–f) sections of knee joints are presented. Scale bars, 200 μm. Statistical analysis was performed using unpaired two-tailed t-test and one-way ANOVA with Tukey-Kramer post-hoc test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 1—figure supplement 1
Mouse meniscal morphogenesis during development.

(A) The morphological overview of meniscus at 1, 2, 3, 4, 8, 12, 24, 48 weeks of age. Scale bars, 1 mm. (B) The meniscal perimeter was quantified. n = 3 mice/age.

Figure 1—figure supplement 2
The density of Gli1+ cells along meniscus surface was measured in mice at different ages.

n = 5 mice/age.

Figure 1—figure supplement 3
Meniscal enthesis and ligamental enthesis regions in joint are enriched with Gli1+ cells.

Gli1ER/Td mice at 12 weeks of age received Tam injections followed by tissue harvest 24 hr later. Knees were sectioned to show meniscal enthesis regions (A) and ligamental enthesis regions (B, C) within the knee joint. MM: medial meniscus; LM: lateral meniscus; ACL: anterior cruciate ligament; PCL: posterior cruciate ligament. Yellow arrows indicate the enthesis regions.

Figure 1—figure supplement 4
Gli1 labels superficial zone cells of mouse and mini-pig meniscal horns.

(A) Immunofluorescence staining of Gli1 (green) on sagittal sections of 12-week-old Gli1ER/Td mouse knee joints. Boxed area in a is enlarged in b. Dashed line indicates the surface of meniscus. Scale bars, 200 μm. F: femur; T: tibia; M: meniscus. Blue: DAPI, Red: Td; Green: Gli1. (B) Representative safranin O/fast green staining (left) and immunohistochemistry staining of Gli1 (right) in the horn area of mini-pig meniscus. Scale bars, 200 μm.

Figure 2 with 2 supplements
Gli1-labeled meniscus cells possess mesenchymal progenitor properties.

(A) Representative immunofluorescence images of Sca1, Cd90, Cd200, PDGFRα, Cd248, and Prg4 staining in 3-month-old Gli1ER/Td meniscus. Scale bars, 200 μm. Boxed areas are shown at high magnification in corresponding panels at the bottom. Dashed lines indicate meniscus surface. Yellow cells are double positive for progenitor marker and Td. F: femur; T: tibia; M: meniscus. Blue: DAPI. (B) Quantification of the expression level of mesenchymal progenitor markers in Gli1+ and Gli1- cells from meniscus. Digested meniscus cells from 3-month-old Gli1ER/Td mice were subjected to flow cytometry analysis. n = 3 independent experiments. (C) CFU-F assay of digested meniscus cells. Td+colonies and Td- colonies were counted from 1 × 104 seeded cells. n = 3 independent experiments. (D) Representative bright-field images of the scratch-wound closure in Gli1+ or Gli1- meniscus cells at 0 and 48 hr. Scale bars, 200 μm. Solid lines indicate the remaining area not covered by meniscus cells. (E) The relative migration rate was measured by the percentage of scratched area being covered by migrated cells at 48 hr. n = 3 independent experiments. (F) Representative adipogenic (AD), osteogenic (OB), and chondrogenic (CH) differentiation images of Gli1+ and Gli1- cells. Cells were stained by Oil Red, Alizarin red, and Alcian blue, respectively. (G) qRT-PCR analysis of lineage markers in Gli1- or Gli1+meniscal cells after being cultured in adipogenic, osteogenic and chondrogenic differentiation media for 1, 2, and 3 weeks, respectively. n = 4 independent experiments. Statistical analysis was performed using unpaired two-tailed t-test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 1
Mesenchymal progenitor markers are enriched in Gli1+ meniscus cells.

Digested meniscus cells from 3-month-old Gli1ER/Td mice were subjected to flow cytometry analysis.

Figure 2—figure supplement 2
Gli1+ cells proliferate faster than Gli1- cells.

(A) Td(Gli1)+ and Td- cells digested from meniscus cells from Gli1ER/Td mice were grown to confluence and then seeded at 50,000 cells/well on day 0. Cell number/well was counted every other day. n = 3 independent experiments. (B) The percentage of Td+ cells from freshly isolated meniscus cells (FI) and from cells being cultured for 7 days (P0) was quantified by flow cytometry. n = 3 independent experiments. Statistical analysis was performed using unpaired two-tailed t-test. Data presented as mean ± s.e.m. **p<0.01, ***p<0.001.

Hh signaling stimulates proliferation and migration of meniscus mesenchymal progenitors.

(A) qRT-PCR analysis of Gli1 mRNA in primary mouse meniscus cells treated with vehicle, GANT-61 (10 μM) or PMA(1 μM) for 48 hr. n = 3 independent experiments. (B) The proliferative ability of primary mouse meniscus cells was up-regulated by PMA and down-regulated by GANT-61 over 8 days of culture. n = 3 independent experiments. (C) Representative bright-field images of the scratch-wound closure in meniscus cells treated with veh, GANT-61 or PMA after 48 hr. Scale bars, 200 μm. Solid lines indicate the remaining area not covered by meniscus cells. (D) The relative migration rate was measured. n = 3–6 independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 4 with 2 supplements
Meniscus injury rapidly expands Gli1-lineage cells.

(A) Schematic cartoon of meniscus shows the sectioning site. M: meniscus; MS: meniscus synovial end; ML: meniscus ligamental end. A pair of scissors indicates the transection site. ACL: anterior cruciate ligament; PCL: posterior cruciate ligament. (B) Male Gli1ER/Td mice received Tam injections (day −5 ~ −1) and meniscus injury (day 0) at 12 weeks of age. Knee joints were harvested at 1, 2, 4, 8, 12 weeks after injury. (C) Representative safranin O/fast green staining (top) and fluorescence images (bottom) of oblique sections of mouse knee joints harvested at indicated time points after injury. Dashed lines outline the meniscus. Scale bars, 200 μm. F: femur; T: tibia; S: synovium; MS: meniscus synovial end; ML: meniscus ligamental end. Red: Td; Blue: DAPI. (D) Repair score was evaluated at indicated time points after meniscus injury. n = 8 mice/group. (E) Cell density in the synovial and ligamental ends of meniscus was quantified at 4 weeks post meniscus injury. n = 5–6 mice/group. (F) The percentage of Td+ cells in the synovial and ligamental ends of meniscus was also quantified. n = 5–6 mice/group. (G) qRT-PCR analysis of Hh signaling component genes in meniscus at 1 and 2 weeks post injury. n = 4 independent experiments. (H) Top panel is a schematic representation of the study protocol. Gli1ER/Td mice at 12 weeks of age were treated with Tam (day −5 to −1), meniscus injury (day 0), and EdU injections (day 9–13). A representative confocal image of knee joint at day 14 is shown below (Scale bars, 250 μm). Boxed areas of synovial and ligamental ends of meniscus (MS and ML, respectively) are shown at high magnification at the bottom (Scale bar, 25 μm). Green: EdU. (I) Top panel is a schematic representation of the study protocol. Gli1ER/Td (Ctrl) or Gli1ER/Td/DTA (DTA) mice were treated with Tam (day −5 to −1) and meniscus injury at 12 weeks of age (day 0). Representative fluorescent images of sagittal and coronal mouse knee joint sections at day 0 without injury are shown below (Scale bars, 200 μm). A: anterior; P: posterior. (J) The percentage of Td+ cells in the anterior horn was quantified. n = 4 mice/group. (K) Representative safranin O/fast green staining (a, b) and fluorescence images (c, d) of oblique sections of mouse knee joints harvested at 12 weeks after injury. Dashed lines outline the meniscus. Scale bars, 200 μm. (L) Repair score was evaluated. n = 5 mice/group. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-hoc test for (D), one-way ANOVA with Tukey-Kramer post-hoc test for (G) and unpaired two-tailed t-test for (E), (F), (J) and (L). Data presented as mean ± s.e.m. *p<0.05, ***p<0.001.

Figure 4—figure supplement 1
Aging diminishes Gli1-lineage cell expansion and the repair ability of meniscus.

(A) Representative safranin O/fast green staining (top) and fluorescence images (bottom) of aged mouse knee joints at 4 weeks after sham or meniscus injury. Gli1ER/Td mice at 12 months of age received Tam followed by meniscus injury. Dashed lines outline the meniscus. Scale bars, 200 μm. F: femur; T: tibia; MS: meniscus synovial end; ML: meniscus ligamental end; Red: Td; Blue: DAPI. (B) Repair score was quantified. n = 3 mice/group. Statistical analysis was performed using unpaired two-tailed t-test. Data presented as mean ± s.e.m. ***p<0.001.

Figure 4—figure supplement 2
Gli1+ cells appear in proliferative cell clusters of degenerated human meniscus.

Representative safranin O/fast green staining (a–d) and immunohistochemistry staining of Ki67 (e–h) and Gli1 (i–l) in human meniscus tissues at different degenerative stages. n = 3 samples/stage. Scale bars, 200 μm.

Figure 5 with 1 supplement
Activation of Hh/Gli1 pathway accelerates mouse meniscus repair.

(A) Schematic representation of the study protocol. WT mice received meniscus injury at 12 weeks of age followed by transplantation of 5000 Gli1+ or Gli1- meniscus cells at the injury site. Knee joints were harvested at 1 and 4 weeks after injury. (B) Representative overview (a–c), safranin O/fast green staining (d–i), and polarizing images (j–l) of mouse knee joints at 4 weeks after injury. Yellow dashed lines outline the overview morphology of injured meniscus. Meniscus is shown attached to tibial plateau. Arrows point to the injury site. Red boxed areas in d-f are shown at high magnification in g-i, respectively. Scale bars, 200 μm. F: femur; T: tibia; MS: meniscus synovial end; ML: meniscus ligamental end. (C) Repair score was evaluated. n = 8 mice/group. (D) Representative confocal images of mouse knee joints at 1 and 4 weeks after injury. Boxed areas in the top panel are shown at a high magnification at the bottom panel. Dashed line outlines meniscus. Scale bars, 200 μm. Blue: DAPI, Red: Td. (E) Schematic representation of the study protocol. Gli1ER/Td mice received Tam injections and meniscus injury at 12 weeks of age (day 0) followed by vehicle and PMA injection. Knee joints were harvested at 1 and 4 weeks after injury. (F) Representative overview (a, b), safranin O/fast green staining (c–f), and polarizing images (g, h) of mouse knee joints at 4 weeks after injury. Yellow dashed lines outline the overview morphology of injured meniscus. Meniscus is shown attached to tibial plateau. Red boxed areas in c and d are shown at high magnification in e and f, respectively. Arrows point to the injury site. Scale bars, 200 μm. (G) Repair score was evaluated. n = 7 mice/group. (H) Representative fluorescence images of vehicle- and PMA-treated mouse meniscus at 1 and 4 weeks after injury. Scale bars, 200 μm. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-hoc test for (C) and unpaired two-tailed t-test for (G). Data presented as mean ± s.e.m. ***p<0.001.

Figure 5—figure supplement 1
Hh agonist treatment does not promote joint calcification.

(A) Representative microCT images of WT mouse knee joints at 3 months post surgery as well as vehicle (Veh) or PMA treatment. (B) The volume of calcified meniscus was quantified. n = 5–6 mice/group. (C) Osteophyte volume was quantified. n = 5–6 mice/group. Statistical analysis was performed using unpaired two-tailed t-test. Red arrows indicate the osteophytes. Data presented as mean ± s.e.m.

Meniscus repair by enhancing Hh/Gli1 signaling delays OA progression.

(A) Representative safranin O/fast green staining of sagittal sections of vehicle-, Gli1+ cell- and PMA-treated mouse knee joints at 8 weeks after sham or meniscus injury. Scale bars, 200 μm. (B) Average thicknesses of uncalcified zone (Uncal.Th.) and calcified zone (Cal.Th.) of the tibial articular cartilage were quantified. n = 8 mice/group. (C) The OA severity was measured by Mankin score. n = 8 mice/group. (D) Von Frey assay was performed at 8 weeks after injury. PWT: paw withdrawal threshold. n = 5 mice/group. (E) Representative safranin O/fast green staining of sagittal sections of vehicle- and PMA-treated mouse knee joints at 8 weeks after sham or DMM surgery. Scale bars, 200 μm. (F) The OA severity was measured by Mankin score. n = 7 mice/group. Statistical analysis was performed using two-way ANOVA with Tukey-Kramer post-hoc test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Author response image 1
CFU-F assay of total digested meniscus cells.

(A) Crystal-violet staining of CFU-F colonies in a dish. (B) Brightfield and fluorescence images of a representative Gli1+ CFU-F colony in the dish.

Author response image 2
Representative TUNEL fluorescent staining of Gli1ER/Td mouse knee joints harvested at 2, 4, or 8 weeks after meniscus injury.

Mice received Tamoxifen injections right before the injury. Boxed area at the top is shown at a high magnification at the bottom (Scale bars, 200 μm). F: femur; T: tibia; MS: meniscus synovial end; ML: meniscus ligamental end. Red: Td; Green: TUNEL; Blue: DAPI.

Author response image 3
Representative confocal images of WT mouse knee joints at 1, 4, and 8 weeks after meniscus injury and injection of Gli1+ cells derived from Gli1ER/Td meniscus.

Boxed areas in the top panel are shown at high magnification at the bottom. Dashed line outlines meniscus. Scale bars, 200 μm. Blue: DAPI; Red: Td.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, Strain background (Mus musculus)Gli1CreERJackson LaboratoryStock No: 007913
Strain, Strain background (Mus musculus)Rosa26 lsl-tdTomatoJackson LaboratoryStock No: 007909
Strain, Strain background (Mus musculus)Rosa26 lsl-DTAJackson LaboratoryStock No: 010527
Sequence-based reagentGli1CreER
Primer 1
Jackson LaboratoryPCR Primer5’-GCGGTCTGGCAGTAAAAACTATC-3’
Sequence-based reagentGli1CreER
Primer 2
Jackson LaboratoryPCR Primer5’-GTGAAACAGCATTGCTGTCACTT-3’
Sequence-based reagentGli1CreER
Primer 3
Jackson LaboratoryPCR Primer5’-CACGTGGGCTCCAGCATT-3’
Sequence-based reagentGli1CreER
Primer 4
Jackson LaboratoryPCR Primer5’-TCACCAGTCATTTCTGCCTTTG-3’
Sequence-based reagentRosa26 lsl-tdTomato
Primer 1
Jackson LaboratoryPCR Primer5’-AAGGGAGCTGCAGTGGAGTA-3’
Sequence-based reagentRosa26 lsl-tdTomato
Primer 2
Jackson LaboratoryPCR Primer5’-CCGAAAATCTGTGGGAAGTC-3’
Sequence-based reagentRosa26 lsl-tdTomato
Primer 3
Jackson LaboratoryPCR Primer5’-GGCATTAAAGCAGCGTATCC-3’
Sequence-based reagentRosa26 lsl-tdTomato
Primer 4
Jackson LaboratoryPCR Primer5’-CTGTTCCTGTACGGCATGG-3’
Sequence-based reagentRosa26 lsl-DTA
Primer1
Jackson LaboratoryPCR Primer5’-CGACCTGCAGGTCCTCG-3’
Sequence-based reagentRosa26 lsl-DTA
Primer 2
Jackson LaboratoryPCR Primer5’-CCAAAGTCGCTCTGAGTTGTTATC-3’
Sequence-based reagentRosa26 lsl-DTA
Primer 3
Jackson LaboratoryPCR Primer5’-GAGCGGGAGAAATGGATATG-3’
Sequence-based reagentRosa26 lsl-DTA
Primer 4
Jackson LaboratoryPCR Primer5’-CTCGAGTTTGTCCAATTATGTCAC-3’
AntibodyRabbit polyclonal Anti-Gli1NOVUS biologicalsNB600-600IF (1:100)
AntibodyRabbit polyclonal Anti-Ki67Abcamab15580IF (1:50)
AntibodyRat monoclonal Anti-Sca1Santa Cruz Biosc-52601IF (1:200)
AntibodyRat monoclonal Anti-Cd200Santa Cruz Biosc-53100IF (1:100)
AntibodyMouse monoclonal Anti-Cd90Santa Cruz Biosc-53456IF (1:100)
AntibodyMouse monoclonal Anti-PDGFRαSanta Cruz Biosc-398206IF (1:200)
AntibodyMouse monoclonal Anti-Cd248Santa Cruz Biosc-377221IF (1:200)
AntibodyRabbit polyclonal anti-Prg4Abcamab28484IF (1:50)
AntibodyRat monoclonal Anti-Sca1BioLegend108131Flow analysis (1:100)
AntibodyMouse monoclonal Anti-Cd90BioLegend202526Flow analysis (1:100)
AntibodyRat monoclonal Anti-Cd200BioLegend123809Flow analysis (1:100)
AntibodyRat monoclonal Anti-PDGFRαBioLegend135907Flow analysis (1:100)
Software, algorithmGraphpad 8.0Statistical AnalysisGraph preparation, statistical analysis

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