Identification of Gli1 as a progenitor cell marker for meniscus development and injury repair
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Abstract
Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using Gli1-CreER tdTomato mice, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1+ cells. Normally, the tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh activator right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 14 2020, follows.
Summary
Injuries of the meniscus are associated with the future development of articular cartilage damage and ultimately osteoarthritis (OA). Prior work in this field has suggest that there are undifferentiated progenitor cells residing in the meniscus and it has been hypothesized that these cells could be harnessed to aid in meniscal healing after injury. The authors provide evidence that Hedgehog activation promotes meniscal healing and identify population of cells that are positive for Gli1, an effector protein in the Hedgehog signaling pathway. Using a combination of approaches, including lineage tracing, in vitro cell culture approaches and cell transplantation experiments, they show that this Gli1 …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 14 2020, follows.
Summary
Injuries of the meniscus are associated with the future development of articular cartilage damage and ultimately osteoarthritis (OA). Prior work in this field has suggest that there are undifferentiated progenitor cells residing in the meniscus and it has been hypothesized that these cells could be harnessed to aid in meniscal healing after injury. The authors provide evidence that Hedgehog activation promotes meniscal healing and identify population of cells that are positive for Gli1, an effector protein in the Hedgehog signaling pathway. Using a combination of approaches, including lineage tracing, in vitro cell culture approaches and cell transplantation experiments, they show that this Gli1 populations contains putative meniscal progenitors involved in meniscus development and healing. Overall, the reviewers found this paper to have high potential and were particularly enthusiastic about the therapeutic potential of Purmorphamine to promote meniscal healing. However, all reviewers felt that the conclusions (and title) of this paper overstated the utility of Gli1 as a marker of de facto meniscal progenitor cells. It is specifically requested that the title of this manuscript be reworded in light of the previous work showing that Gli1 can be found in a number of cell types. In several places, additional work was requested to support the conclusions made in this manuscript. Please see the detailed comments below.
Essential Revisions
The authors describe many of their results as "novel". Gli1 reporter mice have been used extensively in other tissues to non-specifically describe progenitor cells (bone marrow, periosteum, peri-vascular spaces and others). Further, the role of Gli1+ cells in enthesis and and periodontal ligament (PDL) formation and healing has been previously explored. Gli proteins, which have a half-life of minutes-to-hours, may be a relatively unstable foundation for defining cellular identity. While the value of Gli1 as a general Hh reporter is clear, its utility as a putative stem cell marker (Title) does not seem adequately substantiated. The authors must temper their statements on novelty, exclusivity and utility of Gli1. The title of this paper also should be reworded.
The Hedgehog (Hh) signaling manipulation conducted is rather straightforward and some overlapping studies have been performed in murine joints. Many of the experimental results could have been predicted. Other elements that contribute to the superficial nature of the studies are that Gli1 reporter activity is the only marker of Hh signaling examined (for example Gli2/Gli3 are not), and that the abundance and cellular source of an Hh ligand during development or repair is never entertained. Of note, these reporters for Ihh and Shh are available.
It is a stretch to say that Gli1;tdTom labels meniscus progenitor cells (Lines 268-271). There is relative enrichment of Sca1/CD90/CD200/PDGFRa in Gli1+ cells (Fig 2B), yet the vast majority of cells positive for those markers are Gli1-negative (Fig S5). Positive outcomes during in vitro differentiation and scratch assays may primarily result from increased Hh-mediated proliferation. This logic extends all the way through the in vivo experiments (which are quite promising, translationally).
The spatial profile of Gli1-expressing cells in the meniscus is beautifully described, however an interpretation for the superficially restricted zonation of Gli1 reporter activity is not given. Do these superficial cells have more or less cartilage antigen expression? Is there something clearly physiologically different in the Gli1-rich superficial layers that could be determined? Line 401 cites an osteoblast paper to set up the relevance of Gli1+ cells in development of musculoskeletal tissues. However, the meniscus is much more similar to the enthesis and the PDL. The authors should therefore lead with that literature. The PDL literature in particular is not cited and should be added. Also missing are recent enthesis development/regeneration papers (PMID: 30504126, 26141957, and 28219952).
The characterization of Gli1+ and Gli1- FAC sorted cells could be expanded on a bit.
CFU-F images should be provide in addition to quantification. The differentiation studies in Fig 2E are non-quantitative and not convincing. Further, it is a little contradictory that under certain contexts Gli1+ cells form more cartilage (2E), but under other culture conditions they have reduced cartilage markers (2F). These points need to be clarified.
In Fig 5, changes in distribution or survival of Gli1+/- cells may underlie the difference, but survival nor Gli1- cell distribution were not assessed.
Cartilage differentiation within the meniscus appears to be promoted with Gli1+ cell therapy and Purmorphamine. This could be assessed. Similarly, Hh signaling is known to induce osteogenesis. Osteoblastic antigens and/or presence of osteophytes should be assessed for in purmorphamine treated joints.
One topic that is not covered in the paper is the role of Hh signaling in chondrocyte mineralization. This has been well studied in the growth plate (esp. related to PTHrP / IHH feedback loop) and may have relevance to the meniscus as well. The healing studies should consider this carefully, as ectopic mineralization is a possible negative side effect of Hh treatment.
There are a number of places in the results where it is unclear if the authors are talking about Gli+ cells or Gli1-lineage cells. This should be clarified throughout, perhaps with specific nomenclature that defines "Gli1+" as cells that are positive for Gli and "Gli1-lineage" for cells that are descendants of Gli+ cells. Supplemental Figure 1A should be in the main document. Similar schematics in other figures are very useful for understanding the experiment.
What are the temporal expression patterns of Gli1 and other Hh related genes during development and healing? It would be informative to see localized expression (e.g., in situ hybridization) or qPCR expression for healing tissues.
The authors should clarify a number of things with meniscal cell isolation: (a) There are clearly differences in cell phenotype between superficial and deep areas and between attachment and midsection; was this considered for cell isolation? (b) I assume TAM injections were performed and then cells were isolated a few days later via FACS; please clarify details to show that Gli1+ (not Gli1-lineage) cells were characterized. (c) Fig 2: 3-month old mice were used, but again, Gli+ vs. Gli1-lineage cells is not indicated.
The mechanisms by which Gli1+ and Hh treatments work is not explored. Some of the results are counter-intuitive. For example, why would Hh stimulate proliferation if Gli1+ cells if these are thought to be slow turnover resident stem cells? Furthermore, why would Hh stimulation lead to proliferation rather than differentiation, in contrast to what is know in growth plate biology)?
The assessment of healing is qualitative/semi-quantitative (histomorphometry). The authors should perform a more rigorous assessment of healing to demonstrate the effectiveness of the Gli1+ cell and Hh therapies. This should include quantitative outcome(s) such as qPCR, mechanics, etc.
The Gli1+ cell therapy histologic results are impressive. This is surprising because the delivery method was relatively simple. How much cell engraftment was there? Can the authors comment further (or add experiments to elucidate) on how long the cells were present and what their direct involvement was in healing?
The authors show that native Gli1+ cells expand after injury. If this is the case, what is the rationale for adding more Gli1+ cells? Is the idea that the tissue has the capacity to heal but there aren't enough native Gli1+ cells to do the job?
Figures and text jump between methodologies, making interpretation of results difficult. Fig1 shows that superficial cells of the meniscus generally have active Hh signaling 24-hours prior to a variety of postnatal-to-adult timepoints (A, B, E, F), and postnatal Hh signaling drives proliferation of early meniscus cells (C, D). It does not appear to report any long-term pulse/chase lineage tracing experiment as suggested in the text (Lines 223+). If this interpretation is incorrect, perhaps this could be addressed by increased clarity of figures and text (Methods, Results, Figure organization and captions).
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