Identification of a sub-population of synovial mesenchymal stem cells with enhanced treatment efficacy in a rat model of Osteoarthritis

Reviewer #1 (Public review):

Summary:

This work by Al-Jezani et al. focused on characterizing clonally derived MSC populations from the synovium of normal and osteoarthritis (OA) patients. This included characterizing the cell surface marker expression in situ (at time of isolation), as well as after in vitro expansion. The group also tried to correlate marker expression with trilineage differential potential. They also tested the ability of the different sub-populations for their efficacy in repairing cartilage in a rat model of OA. The main finding of the study is that CD47hi MSCs may have a greater capacity to repair cartilage than CD47lo MSCs, suggesting that CD47 may be a novel marker of human MSCs that have enhanced chondrogenic potential.

Strengths:

Studies on cell characterization of the different clonal populations isolated indicate that the MSC are heterogenous and traditional cell surface markers for MSCs do not accurately predict the differentiation potential of MSCs. While this has been previously established in the field of MSC therapy, the authors did attempt to characterize clones derived from single cells, as well as evaluate the marker profile at the time of isolation. While the outcome of heterogeneity is not surprising, the methods used to isolate and characterize the cells were well developed. The interesting finding of the study is the identification of CD47 as a potential MSC marker that could be related to chondrogenic potential. The authors suggest that MSCs with high CD47 repaired cartilage more effectively than MSC with low CD47 in a rat OA model.

Weaknesses:

While the identification of CD47 as a novel MSC marker could be important to the field of cell therapy and cartilage regeneration, there was a lack of robust data to support the correlation of CD47 expression to chondrogenesis. The authors indicated that the proteomics suggested that the MSC subtype expressed significantly more CD47 than the non-MSC subtype. However, it was difficult to appreciate where this was shown. It would be helpful to clearly identify where in the figure this is shown, especially since it is the key result of the study. The authors were able to isolate CD47hi and CD47 low cells. While this is exciting, it was unclear how many cells could be isolated and whether they needed to be expanded before being used in vivo. Additional details for the CD47 studies would have strengthened the paper. Furthermore, the CD47hi cells were not thoroughly characterized in vitro, particularly for in vitro chondrogenesis. More importantly, the in vivo study where the CD47hi and CD47lo MSCs were injected into a rat model of OA lacked experimental details regarding how many cells were injected and how they were labeled. No representative histology was presented and there did not seem to be a statistically significant difference between the OARSI score of the saline injected and MSC injected groups. The repair tissue was stained for Sox9 expression, which is an important marker of chondrogenesis but does not show production of cartilage. Expression of Collagen Type II would be needed to more robustly claim that CD47 is a marker of MSCs with enhanced repair potential.

Reviewer #2 (Public review):

Summary:

This is a compelling study that systematically characterized and identified clonal MSC populations derived from normal and osteoarthritis human synovium. There is immense growth in the focus on synovial-derived progenitors in the context of both disease mechanisms and potential treatment approaches, and the authors sought to understand the regenerative potential of synovial-derived MSCs.

Strengths:

This study has multiple strengths. MSC cultures were established from an impressive number of human subjects, and rigorous cell surface protein analyses were conducted, at both pre-culture and post-culture timepoints. In vivo experiments using a rat DMM model showed beneficial therapeutic effects of MSCs vs non-MSCs, with compelling data demonstrating that only "real" MSC clones incorporate into cartilage repair tissue and express Prg4. Proteomics analysis was performed to characterize non-MSC vs MSC cultures, and high CD47 expression was identified as a marker for MSC. Injection of CD47-Hi vs CD47-Low cells in the same rat DMM model also demonstrated beneficial effects, albeit only based on histology. A major strength of these studies is the direct translational opportunity for novel MSC-based therapeutic interventions, with high potential for a "personalized medicine" approach.

Weaknesses:

Weaknesses of this study include the rather cursory assessment of the OA phenotype in the rat model, confined entirely to histology (i.e. no microCT, no pain/behavioral assessments, no molecular readouts). It is somewhat unclear how the authors converged on CD47 vs the other factors identified in the proteomics screen, and additional information is needed to understand whether true MSCs only engraft in articular cartilage or also in ectopic cartilage (in the context of osteophyte/chondrophyte formation). Some additional discussion and potential follow-up analyses focused on other cell surface markers recently described to identify synovial progenitors is also warranted. A conceptual weakness is the lack of discussion or consideration of the multiple recent studies demonstrating that DPP4+ PI16+ CD34+ stromal cells (i.e. the "universal fibroblasts") act as progenitors in all mesenchymal tissues, and their involvement in the joint is actively being investigated. Thus, it seems important to understand how the MSCs of the present study are related to these DPP4+ progenitors. Despite these areas for improvement, this is a strong paper with a high degree of rigor, and the results are compelling, timely, and important.

Overall, the authors achieved their aims, and the results support not just the therapeutic value of clonally-isolated synovial MSCs but also the immense heterogeneity in stromal cell populations (containing true MSCs and non-MSCs) that must be investigated further. Of note, the authors employed the ISCT criteria to characterize MSCs, with mixed results in pre-culture and post-culture assessments. This work is likely to have a long-term impact on methodologies used to culture and study MSCs, in addition to advancing the field's knowledge about how synovial-derived progenitors contribute to cartilage repair in vivo.

Author response:

We appreciate the reviewers’ thoughtful and constructive feedback, which has provided valuable insights to refine our manuscript. Below, we outline the planned revisions in response to the public reviews.

Response to Reviewer #1

We are grateful for the reviewer’s recognition of our methodological approach and the potential significance of CD47 as a novel MSC marker for cartilage repair. To address the concerns raised:

(1) Clarifying the proteomics data supporting CD47 as an MSC marker

· The manuscript will be revised to clearly indicate where the proteomics data demonstrate elevated CD47 expression in MSCs compared to non-MSCs.

· Additional figure annotations or a supplemental figure may be included to enhance clarity.

(2) Providing further details on CD47hi and CD47lo MSC populations

· Information on the number of isolated CD47hi and CD47lo cells, along with any necessary expansion steps before in vivo use, will be explicitly detailed.

(3) Expanding the characterization of CD47hi MSCs in vitro

· A more comprehensive analysis of the chondrogenic differentiation capacity of CD47hi MSCs will be incorporated to strengthen the findings.

(4) Clarifying experimental details of the in vivo rat OA model

· The methodology section will be updated to specify the number of injected cells and their labeling strategies.

· Representative histological images will be added to support the results.

· To further substantiate the cartilage repair potential of CD47hi MSCs, additional staining for Collagen Type II will be included alongside Sox9 expression.

Response to Reviewer #2

We appreciate the reviewer’s enthusiasm for the study and recognition of its rigor and translational significance. The following revisions are planned to address the feedback:

(1) Addressing additional assessments for OA phenotype in the rat model

· While this study primarily relied on histology, the limitations of this approach will be acknowledged in the discussion.

· The absence of microCT and behavioral assessments will be explained, with suggestions for incorporating these methods in future studies.

(2) Justifying the focus on CD47

· The rationale behind prioritizing CD47 over other proteomics-identified markers will be expanded to provide better context for this choice.

(3) Clarifying MSC engraftment patterns

· The manuscript will include a discussion on whether CD47hi MSCs specifically engraft in articular cartilage or contribute to ectopic cartilage formation (e.g., osteophytes).

(4) Contextualizing findings within recent research on synovial progenitors

· Additional discussion will highlight recent studies on DPP4+ PI16+ CD34+ stromal cells and how the identified MSC populations may relate to these universal fibroblasts.

We are confident that these revisions will strengthen the manuscript and enhance its clarity and impact. The reviewers’ insights have been invaluable, and we look forward to refining the study accordingly.