Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Orthopedic and Rehabilitation Engineering

Orthopedic and Rehabilitation Engineering - Poster Session A

Poster K9 - Characterization of Functional Meniscal Cell Subpopulations via Immunofluorescent Antibody Staining

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The meniscus has poor regenerative properties after injury due to its limited vascularity and sparse distribution of cells. Common treatments like meniscectomies and suture repair do not support new tissue growth and often result in post-traumatic osteoarthritis. This poses a particular risk for pediatric patients, who may permanently lack a critical component of their knee. Given the frequency of meniscus tears and ineffectiveness of traditional treatments, regenerative medicine offers a potential alternative remedy. Cells in the meniscus which may play a role in regeneration after injury have not yet been discovered, and there is limited knowledge of any specific cell subpopulations throughout the meniscus, especially in pediatric tissues. Identifying meniscus cells which can form new tissue via extracellular matrix synthesis is critical for developing effective regeneration-based treatments. Our group previously performed single-cell RNA sequencing of pediatric meniscus tissues, revealing functionally-distinct cell clusters. This work highlights markers of two matrix-synthetic clusters, fibroblast-like and chondrocyte-like cells, due to their potentially significant role in producing and organizing extracellular matrix during tissue regeneration. This project aims to validate and characterize the specific expression patterns of these markers in cultured primary meniscus cells, a necessary step toward our ultimate goal of identifying matrix-synthetic meniscus cells. By confirming the association of characteristic protein markers with specific cell phenotypes, we can apply the results of our single-cell analysis to isolate and examine pediatric meniscus cells based on their expected regenerative capabilities in injury contexts.

Materials and

Methods:  Primary meniscus cells were obtained from a single donor (Male, Age 17, Children’s Mercy Hospital, IRB Exemption #STUDY00000746) following partial meniscectomy after lateral meniscus tear. Tissue was digested using collagenase and harvested cells were cryopreserved. The protein products of selected markers for these clusters will be analyzed in this study: MXRA8 and PDGFRβ are associated with fibroblast-like cells, TNFRSF11b has an association with chondrocyte-like cells, and CD44 was chosen as a general marker for meniscus tissue (Table 1). The cells were cultured in a tissue culture-grade 24-well plate, fixed at Passage 4, and stained for the targets listed using standard immunofluorescent antibody protocols (1° antibody dilution of 1:1000 with 1 hr 50 min incubation time, 2° antibody dilution of 1:5000 with 1 hr incubation time). Stained cells were then imaged using the ECHO Revolve widefield fluorescence microscope, and target marker expressions were visualized and quantified by use of ImageJ/FIJI and CellProfiler.

Results, Conclusions, and Discussions: The overall goal of the antibody staining panel was to validate the presence of potential meniscus cell subpopulation-specific markers and analyze their feasibility in future cell isolation and sorting experiments. The panel included control wells for non-specific target proteins and species isotypes that demonstrated the efficacy of targeted markers. Stains targeting nuclei (DAPI) and actin filaments (Phalloidin) displayed uniform appearance between each well. MXRA8, which has not previously been characterized in meniscus tissues, displayed a less homogenous signal morphology within and across multiple wells compared to other markers studied (Figure 1). This marker had varied expression patterns and localizations, including clusters of strong concentrated expression near the nucleus and more dispersed and irregular signals throughout the cell membrane. Due to this heterogeneous staining pattern between otherwise morphologically similar cells within a single image, MXRA8 is a strong candidate for isolating meniscus cells based on their potentially unique functions as indicated by the presence or absence of this target marker. Although varying in signal intensity and robustness, PDGFRβ, TNFRSF11b, and CD44 all demonstrated seemingly uniform expression within the cell membrane. PDGFRβ and TNFRSF11b appear to be present within all cells, but not at the level of intensity as CD44. Thus, CD44 proves to be a promising positive control marker for the heterogeneous meniscal cell population in future analysis, due to its seemingly ubiquitous nature. These results emphasize the importance of characterizing individual cells as a necessary next step for assessing the regenerative capabilities within meniscus tissue. While CD44 was identified as a strong positive control marker for potentially distinct meniscus cells, MXRA8 was established as a potential functionally-specific cell sorting factor. Future studies will utilize primary meniscus cells sourced from patients with diverse characteristics, including sex and age, to continue validating the applicability of these and other markers. This knowledge is critical for performing robust cell isolation and subsequent in vitro studies. Once isolated, we will conduct studies in meniscus tissue-mimetic environments to gain an understanding of the matrix remodeling capabilities of functionally-distinct cell subpopulations with the hope of regenerating reliable tissue after meniscus injury in pediatric patients.

Acknowledgements (Optional):  I would like to acknowledge the members of the TARE Lab at the University of Washington, Seattle. I would also like to thank the advisors of the Summer 2024 REU at the Institute of Stem Cell and Regenerative Medicine.