Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus

Abstract

Purpose: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging.

Methods: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin.

Results: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44⁺CD105⁺CD29⁺CD90⁺ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31⁺ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed.

Conclusions: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone.

Clinical relevance: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Fig 1.

Research design. (1) The first aim of this study was to characterize and identify the resident stromal progenitor cell population in all 3 zones in freshly harvested human cadaveric menisci. (2) The second aim was to characterize the vascularity of the menisci, using histology, immunofluorescence (IF), and 3-dimensional (3D) light sheet fluorescence microscopy. (aSMA, alpha smooth muscle actin; CFU, colony-forming unit; H&E, hematoxylin and eosin; MSC, mesenchymal stromal cell; MTC, Masson’s trichrome.)

Fig 2.

Evidence of resident stromal progenitor cells in the WW of human meniscus. (A) Dissection of meniscal zones for cell isolation. (B) Box and whisker plots of cell yields of medial or lateral menisci normalized to tissue wet weight. Lines display median values. (C) Colony formation of isolated cells in vitro. Meniscal cells from all zones were clonogenic in culture. No significant differences were found between zones when assessed by colony-forming unit assays (P > .05). (LL, left lateral (meniscus); LM, left medial (meniscus); RR, red‒red; RW, red‒white; WW, white‒white.)

Fig 3.

Identification of freshly isolated meniscus cells versus cultured controls using flow cytometry. (A) Flow cytometry analysis of cells from the 3 meniscal zones displayed presence of 2 distinct subpopulations of cells immediately after isolation (top panel). One subpopulation was positive to MSC surface markers and the other population was negative. Meniscal cells that were selected using plastic adherence and cultured to P2 were all positive for all MSC markers similarly to BM-MSCs (bottom panel). (B) Quantification of individual cell surface markers showing the proportion of each marker per zone. All 4 markers showed greater expression in the WW zone compared to RR and RW (P < .05). (C) Proportion of CD105⁺CD44⁺CD29⁺CD90⁺ cells per zone, P < .05. (BM-MSC, bone marrow-derived mesenchymal stem cell; MSC, mesenchymal stromal cell; P2, passage 2; RR, red‒red; RW, red‒white; WW, white‒white.)

Fig 4.

Multilineage differentiation potential of meniscal stromal cells. (A) ALP activity after 1 week of osteogenic induction. Controls were cultured for 1 week without osteogenic media (*P < .05, ***P < .001) (B) Col1 expression after 3 weeks of osteogenic induction (P < .05). (C) Adipogenesis was induced for 5 weeks with adipogenic supplements. BM-MSCs were treated under the same conditions and were used as assay positive controls. Red-Oil O stains lipid droplets in red. Bars represent 50 μm. (D) Chondrogenesis was induced in transwells for 3 weeks resulting in disk formation. Disks were processed histologically, sectioned and whole-slide scans attained. Alcian blue was used to determine presence of proteoglycans in the disks. BM-MSCs were treated under the same conditions and were used as assay controls. Bars represent 50 μm. (ALP, alkaline phosphatase; BM-MSC, bone marrow-derived mesenchymal stem cell; RR, red‒red; RW, red‒white; WW, white‒white.)

Fig 5.

Limitations of simple 2-dimensional histology approaches to visualize the vasculature of the human meniscus. (A) Schematic Illustration of the three meniscal zones: red‒red (RR), red‒white (RW), white‒white (WW). (B) Full-thickness cut of the meniscus stained with hematoxylin & eosin (H&E) (left) and Masson’s Trichome stain (MTC) (right), and greater magnification (bottom panels) of boxed areas shows relatively low cellularity throughout the RW and WW zones. Top bars represent 1 mm, in bottom panels bars represent 100 µm.

Fig 6.

Immunofluorescent triple staining with lectin (green), CD31 (orange), alpha smooth muscle actin (αSMA; magenta), and DAPI (blue) in all 3 zones using fluorescent microscopy and imaging with 2 different microscopes. Left images from whole-slide scans were taken (20× scan, Leica). Right, orange boxes from left images were identified using a Nikon Eclipse microscope and imaged at higher magnification (40×). Whole-slide scans allow visualization of the exact region imaged (left images, top right; yellow arrows). Areas were assessed using QuPath software to accurately quantify the distances between regions. The RW snapshot shown is at the bottom border of the RW zone of the whole-slide scan and was chosen in order to show the continuity of the scan. Bigger vessels with αSMA lining and lectin/CD31 can be found in both RR and RW zones. However, in WW zone only lectin/CD31/DAPI positive staining was found, suggesting presence of relatively smaller vessels, such as capillaries in this area. Scales bars represent 50µm. (RR, red‒red; RW, red‒white; WW, white‒white.)

Fig 7.

Successful clearing of human meniscus with modified uDISCO procedure and nucleic staining with To-Pro-3 and the endothelial marker CD31. (A) Fully transparent slices of human meniscal tissue cleared with a modified uDisco approach. On the left, a quarter of fully cleared meniscus. On the right, a slice before and after clearing (B). 2D slice of cleared meniscus tissue stained with only with To-Pro-3, displaying cells distributed in all 3 zones (C). Three-dimensional reconstruction of meniscus slices after imaging with light sheet fluorescence microscopy. Double-labeling with To-Pro-3 (red, pan-nuclei marker) and CD-31 (green, endothelial marker) indicates presence of vessels of various sizes in all zones. Bigger vessels were found spanning the RR to RW zones, confirming 2D results. Blood vessels are present in the WW zone. Upper and bottom panels display different views of the same slice. Left; merged channels (red and green), middle; red channel (To-Pro-3), right; green channel (CD31). Arrows point to vessels. Bar represents 500 µm. (2D, 2-dimensional; RR, red‒red; RW, red‒white; WW, white‒white.)