Summary

Meniscus progenitor cells are present in the mature meniscus and are characterized by high chondrogenic capacity, expression of MSC markers, high colony forming ability, high proliferative capacity and the ability to form meniscus matrix upon differentiation, therefore they show promise for use in meniscus tissue engineering.

Abstract

Purpose

Due to the limited regenerative capacity of the meniscus, (partial) meniscectomy is often the surgical treatment of choice. Consequential loss of contact area and hence abnormal distribution of forces predispose to osteoarthritis. Therefore, there is an unmet clinical need for meniscus regeneration. Recently the presence of mesenchymal stem cell (MSC)-like cells or progenitor cells in different intra-articular tissues has been shown. Progenitor cells of the meniscus could be a treatment target and a possibly superior cell type for meniscus tissue-engineering. Therefore, the purpose of this study is to isolate progenitor cells from osteoarthritic meniscus tissue and characterize these cells according to the MSC guidelines by the International Society for Cellular Therapy (ISCT).

Methods

Osteoarthritic menisci from patients undergoing total knee arthroplasty were digested and progenitor cells were selected by differential adhesion to fibronectin and cultured op to passage 4. Trilineage differentiation, population doubling time, and colony forming capacity were assessed, and the total meniscus population at passage 2 and 4 was used as control (n=5 donors, 3 technical replicates). The colony forming ability of meniscus cells derived from inner and outer zone was compared at passage 0. Expression of positive (CD105, CD73 and CD90) and negative (CD45, CD34, CD11b, CD79A, and HLA-DR) MSC markers was measured by flow cytometry. To stimulate redifferentiation, meniscus and progenitor cells were pellet-cultured for 4 weeks in DMEM with 2% human serum albumin, 2% insulin-transferrin-selenium-ethanolamine and antibiotics in absence of growth factors or with the addition of 10 ng/mL TGFß. Chondrogenic differentiation was assessed by gene expression (RT-qPCR), DNA content (PicoGreen assay) and release and deposition of glycosaminoglycans (dimethylmethylene-Blue (DMMB) and Safranin-O), and deposition of collagen (hydroxyproline assay).

Results

Both progenitor and meniscus cells demonstrated osteogenic and adipogenic differentiation. Only meniscus progenitors showed glycosaminoglycan deposition indicating chondrogenic differentiation. At passage 0, a higher colony forming ability was seen in progenitors isolated from the outer zone compared to the inner zone. Colony forming ability of progenitor cells at passage 4 was higher than that of meniscus cells at passage 2 and 4. Population doubling times of progenitors were lower than that of meniscus cells, indicating a higher proliferation rate. Of the progenitor cells, 3/5 donors adhered to the surface marker profile matching ISCT MSC criteria. Gene-expression and biochemical assays indicated a comparable proteoglycan and collagen release and deposition in progenitor cells and meniscus cells. COL2A1 expression of progenitors was higher than that of the total meniscus population with addition of TGFß to the culture media.

Conclusions

This is the first study to show that progenitor cells are present in the osteoarthritic human meniscus based on the standard set by the ISCT. Progenitor cells express MSC markers and have trilineage potential and importantly possess a higher chondrogenic capacity than the total meniscus cell population. Furthermore, progenitor cells have high colony forming ability and proliferative capacities, while retaining their ability to form meniscus extracellular matrix upon redifferentiation even after passage 4. Therefore, meniscus-derived progenitor cells are a promising cell type for meniscus tissue-engineering and a potential treatment target.