Isolation, characterization and chondrogenic differentiation of adult stem cell-derived MUSE-cells

Abstract

Articular cartilage is coating the layers of freely movable joints, enabling a smooth surface and acts resisting to forces. The tissue is aneural and avascular, and has a poor ability to selfrenew in cases of tissue damage. Therefore, cartilage lesions often lead to degenerative disorders such as osteoarthritis (OA). OA is considered the most common form of arthritis affecting people worldwide, causing pain and physical disability. Approaches in cartilage regeneration, especially the use of mesenchymal stem cells (MSCs), have been promising, yet limited. Finding a the most suitable cell type for transplantation strategies is still matter of debate. The recent discovery of a pluripotent stem cell type that represent a minor fraction of the stromal cells present in tissues (MUSE-cells) offer an attractive alternative that deserve to be investigated. The main objective of this study was to establish protocols for the isolation and characterization of MUSE-cells from Hoffa’s fat pad (HFP) and umbilical cords (MC), and to compare the chondrogenic differentiation potential between the MUSE- and non-MUSE-cell populations. MUSE-cells were isolated from the total pull of mesenchymal stem cells by cell sorting, using the embryonic marker SSEA-3 as specific cell surface antigen. Scaffold-free 3D cultures maintained in chondrogenic conditions were used to induce cartilage differentiation. Single cell cluster formation assays were used for functional characterization of MUSE. Pluripotent NTERA-2 cells were used as positive control. Mesenchymal cells displaying phenotypic characteristics of stem cells (MSCs) were successfully isolated from fresh tissues. Scaffold-free spheroids of HFP-MSCs showed a more intense Alcian blue (matrix) staining and had better cartilage-like morphology than those formed from mixed cord MSCs (MC-MSCs). SSEA-3+ MUSE-cells could be identified and isolated from HFP (8% of total MSCs) but were nearly undetectable in MC (0.8% of total MSCs). Phenotypic characterization of sorted cells after cell expansion, and functional characterization by single cell cluster formation abilities confirmed the pluripotent nature of the cells. IV We have demonstrated that the adipose tissue of the infrapatellar pocket (HFP) is a good source of MSCs, with the ability to produce cartilage-like spheroids, and contain a fraction of SSEA-3+ cells (MUSE-cells) with the ability to self-renew. This cell subtype was also highly positive for the pluripotency marker SSEA-4. MC-MSCs on the other hand, did not manage to produce spheroids with properties similar to those of native cartilage, and had not SSEA-3+ MUSE-cells. The chondrogenic abilities of MUSE- and non-MUSE-cells from HFP is under investigation at the time of writing this thesis.

Keywords: Articular cartilage, Articular cartilage disorders, Multilineage-differentiating stress enduring (MUSE) cells, Regenerative medicine, Hoffa’s fat pad, Umbilical cord, Chondrogenesis, Mesenchymal stem cells, SSEA-3, SSEA-4, Cell sorting.