Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine due to their capability of differentiating and self-renewing [1]. These cells are being isolated from different tissues, including bone marrow, dental pulp, adipose tissue, etc.. Menstrual blood MSCs (MenSCs) are much less characterized, however, they are known to possess more pronounced stem cell properties, as compared to classical bone marrow MSCs (BMMSCs). MenSCs exhibit a higher proliferation rate and a broader differentiation capacity [2,3]. Although MenSCs is an attractive alternative to BMMSCs, their use for cartilage tissue regeneration is still under consideration and research. Also, MenSCs are known to possess large amounts of growth factors responsible for an effective endometrium regeneration, which might be a potential co-stimulant for other tissue regeneration [2,3].
The aim of the study is to evaluate the chondrogenic differentiation capacity of MenSCs and their ability to stimulate BMMSCs chondrogenesis in co-culture conditions, analyzing levels of chondrogenesis-related growth factors.
This study was performed in compliance with all bioethical requirements. Cells were isolated from healthy human menstrual blood (n=3) and bone marrow (n=3) samples. MenSCs and BMMSCs were characterized according to stem cell surface marker expression (flow cytometry) and adipogenic/osteogenic differentiation capacity (Oil-Red/Alizarin). Chondrogenic differentiations of MenSCs and BMMSCs were stimulated with transforming growth factor-beta (TGF-β3), activin A, insulin-like growth factor 1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and their combinations. The results of this study indicate that TGF-β3 significantly stimulated chondrogenic response in BMMSCs, as compared to other growth factors, while chondrogenic differentiation of MenSCs was much weaker and stimulated with combination of activin A and TGF-β3. For co-culture conditions, MenSCs were seeded in 24 well plates, while BMMSCs were seeded in 0.4 μm membrane inserts placed into wells with seeded MenSCs. After co-cultures with MenSCs, secretion of TGF-β3, activin A, BMP-2 and IGF-1 was analyzed (ELISA) and chondrogenic differentiation of BMMSC pellets was evaluated according to cartilage oligometric matrix protein (COMP) cultured in chondrogenic medium for 3, 7, 21 days, and immunohistochemical analysis of collagen type II. The results demonstrated that chondrogenic differentiation of BMMSCs was more pronounced in co-culture with MenSCs, as compared to single BMMSCs cultures, which have resulted in a significant secretion of COMP and production of collagen type II in BMMSCs chondrogenic pellets. The amounts of growth factor release (TGF-β3, activin A and IGF-1) was also higher in co-culture conditions, which demonstrated the importance of cell co-stimulation.
In conclusion, MenSCs may turn out to be a promising population of stem cells for the development of cell-based technologies to stimulate chondrogenesis in vitro.