Menstrual blood-derived mesenchymal stem cells as a potential tool for cartilage regeneration

Abstract

Purpose: Human mesenchymal stem cells (MSCs) are multipotent cells found in almost all human tissues. These cells are of great interest in regenerative medicine due to their high potential to differentiate into several directions including osteocytes, adipocytes and chondrocytes. Menstrual blood has been proposed as one of the perspective MSCs sources (MenSCs), due to their ease of access, as compared to the classical bone marrow MSCs (BMMSCs). As MSCs exhibit high chondrogenic differentiation potential, they ideally suit for therapeutic use in cartilage regeneration. Transforming growth factor β (TGF-β) has been considered as one of the major factors for induction of chondrogenic response in BMMSCs, however a member of the TGF-β superfamily Activin A also seems to contribute in the early stages of MSC chondrogenesis. Activin A seems necessary for the maintenance of self-renewal and pluripotency of MSCs. Furthermore there is an increasing body of evidence that Ca2+-dependent regulatory mechanisms can influence differentiation potential of chondrogenic progenitor cells, whereas Activin A was shown to increase free calcium concentrations in cells. Since Activin A also regulates menstrual cycle, we hypothesized that it may influence chondrogenic differentiation of MenSCs. The aim of this study was to characterize MenSCs and their chondrogenic differentiation capacity using two different growth factors - TGF-β3 and Activin A. The data was compared to the classical BMMSCs. Methods: BMMSCs and MenSCs were isolated from different healthy donors. Different cell surface markers (CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD271, CD106, CD34, CD10, Stro1, Notch1) were analysed by flow cytometry. Cell proliferation capacity was determined by CCK-8 proliferation kit. Intracellular calcium was measured using fluorescent dye Cal-520. Adipogenic differentiation capacity was determined by Oil-Red staining and osteogenic differentiation by Alizarin Red staining. Chondrogenic differentiation (Activin A and TGF-b3 stimulation) was investigated by immuno- and histochemical staining with collagen II antibodies and safranin O, respectively. Results: In contrast to BMMSCs, MenSCs exhibited higher expression of CD10 and Notch1, and higher proliferative capacity. Furthermore, MenSCs exhibited higher level of intracellular calcium concentration as compared to BMMSCs. Adipogenic differentiation of MenSCs was weaker, whereas osteogenic differentiation capacity was higher compared to BMMSCs. Chondrogenic differentiation in BMMSCs was stronger with TGF-β3 than with Activin A, while MenSCs chondrogenic differentiation was remarkably more pronounced with Activin A than with TGF-β3. Conclusions: Taken together, our results demonstrate that MenSCs exhibit similar stem cell properties to BMMSCs, better proliferation potential and different regulatory mechanisms in chondrogenesis in vitro. Menstrual blood seems a convenient stem cells source for isolation of MSCs with chondrogenic differentiation potential, as does not require any invasive surgical interventions, and thus provides an option of repeated sample collection in the same donor, which make MenSCs a perspective tool in cartilage regenerative medicine.