Evaluation of osteoblastic differentiation of human Mesenchymal Stem Cells extracted from Wharton’s Jelly Umbilical Cords for the treatment of osteoporosis

Abstract

In regenerative medicine, obtaining Mesenchymal Stem Cells (MSCs) from Wharton's jelly (WJ) in the umbilical cord offers a potential path toward tissue regeneration. The unique qualities of WJ-MSCs, such as their self-renewal capacity, multilineage differentiation, safety profile, high rate of proliferation, and immunological compatibility, make them an attractive source for regenerative applications and treatment of osteoporosis. In contrast to costly and ready-made osteogenic media, this work aims to introduce WJ-MSCs to an osteogenic differentiation medium made of dexamethasone, ascorbic acid, and beta-glycerophosphate. We also examined the impact of the osteoblastic differentiation medium on the WJ-MSCs that were transformed into osteoblastic-like cells, which are being tested for their capability for bone repair. The study included several cell passages (P0, P1, and P2) and evaluated cell growth, confluence, and morphological changes over time. According to the findings, the proliferating MSCs reached an 80% confluence and covered nearly 80% of the culture surface after five weeks of development. From P0 to P2, there was a progressive rise in the quantity of MSCs, indicating successful proliferation and development over several passages. The identification of these cells as MSCs was confirmed by flow cytometry analysis; they consistently expressed mesenchymal markers (CD44, CD73, and CD90) and lacked hematopoietic markers (CD34 and CD45). WJ-MSCs showed a change in shape from fibroblastic to spheroid after being stimulated with osteogenic differentiation media, indicating that they had differentiated into osteoblastic-like cells. The medium, which contains dexamethasone, ascorbic acid, and -glycerophosphate, plays a crucial role in inducing osteogenesis by activating essential osteogenic transcription factors and genes. These osteoblastic-like cells also had significant Runx2 and ALP expression levels, two essential markers for osteoblasts. Unexpectedly, pluripotent markers, including Oct4 and Nanog, were also found, indicating that pluripotent traits remained present after osteoblastic commitment. The presence of these pluripotent markers may significantly impact the use of WJ-MSCs in tissue engineering and regenerative medicine. Additional research revealed alterations in surface marker expression, with lower levels of CD44 and CD73 indicating downregulation of MSCs after induction. On the other hand, there was a significant rise in CD34 expression, which is not generally connected to MSCs. The persistence of CD90 expression demonstrated the effective conversion to osteoblast-like cells. As WJ-MSCs successfully differentiated into osteoblast-like cells, Alizarin Red staining revealed the mineralization capability of these cells. These findings establish the framework for future developments in the area by highlighting the potential of WJ-MSCs and their flexibility for regenerative therapy.