Damage to articular cartilage often results in inefficient repair by local chondrocytes, an outcome that is further impacted upon by the intrinsic lack of vascularity in this tissue and the ever-present wound-healing response post injury. The limited healing potential of this tissue has resulted in knee cartilage degeneration gaining significant clinical focus for many years. Mesenchymal stromal cells (MSCs) have emerged as a promising cell source for the regeneration of cartilage, as they possess chondrogenic differentiation potential and are easily expanded ex vivo. Bone marrow (BM) is currently the major source of MSCs (hBMSCs), and to date, the most extensively characterized for cartilage tissue engineering purposes. However, cells with similar characteristics have been isolated from a wide range of prenatal and postnatal tissues.
Human placenta is considered one of the more clinically relevant sources of MSCs, due to its wide availability, and the high ex vivo proliferation, differentiation potential and low immunogenicity exhibited by mesenchymal progenitors isolated from this tissue. The overall aim of this thesis was thus to investigate the potential of placenta derived MSCs as an alternative source of progenitors for cartilage tissue engineering applications. In order to confirm this potential, studies detailed in the chapters of this thesis investigated the impacts of variations in critical microenvironmental conditions, including soluble factors provision and oxygen tension, on their ability to drive and sustain chondrogenic differentiation of MSCs isolated from the chorionic mesodermal layer of human term placenta’s amniochorionic membrane.
The first study describes the isolation of mesenchymal progenitors from the amniochorionic fetal membrane of human term placenta and their comparative characterization to human hBMSCs. We determined that amnion (hAMSCs), chorion (hCMSCs) and bone marrow (hBMSCs) derived MSCs shared the expression of typical mesenchymal and pluripotent markers. Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. We found a differential expression of cell-cell and cell matrix receptors in these three sources. In addition, hCMSCs displayed a strong chondrogenic and osteogenic response, but very limited capacity for adipogenic conversion, illustrating the applicability of these cells for cartilage regeneration.
Having demonstrated the strong chondrogenic potential of hCMSCs, we next investigated the effect of two members of the Transforming Growth Factor beta (TGFβ) superfamily for inducing chondrogenesis: TGFβ-3 and Growth and Differentiation Factor 5 (GDF5). We determined that undifferentiated hCMSCs express differentiation markers for cartilage, fibrocartilage and vi tendonogenic differentiation. TGFβ3 and GDF5 induced chondrogenesis in hCMSCs, promoting sulphated GAG’s synthesis and extracellular cartilaginous matrix formation. Both factors stimulated the upregulation of chondrogenic, fibrochondrogenic and tendonogenic differentiation markers, confirming the utility of hCMSCs for fibrocartilage and tendon tissue engineering strategies.
Following on from this, we thereafter studied the effect of oxygen tension on the expansion and chondrogenic potential of hCMSCs. We determined that hypoxic expansion of hCMSCs preserved hCMSCs immunophenotype, senescence status and integrin expression profile. hCMSCs proliferated faster under hypoxia, leading to increased cell yields, which was associated to an enhanced progression through the cell cycle and a differential expression of the cell cycle regulators. In addition, hCMSCs preconditioned under hypoxia or normoxia similarly differentiated towards chondrocytes, whether differentiated under hypoxic or normoxic conditions. Taken together, these results allowed us to conclude that hypoxic ex vivo expansion and differentiation is beneficial for the application of hCMSCs in cartilage tissue engineering applications.
The isolation of chondrogenic-committed progenitors is currently one of the main challenges in cellular therapy for cartilage repair. We investigated the feasibility of using N-CAM as a suitable biomarker for identifying chondrogenic progenitors from hCMSCs using fluoresce activated cell sorting. We found that NCAM+ sorted hCMSCs expressed higher levels of early osteogenic and chondrogenic markers in comparison to NCAM- hCMSCs, indicative of a distinctive potential for bone and cartilage differentiation. Upon in vitro differentiation, we determined that both cell subsets underwent osteogenesis and chondrogenesis, but no significant differences were found between both cellular subsets.
Collectively, these studies demonstrate the feasibility of using hCMSCs for cartilage tissue engineering strategies, and lay the foundation for further development towards their clinical application. It is hoped that the insights provided by this thesis will aide in the development of improved cellular therapy approaches aimed at cartilage regeneration.