M.Sc Thesis

M.Sc StudentDomev-Cohen Hagit
SubjectDirected Differentiation of Human Embryonic Stem Cells
towards Mesenchymal Stem Cells
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Joseph Itskovitz
Full Thesis textFull thesis text - English Version


Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells that can be derived from various adult and fetal tissues. Additionally to their importance for research, MSCs constitute a potentially powerful tool in regenerative medicine due to their extensive proliferative potential, their ability to differentiate into various cell lineages and their special immunomodulatory properties. MSCs are defined by a combination of characteristics; MSCs are plastic-adherent, express the surface markers CD73, CD105 and CD90, lack the expression of the hematopoietic markers and differentiate into osteoblasts, adipocytes and chondrocytes in-vitro. The availability of tissues for the isolation of adult or fetal MSC remains limited and requires invasive procedures. Therefore human embryonic stem cells (hESCs) can potentially provide alternative, unlimited and reproducible source of identical MSCs, which also circumvent the need for risky invasive techniques. The successful use of hESC- derived MSCs for clinical applications will require strict control of the cells' differentiation process and the isolation of pure populations of the desired cells. In this research, we present two simple, efficient and reproducible directed differentiation systems of hESCs towards MSCs: co-culture with murine OP9 stromal cells or feeder layer-free, which successfully allow the isolation of a pure population of MSC using fluorescent-activated cell sorting (FACS). Cells were purified from two different hESC lines based on the expression of the surface antigen CD73. In comparison to previous studies in the field, our protocol generates a high percentage of cells expressing the mesenchymal marker CD73 a fact that allows us to isolate highly enriched populations of CD73+ cells very easily.  In contrast to other protocols, our system is simple and induces mesenchymal differentiation based on a one-step process that does not require any enzymatic or mechanical passaging or costly growth factors. Our purified CD73 positive cells from both co-culture and feeder-free systems were plastic-adherent, expressed comprehensive set of markers such as CD105, CD90, CD29 and CD44 and were devoid of the hematopoietic markers CD45 and CD34 as well as for the endothelial marker CD31. The MSCs generated by our protocol had multilineage potential and differentiated into adipocytes, osteoblasts and chondrocytes in culture.The established feeder-free system which was found to be more efficient for the derivation of MSC from hESCs, also eliminates the risk of contamination by xenozootic infectious agents, thus making it desirable and applicable for clinical purposes. Therefore, our MSC can serve for both research and clinical applications, following examination of their immunological properties.