M.Sc Thesis

M.Sc StudentWilli Anteer Lubna
SubjectInduced Differentiation of Human Embryonic Stem Cells into
Mesenchymal Stem Cells in Suspension
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Joseph Itskovitz


             Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are a subset of non-hematopoietic stem cells that can be isolated from various adult and fetal tissues such as bone marrow, fat tissue and cord blood. MSCs constitute a potentially powerful tool in regenerative medicine and research due to their ability to differentiate into various cell lineages, their extensive proliferative potential, and their special immunomodulatory properties. Following the development of preclinical studies, MSCs have been shown to be effective in the treatment of different diseases in clinical trials. MSCs are defined by a combination of physical, morphological, phenotypic and functional characteristics; MSCs are plastic-adherent, express several surface markers, lack the expression of the CD45 and CD31 markers, and differentiate into osteoblasts, adipocytes and chondrocytes in-vitro. Human embryonic stem cells (hESCs) can potentially provide an alternative, unlimited and reproducible source of MSCs, which also circumvent the need for risky invasive techniques for their isolation. Recently, there have been many efforts to switch from two-dimensional (2D) to three-dimensional (3D) systems since the thoughts are that 3D, or suspension, culture conditions reflect the in-vivo situation more accurately. Nevertheless, the successful use of hESC-derived MSCs for clinical applications in a 3D system will require tight control of the cells' differentiation process and isolation of pure populations of the desired cells without adhesion.

            Accordingly, our research hypothesis is that we will be able to isolate MSCs in an earlier development stage, in suspension, with the ability to mature to multipotent MSCs. Here, we present an efficient 3D system of culturing and differentiating hESCs towards MSCs, which successfully allow the isolation of a pure population of MSCs using fluorescent-activated cell sorting (FACS). Interestingly, surface antigen CD73 (adhesion molecule) known to be expressed by MSCs was not detected in cell culture in suspension. Cells were purified from hESCs principally based on the negative expression of the pluripotent marker surface antigen SSEA4. We demonstrate that hESCs differentiate to MSCs through an early intermediate precursor CD105 SSEA-4- CD31- with a high rate, about 28%. This allows us to isolate highly enriched populations of CD105. This population is described as putative MSC Progenitors.


In this research, the hESC- derived MSCs cultured in suspension are multipotent and were able to differentiate into the three MSC-linages in the appropriate "home-made" media: osteocytes, chondrocytes and adipocytes. Furthermore, they completely lost pluripotent markers and were devoid of the hematopoietic marker CD45 as well as the endothelial marker CD31. Thus, we succeeded in directed differentiation of pluripotent hESCs into multipotent MSCs with high self-renewal potential in 3D system by developing an effective, simple and reproducible culture method. Freeze and thaw cycles did not affect the cells' features, and resulted in high survival rates.

            In summary, our work provides the first proof of concept that MSCs, adherent cells by nature, can be differentiated from hESCs and propagated in suspension culture. The putative MSC progenitors derived in this dynamic culture, once adhered to the matrix, display the typical multipotency of mature MSCs.