WP3 - Identification of a Novel MSC Surface Receptome

Extensive characterization work has been carried out on the identification of the mesenchymal stem cell (MSC) phenotype but no one marker or unique set of markers has been identified that distinguishes a pure, native MSC population from other cell types with intrinsic MSC qualities, such as stem cell precursors, endothelial or epithelial cells, T lymphocytes, B lymphocytes, natural killer cells, macrophages or monocytes, granulocytes, dendritic cells, platelets and erythrocytes.

In 2006, the International Society for Cellular Therapy (ISCT) published a position paper proposing a minimal set of standards to define human MSCs. In addition to plastic adhesion properties and multipotent differentiation potential, the ISCT proposed a specific surface antigen expression profile that should include, at least: ≥95% positive for CD105 (SH2), CD73 (SH3/4) and CD90 (Thy-1) and ≤ 2% expression of CD45, CD34, CD14/CD11b, CD79α/CD19 and HLA-DR.

However, this list is by no means exhaustive. Additional markers that have been associated with MSC identification include CD44, CD133, CD49a, LNGFR, CD10, CD13, BMPRIA and STRO-1 antigen molecule, in addition to adhesion molecules CD106 (VCAM-1), CD166 (ALCAM), ICAM-1 (intracellular adhesion molecule) and CD29. MHC1+, MHCII-, CD40-, CD80-, CD86- markers are linked to MSC immunosuppression and immunomodulation. MSCs have been shown to express intermediate levels of major histocompatability complex (MHC) class I but not human leukocyte antigen (HLA) class II on the cell surface.

Chemokines, growth factors and adhesion molecules and their cell surface receptors influence MSC proliferation, differentiation and migration. Culture expansion conditions, such as donor profile, basal medium, foetal calf serum, growth factors, cell plating density, passaging density and plastic surface quality can have an effect on the surface marker expression profile of the MSC. Identification of specific adhesion molecules on the MSC surface could allow for the selection of the most suitable plastic surface quality. Any change in media may influence proliferation and differentiation potency.

Safety, reproducibility and quality are critical for MSC application in humans. A standardized protocol for MSC culturing for human clinical application requires a stable composition to ensure good reproducibility during manufacturing. Herein lies the importance of a unique surface marker expression profile.

In WP 3, flow cytometry analysis will be used to identify, characterize and quantify the expression of human cell surface markers on our MSC population cultured using a standardized protocol. The objective is to develop a novel database of receptors that is specific to our standardized population of MSCs, and consequently, to identify growth factor combinations leading to a better understanding of the optimal parameters required for proliferation and differentiation of these MSCs in a serum free media. This novel database should also allow for more consistent and more specific clinical release criteria for the manufactured MSCs.

Identification of a unique expression profile that remains consistent throughout the culturing process will allow for direct MSC isolation, provide specificity at each step of the manufacturing process and allow for more stringent quality control processes.