Mesenchymal stromal/stem cells : expansion, engraftment and immune modulation

Due to their production of hematopoietic growth factors and their immunomodulatory capacity, bone marrow-derived mesenchymal stromal/stem cells (MSC), are a promising tool in the procedure of hematopoietic stem cell transplantation (HSCT).

Seven patients undergoing allogeneic HSCT were co-infused with MSC. Three of the patients were regrafted after a previous rejection. Either HLA-identical or haploidentical MSC were given at the time of HSC infusion. Engraftment was successful in all patients with no untowards effects.

Large-scale expansion of MSC intended for clinical use, requires standardized and optimized handling of the cells ex vivo. For safety and regulatory reasons, we explored if fetal calf serum (FCS) in the culture medium could be replaced by human blood group AB serum. Characteristics and immune suppressive function of MSC were not affected by culturing MSC in the alternative serum, and the cell yield was slighlty improved.

Cryopreservation of bone marrow derived mononuclear cells negatively influenced the final MSC yield. An increased number of MSC was achieved by increasing the FCS levels above 10% and decreasing the replating density. The suppressive effect of MSC was similar after repeated passaging. Cell viability and immunosuppression in vitro remained high even after several years storage in liquid nitrogen. Mixing MSC from several donors improved suppression.

When selecting MSC for immunosuppressive therapy, it would be helpful to have defined markers relating MSC characteristics to their immunosuppressive function. We compared phenotype, growth characteristics and gene expression of MSC with differing immunosuppressive capacity, derived from two donors. MSC from donor 1 consistently suppressed proliferation of activated lymphocytes. This was in contrast to MSC from donor 2, where MSC had no or little inhibition or a stimulating effect. Furthermore, single cell derived cultures from the suppressive donor generated suppressive and non-suppressive clones, indicating that the suppressive phenotype is not inherent to the MSC donor. However, phenotypic differences between all cells from the two donors were too diverse to be efficient for prospective isolation of MSC with suppressive capacity. Thus, the only currently reliable means of validating MSC remain the proposed set of standards focusing on the cells adherence to plastic, the phenotype and trilineage potential. Functional testing by in vitro assays is required to assess the immunosuppressive function.

List of scientific papers

I. Le Blanc K, Samuelsson H, Gustafsson B, Remberger M, Sundberg B, Arvidson J, Ljungman P, Lönnies H, Nava S, Ringden O (2007). "Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells." Leukemia 21(8): 1733-8. Epub 2007 May 31
https://pubmed.ncbi.nlm.nih.gov/17541394

II. Le Blanc K, Samuelsson H, Lönnies L, Sundin M, Ringden O (2007). "Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum." Transplantation 84(8): 1055-9
https://pubmed.ncbi.nlm.nih.gov/17989613

III. Samuelsson H, Ringden O, Lönnies H, Le Blanc K (2009). "Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells." Cytotherapy 11(2): 129-36
https://pubmed.ncbi.nlm.nih.gov/19152151

IV. Samuelsson H, Ringden O, Kumagai-Braesch M, Rosendahl K, Le Blanc K (2009). "Characterization of clonal and polyclonal mesenchymal stromal cells with different immunosuppressive capacity." (Manuscript)