Mesenchymal stromal/stem cells : expansion, engraftment and immune modulation
Author: Samuelsson, Håkan
Date: 2009-04-28
Location: Föreläsningssalen C1.87, Karolinska Universitetssjukhuset, Huddinge
Time: 09.00
Department: Institutionen för laboratoriemedicin / Department of Laboratory Medicine
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thesis.pdf (535.2Kb)
Abstract
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.
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 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
Pubmed
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
Pubmed
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
Pubmed
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)
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
Pubmed
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
Pubmed
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
Pubmed
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)
Issue date: 2009-04-07
Rights:
Publication year: 2009
ISBN: 978-91-7409-406-0
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