Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity

Aalto University, P.O. Box 11000, FI-00076 Aalto www.aalto. fi Author Pietilä Mika Name of the doctoral dissertation Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity Publisher School of Chemical Technology Unit Department of Biotechnology and Chemical Technology Series Aalto University publication series DOCTORAL DISSERTATIONS 161/2012 Field of research Adult Stem Cell Biology Manuscript submitted 17 August 2012 Date of the defence 5 December 2012 Permission to publish granted (date) 30 November 2012 Language English Monograph Article dissertation (summary + original articles) Abstract Human mesenchymal stem cells (hMSCs) are a heterogenous population of multipotent adult stem cells that can be used to treat many degenerative and immunological diseases. Currently there is a lack of a specific definition for hMSCs. This is a major problem as standardization of hMSC products is therefore currently impossible. Accordingly, the current thesis focuses on detailed mitochondrial and ultrastructural characterization of hMSC from different origin. The results show that the increase in mitochondrial mass, mitochondrial reactive oxygen species and activation of the antioxidant defense system occurs during early osteogenic differentiation. Therefore these parameters could be utilized in parallel with current immunophenotype characterization to detect and isolate hMSCs with different metabolical status, which may reflect distinct developmental stages of hMSCs. hMSCs should posses high viability and functionality to mediate the healing effect to the site of injury or inflammation. Therefore sensitive and especially rapid quality control tools are urgently needed. The present thesis demonstrates that monitoring the mitochondrial energy state could be utilized as a viability and potency measure for hMSCs. Specifically, sensitivity of the JC-1 dye for the mitochondrial inner membrane potential to detect oxidative stress and microbial infection induced apoptosis was clearly superior when compared to traditional methods. Moreover, the mitochondrial inner membrane potential correlated with the osteogenic differentiation potency of hMSCs and could be utilized as a potency assay for hMSCs intended for cellular therapies. The immunosuppressive activity of hMSCs has been demonstrated both in vivo and in vitro. In the present study, a macrophage model was established to study the role of the CD200CD200r axis in hMSCs mediated immunosuppression of macrophages. hMSC lines with higher positivity for CD200 showed more efficient inhibition of TNF-alpha secretion from THP-1 macrophages when compared to hMSC lines with low CD200 positivity. Moreover, the role of CD200-CD200r axis in hMSC mediated immunosuppression was confirmed by blocking CD200 from the cell surface of hMSCs. The results from the present thesis suggest that mitochondrial analysis could be used to manufacture pure and viable hMSC products, essential parameters for highly functional hMSCs. These studies also show that hMSC could be used to ameliorate inflammatorial conditions by suppressing the pro-inflammatorial acitivity of macrophages via the CD200CD200r axis.Human mesenchymal stem cells (hMSCs) are a heterogenous population of multipotent adult stem cells that can be used to treat many degenerative and immunological diseases. Currently there is a lack of a specific definition for hMSCs. This is a major problem as standardization of hMSC products is therefore currently impossible. Accordingly, the current thesis focuses on detailed mitochondrial and ultrastructural characterization of hMSC from different origin. The results show that the increase in mitochondrial mass, mitochondrial reactive oxygen species and activation of the antioxidant defense system occurs during early osteogenic differentiation. Therefore these parameters could be utilized in parallel with current immunophenotype characterization to detect and isolate hMSCs with different metabolical status, which may reflect distinct developmental stages of hMSCs. hMSCs should posses high viability and functionality to mediate the healing effect to the site of injury or inflammation. Therefore sensitive and especially rapid quality control tools are urgently needed. The present thesis demonstrates that monitoring the mitochondrial energy state could be utilized as a viability and potency measure for hMSCs. Specifically, sensitivity of the JC-1 dye for the mitochondrial inner membrane potential to detect oxidative stress and microbial infection induced apoptosis was clearly superior when compared to traditional methods. Moreover, the mitochondrial inner membrane potential correlated with the osteogenic differentiation potency of hMSCs and could be utilized as a potency assay for hMSCs intended for cellular therapies. The immunosuppressive activity of hMSCs has been demonstrated both in vivo and in vitro. In the present study, a macrophage model was established to study the role of the CD200CD200r axis in hMSCs mediated immunosuppression of macrophages. hMSC lines with higher positivity for CD200 showed more efficient inhibition of TNF-alpha secretion from THP-1 macrophages when compared to hMSC lines with low CD200 positivity. Moreover, the role of CD200-CD200r axis in hMSC mediated immunosuppression was confirmed by blocking CD200 from the cell surface of hMSCs. The results from the present thesis suggest that mitochondrial analysis could be used to manufacture pure and viable hMSC products, essential parameters for highly functional hMSCs. These studies also show that hMSC could be used to ameliorate inflammatorial conditions by suppressing the pro-inflammatorial acitivity of macrophages via the CD200CD200r axis.