Development of methods for the in-vitro assessment of the adipogenesis and immunogenicity of human multipotent mesenchymal stromal cells

Adult mesenchymal stromal cells (MSCs) have enormous potential in tissue engineering and regenerative medicine applications. MSC have also been reported to be immune-privileged. The aim of this study was to develop methods to test two related hypotheses. Firstly, that MSCs will not directly stimulate allogeneic lymphocytes in classical lymphocyte proliferation assays carried out over 5-7 days due to lack of expression of immune-regulatory molecules (CD40, CD80, CD86 and MHC Class-II) but may be capable of the stimulation of allogeneic lymphocytes when cultured over extended time periods that allow the indirect pathway of antigen presentation. Secondly, that following differentiation of MSCs into mature differentiated adipose cells, the cells would become immunogenic or increase their immunogenicity, as determined by the stimulation of allogeneic peripheral blood mononuclear cells in lymphocyte transformation assays. MSCs from 4 human bone marrow donors were isolated, expanded and characterised and shown to conform to the ISCT definition of MSC through FACs analysis of key antigen expression and trilineage differentiation. MSCs (from donors 1-3) were differentiated into adipocytes, and current methodology for assaying adipogenesis was evaluated. A new fluorescent microplate assay was developed using DAPI to normalise for cell number and Nile red to stain for intracellular lipids, to generate a ratio of adipogenesis. Peripheral blood mononuclear cells (PBMCs) from 6 volunteers were isolated, in-vitro assay conditions for performing lymphocyte transformation assays (LTAs) were optimised, and the duration of the assays extended to 16-21 days. It was shown that undifferentiated MSCs stimulated PBMCs in all except 2 out of 24 cases, with the highest stimulation typically post-day 7 with stimulation indices as high as 36 observed. It was shown that MSCs differentiated into adipocytes also stimulated PBMCs with higher stimulation indices observed in differentiated MSCs compared to the undifferentiated counterparts. It was concluded that MSC, whether undifferentiated or differentiated into adipose cells, are indeed capable of stimulating a proliferative response in allogeneic lymphocytes. Therefore, the use of allogeneic MSCs in the traditional engineering setting may be limited, and a cautious approach should be taken before allogeneic MSCs are used in the regeneration or creation of tissue replacements for clinical use.