Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing

Abstract

We have previously demonstrated that cryopreservation and thawing lead to altered MSC functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous PBMCs and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of HSA with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ pre-licensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an IDO inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use.

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

“…To test whether thawing affects the responsiveness of MSC preparations, we examined the secretome signature of MSCs previously cryopreserved and then thawed and activated by co-culture with PBMC responders. Consistent with previous studies (Chinnadurai et al, 2016; Moll et al, 2014), thawed MSCs were defective in inhibiting T cell proliferation (Figure 4A), and here we further show that thawed MSC secretome response failed to exhibit any correlation with T cell proliferation (Figures 4B–4F; Figure S3; Table S3), and the R 2 correlative values of T cell proliferation with secretome signature are significantly lower than the values derived from fit MSCs (Figures 4G and 4H). …”

Extracellular vesicles from bone marrow‐derived mesenchymal stromal cells support ex vivo survival of human antibody secreting cells

“…Human MSC were isolated from BM aspirates collected from the iliac crest of consenting volunteer subjects [52]. BM aspirates were diluted 1:2 with PBS and layered onto a Ficoll density gradient to isolate mononuclear cells.…”

Alterations of Multipotent Mesenchymal Stromal Cells Induced by Interaction with Allogeneic Lymphocytes In Vitro

“…Thus, in the case of cell therapy, it may be most effective to administer IFN-γ-activated MSCs on the first day after priming. The effect of the exposure to IFN-γ in vitro explains the enhanced therapeutic activity of MSCs in the model of colitis [28], as well as their better activity after freezing and thawing [12].…”

“…IFN-γ stabilized the MSCs, and the proportion of viable cells was significantly higher in MSCs-γ after 4 days of co-culture with lymphocytes. Perhaps the effect of IFN-γ on the stabilization of MSCs also helps to maintain their immunomodulatory properties after cryopreservation [12]. …”