Bone Marrow Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Normal Phenotype and Immunomodulatory Capacity

HCT is the only curative therapy for SCD, but engraftment remains a significant barrier for the majority of patients who lack a matched donor. Based upon dual function in promoting hematopoiesis and immunomodulation, MSCs are an attractive therapy to modulate engraftment post-HCT. Studies have revealed limitations in using cryopreserved, random donor MSCs; this suggests that fresh, autologous MSCs could circumvent these limitations, thus increasing the potential of MSCs to enhance engraftment. MSCs were expanded ex vivo from BM samples obtained from pediatric SCD patients and healthy adult volunteers. Cells were counted using an automated cell counter, and time (hrs) for MSCs to double (P0 to P1) was calculated. MSC immunophenotype was determined by flow cytometry. T cell proliferation was assessed by flow cytometry following co-culture of PBMCs (healthy donors or SCD) ±MSCs for 4 d with anti-CD3/CD28 Dynabeads. IDO expression was determined by RT-PCR on MSCs ±IFN-γ. Expression of hematopoietic factors (48 chemokines/growth factors) was determined by qRT-PCR performed on MSCs ±IFN-γ using Fluidigm array. PCR data were normalized and analyzed with SAS/JMP Genomics software. Doubling time was 42.70±5.78 hrs for SCD samples, comparable to published healthy controls. SCD MSCs displayed an immunophenotype and expressed IDO (±IFN-γ) consistent with ISCT guidelines, with no significant differences compared to CTR (Figure 1; p>0.05). Following co-culture at varying concentrations, both CTR and SCD MSCs equally suppressed proliferation of CTR and SCD T cells in a dose-dependent manner (Figure 2; p>0.05). While a significant difference was detected between CTR and SCD MSC suppression of 3rd party T cells (p<0.05), this was dependent on inclusion of SCD001, which had robust suppression of T cell proliferation; when this sample was removed, no significant difference was found (p>0.05). By Fluidigm array, no differences in the expression of hematopoietic factors were seen between SCD and CTR MSCs (p>0.05); stimulation with IFN-γ resulted in upregulation of CX3CL1, IL7, IL12, IL15, FLT3L, and angiopoietin in both CTR and SCD MSCs. MSCs from SCD patients can be expanded ex vivo to clinically relevant doses and have a phenotype and function comparable to CTR MSCs. These in vitro studies demonstrate the feasibility of expanding functional marrow-derived MSCs from SCD patients prior to HCT and support a planned phase I clinical trial of autologous MSCs to enhance haploidentical stem cell engraftment in SCD patients. Future studies are planned to evaluate alternative cytokine regimens to augment MSC expression of factors important in hematopoiesis, as assessed by Fluidigm array. These and future investigations could provide evidence that autologous MSCs are superior to cryopreserved, HLA-unmatched MSCs and evidence of pharmacological superiority of cytokine-licensed MSCs.Figure 2Suppression of allogeneic (A) and SCD (B: representative) T cell proliferation by CTR and SCD MSCs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)