ABSTRACT
Background & Aim: The pro-angiogenic, immunoregulatory and anti- inflammatory properties of MSCs are being exploited for the development of cellular therapies, including the treatment of graft versus host disease (GvHD), inflammatory bowel disease and COVID-19. SNBTS have developed a GMP process to bank umbilical cord MSCs (UC-MSCs) whereby we can reliably bank 100 vials of 10 million P2 UC-MSCs per cord. Each of these vials can be extensively expanded and stored for specific applications. The ultimate aim of the bank is for off-the-shelf clinical use, e.g., in GvHD or as an adjuvant therapy in Islet transplantations. Methods, Results & Conclusion(s): During process development, different basal media and supplements were screened for proliferation and MSC marker expression. Cells grown in promising media combinations were then tested for tri-lineage differentiation (identity), their chemokine/cytokine expression and T-cell inhibition (function) assessed. Medium selected for further GMP development and scale up was ultimately determined by all round performance and regulatory compliance. GMP-like UC-MSCs were shown to have immune-modulatory activity in T-cell proliferation assays at 4:1 or 16:1 ratios. Co-culture of UC-MSCs and freshly isolated leukocytes, +/- the immune activating agent LPS, show a dose dependent survival effect on leukocytes. In particular, neutrophils, which are normally very short lived in vitro demonstrated increased viability when co-cultured with UCMSCs. The survival effect was partially reproduced when UC-MSC were replaced with conditioned medium or cell lysate indicating the involvement of soluble factors. This improved neutrophil survival also correlates with results from leukocyte migration studies that demonstrate neutrophils to be the main cell type attracted to MSCs in in vitro and in vivo. Genetic modification of UC-MSC may improve their therapeutic potential. We have tested gene editing by CRISPR/Cas9 technology in primary UC-MSCS. The CXCL8 gene, highly expressed in UC-MSC, was targeted in isolates from several different donors with editing efficiencies of 78-96% observed. This translated to significant knockdown of CXCL8 protein levels in resting cells, however after stimulation levels of CXCL8 were found to be very similar in edited and non-edited UC-MSCs. This observation requires further study, but overall the results show the potential to generate future banks of primary UC-MSCS with genetically enhanced pro-angiogenic, immunoregulatory and/or anti-inflammatory activities.Copyright © 2023 International Society for Cell & Gene Therapy
ABSTRACT
Severe acute respiratory syndrome coronavirus (SARS-CoV) possesses high replicative capacity and pathogenicity than the classical coronavirus. However, the factors that lead to enhanced replication and pathogenicity remain unclear. Among the non-structural proteins of SARS-CoV, non-structural proteins 9 (NSP9) is known to most likely be involved with viral RNA synthesis. In this study, we investigated the effect of NSP9 on the human upper respiratory system using tonsil-derived mesenchymal stem cells (TMSCs). In order to confirm the significant toxic effect, NSP9 were treated to TMSCs in various concentrations as follows;0.01 ,0.05 ,0.2, 0.4, and 0.8 ug/ml. Cell proliferation assay and LIVE/DEAD assay revealed that NSP9 was most toxic to TMSCs at a concentration of 0.2 ug/ml. In addition, NSP9 treated TMSCs showed a pattern of decreased metabolic efficiency in mitochondria. The phosphoinositide 3-kinase (PI3K) and phosphorylated Akt protein markers were compared to confirm that it affects cell proliferation by reducing the metabolic rate. These results showed that NSP9 inhibits PI3K/Akt signaling proteins related to cell proliferation in TMSCs and induces toxicity by decreasing the cellular metabolic rate. Taken together, the NSP9, possessing toxicity to TMSCs, can affect to upper respiratory system by inhibiting cell proliferation and metabolic rate.
ABSTRACT
Background: People living with HIV (PWH) may represent a high-risk group for adverse clinical outcomes from SARS-CoV-2. The duration of protection from SARS-CoV-2 and emerging variants of concern (VOC) infection in PWH following vaccination is unclear. Furthermore, the role of preexisting SARS-CoV-2 immune responses, likely acquired from prior exposure to circulating human coronaviruses (HCoVs), on vaccine-mediated immunity remains to be determined. Understanding the kinetics of immune responses to SARS-CoV-2 and VOCs, and the impact of preexisting SARS-CoV-2 immunity on vaccine-mediated immune responses will be critical in informing COVID-19 vaccination policies in PWH. Methods: In this sub-study of the Phase II/III COV002 trial (open-label, non-randomised clinical trial ID: NCT04400838), 54 HIV+ male participants on antiretroviral therapy (undetectable viral loads, CD4+ T cells >350 cells/ul) and 50 HIV-sex and age-matched controls received two doses of ChAdOx1 nCoV-19 (AZD1222) 4-6 weeks apart and were followed for 6 months. Immune responses to vaccination were determined by ELISA (standard and MSD assay), neutralisation, ACE-2 inhibition, IFNγ ELISpot, activation-induced marker (AIM) assay and T cell proliferation assays. Results: 6 months after vaccination, antibody IgG levels to SARS-CoV-2 S and RBD proteins, ACE-2 inhibition and T cell responses to S protein were significantly higher than baseline (Table 1). Both humoral and cell-mediated immunity waned over time, but with no significant difference compared with HIV-individuals vaccinated with the same regimen. T and B cell-mediated immune responses to VOCs α, β, γ, and δ were detectable, although at lower magnitudes than wild type. Prior exposure to circulating β coronavirus HKU1 and OC43 was associated with measurable proliferative SARS-CoV-2 T cell response at baseline and a higher magnitude of post-vaccine T cell responses. Conclusion: Our data demonstrate no significant difference in ChAdOx1 nCoV-19 vaccine-mediated immune responses by HIV status. For all groups, we show waning but detectable immune responses against SARS-CoV-2 and VOCs 6 months after vaccination supporting the on-going policy to vaccinate against SARS-CoV-2 and reinforces the argument for long-term monitoring of responses.