Biology subjects

Forsyth, N. R.

Publications and source records attributed to Forsyth, N. R..

4 recordsLinked to original sources

bioRxiv2022

Physoxia influences global and gene-specific methylation in pluripotent stem cells

Pluripotent stem cells (PSC) possess unlimited proliferation, self-renewal, and a differentiation capacity spanning all germ layers. Appropriate culture conditions are important for the maintenance of self-renewal, pluripotency, proliferation, differentiation, and epigenetic states. Oxygen concentrations vary across different human tissues depending on precise cell location and proximity to vascularisation. The bulk of PSC culture-based research is performed in a physiologically hyperoxic, air oxygen (21% O2) environment, with numerous reports now detailing the impact of physiologic normoxia (physoxia) low oxygen culture in the maintenance of stemness, survival, morphology, proliferation, differentiation potential, and epigenetic profiles. Epigenetic mechanisms affect multiple cellular characteristics including gene expression during development and cell fate determination in differentiated cells. We hypothesized that epigenetic marks are responsive to a reduced oxygen microenvironment in PSCs and their differentiation progeny. Here, we evaluated the role of physoxia in PSC culture, the regulation of DNA methylation (5mC and 5hmC), and expression of regulatory enzymes DNMTs and TETs. Physoxia enhanced the functional profile of PSC including proliferation, metabolic activity, and stemness attributes. PSCs cultured in physoxia revealed significant downregulation of DNMT3B, DNMT3L, TET1, and TET3 vs. air oxygen, accompanied by significantly reduced 5mC and 5hmC levels. Downregulation of DNMT3B was associated with an increase in its promoter methylation. Coupled to above we also noted decreased HIF1A but increased HIF2A expression in physoxia cultured PSCs, versus air oxygen. In conclusion, PSCs display oxygen-sensitive methylation patterns that correlate with transcriptional and translational regulation of the de novo methylase DNMT3B.

cell biology↗

bioRxiv2022

Hypoxic conditions promote a proliferative, poorly differentiated, and pro-secretory phenotype in COPD lung tissue progenitor cells in vitro

Chronic obstructive pulmonary disease patients experience variable symptoms dependent on the presence of an emphysematous versus a chronic bronchitis phenotype. Both presentations can be associated with lung tissue and systemic hypoxia, at its most severe leading to Cor pulmonale. Despite this, minimal attention has been given to the effects of hypoxia at the cellular disease level. We isolated and cultured progenitor cells from the distal lung tissue of a 64 year-old, male, emphysematous donor in ambient (21%) and hypoxic (2%) oxygen conditions. Proliferative capacity was determined on collagen coated culture plastic and growth-inactivated 3T3-J2 co-cultures. Epithelial (E-cadherin and pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expression were examined. Expanded cells were differentiated at air-liquid interface and ciliated, mucous producing, and club cell populations identified. Isolated cells were positive for the epithelial, pan-cytokeratin and E-cadherin, and progenitor, TP63 and cytokeratin 5, cell markers at isolation and again at passage 5. Passage 5 expanded cells in hypoxia had increased the proportion of TP63 expressing cells by 10% from 51.6 {+/-} 1.2% to 62.6 {+/-} 2.3% (p [≤] 0.01). Proliferative capacity was greater in 3T3J2 co-cultured cells overall and in 2% oxygen this supported the emergence of a proliferation unrestricted population with a limited differentiation capacity. Cells expanded on collagen I in either oxygen underwent differentiation having been expanded with the production of ciliated cells positive for {beta}IV tubulin, and mucin 5ac, mucin 5b and CC10 positive secretory cells. Epithelial barrier formation was reduced significantly (p [≤] 0.0001) in hypoxia-expanded cells compared to normoxia. qRT-PCR showed higher expression of mucins in 2% expanded cells, significantly so with MUC5B (P [≤] 0.05) although mucin protein secretion was greater in 21% expanded cells. Concomitantly these results demonstrate that hypoxia promotes a proliferative phenotype while reducing the overall differentiation capacity of the cells. Further, the retained differentiation potential becomes skewed to a more secretory phenotype demonstrating that hypoxia may be contributing to disease symptom and severity in COPD patients.

physiology↗

bioRxiv2021

THERAPEUTIC EFFECTS OF HYPOXIC AND PRO-INFLAMMATORY PRIMING OF MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES IN INFLAMMATORY ARTHRITIS

Novel biological therapies have revolutionised the management of Rheumatoid Arthritis (RA) but no cure currently exists. Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Th17:Treg homeostatic balance. Priming of MSCs in EV production can be applied to alter the therapeutic efficacy however normoxic EVs present the optimal strategy for broad therapeutic benefit. The varied outcomes observed in MSCs priming may promote EVs optimised for therapies targeted for specific therapeutic priorities. EVs present an effective novel technology with potential for cell-free therapeutic translation.

immunology↗

bioRxiv2020

MSCs from syndecan-3 null mice exhibit enhanced adhesion to collagen type I, hyperactivation of the AKT pathway and increased efficacy in inflammatory arthritis

1Rheumatoid arthritis (RA) is a debilitating and painful inflammatory autoimmune disease characterised by the accumulation of leukocytes in the synovium, cartilage destruction and bone erosion. The immunomodulatory effects of bone marrow derived mesenchymal stem cells (MSCs) has been widely studied and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology. MSCs isolated from bone marrow of wild type and Sdc3-/- mice were used to assess immunophenotype, differentiation, adhesion and migration properties and cell signalling pathways. While both cell types show similar differentiation potential and forward scatter values, the cell complexity in wild type MSCs was significantly higher than in Sdc3-/- cells and was accompanied by lower spread surface area. Moreover, Sdc3-/- MSCs adhered more rapidly to collagen type I and showed a dramatic increase in AKT phosphorylation, accompanied by a decrease in ERK1/2 phosphorylation compared with control cells. In a mouse model of antigen-induced inflammatory arthritis, intraarticular injection of Sdc3-/- MSCs yielded enhanced recovery compared to injection of wild type MSCs. In conclusion, our data suggest that syndecan-3 regulates MSC adhesion and efficacy in inflammatory arthritis, likely via induction of the AKT pathway.

immunology↗