Endothelial cells protecting oligodendrocyte progenitor cells from TNFα induced damage

Project Outline

The United Nations General Assembly has declared 2021-2030 the Decade of Healthy Ageing. Aging is associated with a build-up of damaging inflammation in the brain, which potentially contributes to cellular senescence, neuronal and cognitive decline. Studies have shown that overall levels of inflammatory markers are higher in aged brain, including tumour necrosis factor alpha (TNFα), an important neuroinflammatory factor.

Myelin, containing a high content of lipids, plays an important role by ensuring rapid conduction and providing trophic support to the axons. In the central nervous system (CNS), age-related changes in myelin physiology may lead to myelin degeneration, and ultimately neurodegeneration. Myelin forming oligodendrocytes (OL) are renewed and regenerated from their progenitor cells (OPC), this regeneration process is vital for myelin maintenance, but it declines with age. TNFα is required for OPC proliferation and remyelination. However, TNFα interferes with OL differentiation and causes their death. Overall, persistent and high level of TNFα can induce OL and myelin pathology in the neuroinflammatory environment.

Endothelial cells (EC) are essential in the development, maintenance and regeneration of myelin1. Cerebral ECs directly secrete factors, which support OPC proliferation and differentiation. We have shown that human induced pluripotent stem cell (iPSC)-derived ECs (iPSC-EC) protect rat OPCs from TNFα-induced cell death and the effect is associated with BDNF-mTORC1 pathway; this mechanism might contribute to the protective effects from iPSC-ECs on myelin damage and enhanced remyelination2. It is still unclear how ECs protect OL linage cells from TNFα-induced damage on their survival and function in myelin homeostasis as in aged CNS

Aim

We will explore the roles of ECs in protecting OPCs from neuroinflammation-induced damage modelling by human iPSC-derived cells.

Methods

The methods include using iPSC technology to produce human ECs (iPSC-ECs) and iPSC-derived OPCs (iPSC-OPC) to model ECs interaction with OPCs and OLs in inflammatory environment. We have established a platform of iPSC-ECs co-culture with rat OPCs, we have demonstrated the beneficial effects from iPSC-ECs in promoting OL lineage progression and an anti-inflammatory property on OPC survival2. We have also successfully differentiated iPSC into OPCs and OLs.

Objectives

To set up the co-culture system of iPSC-ECs (in culture insert) with iPSC-OPCs. iPSC-EC and iPSC-OPC lines will be differentiated from human iPSCs by the established (or commercial) methods.

To investigate the anti-inflammation role of iPSC-ECs on protecting iPSC-OPCs from TNFα-induced cell death and damaged proliferation/differentiation, examined by immunocytochemistry and cell assays. RNA-sequencing will be used to identify the related signalling pathways. The redox status and lipid oxidation of OPCs and OLs will be analysed by LC-MS/MS methods.

To assess the anti-inflammation effects of iPSC-EC on myelination. A triple-culture system will be set-up containing iPSC-OPCs, iPSC-neurons and iPSC-ECs. Myelination by iPSC-OPCs to neuronal axons will be observed after exposure to TNFα, with or without crosstalk with iPSC-ECs.

Outcome

The results will gain new insight into how ECs protect OPCs and OLs against inflammatory attack, which might help to restore resilience of myelin homeostasis in ageing, hence to improve healthy span. Cerebral ECs hold therapeutic potential to prevent myelin damage in ageing.

The student will benefit from engaging our groups’ cutting-edge research and learning multi-disciplinary knowledge on myelin protection by using iPSC technology to build up leadership in future biomedical career.

References

1. Miyamoto et al 2014 Crosstalk between cerebral endothelium and oligodendrocyte. Cell Mol Life Sci. 2. Ma et al 2023 Human iPSC-derived endothelial cells promote CNS remyelination via BDNF and mTORC1 pathway. Glia.

Techniques

• Cell biology: cell culture, optical and fluorescence microscopy, cell growth/viability and cell mobility/invasion assays, cell irradiation and DNA damage and apoptosis assays
• Molecular biology: gDNA and RNA isolation, PCR, RT-qPCR, site-directed mutagenesis, molecular cloning, CRISPR genome editing, RNA-Seq
• Biochemistry: fractionation and protein isolation, Western blotting, co-immunoprecipitation (Co-IPs), siRNA knock-downs, chromatin immunoprecipitation (ChIP) and Re-ChIP, Cut&Run assays
• Computational techniques: RNA-Seq and ChIP-Seq analysis, genomic data analysis (DNA mutations, DNA methylation, mRNA and miRNA expression), gene enrichment and pathway mapping