Lipid Metabolic Readouts in Humanized Models of Neuroimmune Function

Microglia plays a dual role in the exacerbation or resolution of neuroinflammation through innate immune functions like cytokine secretion and phagocytosis. Experimentally, microglial functions can be inferred from transcriptomic and proteomic data. In the clinic however, information is limited to the few biomarkers that can be collected noninvasively. Imaging probes for TREM2, TSPO, and APOE serve as clinical indicators of neuroinflammatory pathologies, but less appreciated is that these proteins share similar functional roles in lipid metabolism across neural tissue. Microglia undergo significant changes in their metabolism during inflammation and neurodegeneration. Neurometabolic profiles, can be readily collected from cells in culture using nuclear magnetic resonance (NMR) in samples, and even measured non-invasively in living animals and human subjects through magnetic resonance spectroscopy (MRS) potentially bridging basic and clinical research. Despite the clinical accessibility of this readout, effectively interpreting metabolic information between models remains a significant challenge. An experimental platform for molecular-specific perturbations that could be resolved consistently across scales would connect accessible neurometabolism with underlying neuroimmune function. NMR has unexplored potential for assaying metabolic changes, providing insights into microglia function, and the progression of neurodegenerative diseases.
We have established a pipeline of human ESC-derived microglia for in culture assays and for combination with human brain organoids for neurometabolic profiling. Microglia were derived from a hematopoietic lineage, and flow cytometry validation demonstrated essential markers of microglia differentiation, with 80% co-expression of TREM2 and CD45 and more than 50% CD11b expression. Microglia were then introduced to 5-week-old human brain organoids, consisting of astrocytes and neurons. Our tri-culture brain organoids were validated by immunohistochemistry for neuron (Neun) astrocyte (GFAP) and microglia (IBA1) markers. For broader transcriptional signatures, organoids were harvested for RNAseq, and a final cohort was solvent extracted into lipid and water-soluble fractions for liquid NMR. NMR was deconvolved through ASICS method into relative concentrations of detectable metabolites for differential analysis and pathway enrichment using the KEGG database. The ASICS method automatically identifies and quantifies all present metabolites in NMR spectra, thereby ensuring consistency in data analysis.
The potential for ESC derived microglia and tri-culture organoids to serve as a more optimal humanized experimental platform needs critical evaluation. Further work will profile whole organoids against mouse biopsy using high-resolution magic angle spinning solid-state NMR and clinical MRS data to directly compare neurometabolism from cells in culture to living human brains.