Immuno-regulation of brain region-specific organoids with isogenic microglia-like cells

Introduction: : Microglia are the resident innate immune cells of the central nervous system (CNS). Microglia cells in the brain express many risk genes of Alzheimer’s disease (AD). Impaired interactions between microglia and CNS cells, are increasingly linked to neurodegenerative and neurodevelopmental disorders such as AD. Findings showed that induced pluripotent stem cell (iPSC)-derived microglia-like cells (MGC) and human microglia are comparable. Recently. brain organoids co-cultured with iPSC-derived MGC emerged as attractive model systems for drug discovery. To date, the characteristics of isogenic MGC-containing brain organoids in modeling neurodegeneration and cell-cell communications have not been well investigated.
Extracellular vesicles (EVs) are lipid membrane bounded vesicles that can be secreted by basically all kinds of cells. Their rich cargo makes them a promising class of therapeutic candidate. Microglia-derived EVs were shown to alleviate neurodegeneration. miR-711-containing EVs inhibited 1,4,5-trisphosphate 3-kinase B (Itpkb) and further repressed Tau phosphorylation, this led to reduced score of neurological deficits and improved cognitive function in mild traumatic brain injury mouse.
In this study, the role of the EVs in regulating neural degeneration was evaluated in MGC-containing brain organoids. To induce neural degeneration, two types of EVs from the AD patient-derived brain organoids, with APOE4 mutation for sporadic AD and presenilin 1 (PS1) mutation for familial AD, respectively, were isolated and used to treat co-cultured brain organoids and MGC. Then the EVs from M0 MGC, M2 MGC, and the co-cultured brain organoids (CO) derived from healthy human iPSCs were evaluated for the ability to reduce neural inflammation and degeneration.

Materials and

Methods: : In this study, MGC were generated from human iPSCs and characterized by flow cytometry, and changes in gene expression in response to stimulation. MGC expressed microglial markers and were responsive to Aβ42 and dexamethasone stimulation. Then MCGs were co-cultured with induced forebrain cortical organoids (iFCo). The co-cultures were characterized by immunocytochemistry and gene expression as well the response to the neural degeneration stimuli. The co-cultured brain organoids expressed neuronal markers and synaptic markers, indicating a functional signal transduction. The presence of brain organoids and MGC mutually promoted the expression of neural marker and microglia marker in the co-culture. An in vitro AD model was induced by EVs from brain organoids differentiated from AD patient-derived iPSCs: APOE ε4/ε4 (APOE4) for sporadic AD and PS1 for familial AD. Further, an IL-4 stimulation was applied to MGC to induce M2 phenotype. Media from M0 and M2 MGC as well as co-cultures were collected for EV isolation and these EVs were evaluated in the AD model at different doses (1000 or 5000 particles/cell). Enzyme-Linked Immunosorbent Assay (ELISA) was performed for determining the secreted cytokines BDNF and IL-6. mRNA expression was quantitatively analyzed for immunometabolism and neuroinflammation as well as neural degeneration markers. For example, TREM2 is triggering receptor expressed on myeloid cells 2 and related to an increased risk of late-onset AD. The glycolytic pathway is critical for cellular energy production, transforming glucose into pyruvate while generating ATP and NADH, essential for various cellular functions in the context of neurodegenerative diseases.

Results, Conclusions, and Discussions:: Two types of EVs from the AD patient-derived brain organoids (APOE4 and PS1) were used to treat co-cultured brain organoids and MGC. CASS4 and MINK1 were enhanced by APOE4 EV treatment in both co-culture and MGC. UNC5C was increased by APOE4 EVs in co-culture, but it was increased by PS1 EVs in MGC. CD2AP had elevated level in MGC by APOE4 EV treatment but was decreased in co-culture by both APOE4 and PS1 EV treatment. Similarly, PS1 EV induced stronger TREM2 expression in MGC but inhibited TREM2 in co-culture. Pentose phosphate pathway genes 6PGP, G6PD, TALDO1, and TKTL1 were all reduced by PS1 EVs in co-culture; 6PGD and TALDO1 were inhibited in MGC by PS1 EVs. APOE4 EVs induced strong pro-inflammatory gene expression in MGC, but these genes remained constant level in co-culture except for the reduced iNOS. In co-culture, APOE4 EVs slightly decreased BDNF secretion and increased IL-6 secretion, which indicated a more responsive change.
The APOE4 EV induced co-culture was treated with healthy EVs from M0 and M2 MGC, as well as co-culture (CO) (Figure 1). These EVs expressed exosomal markers and presented cup-shaped morphology. ELISA results showed that different EVs did not affect BDNF secretion, but IL-6 was significantly increased by M0 1000, M0 5000, M2 5000, and CO 5000. This indicated that 5000 particles/cell may be overdosed, so the dose of 1000 particles/cell was selected. M0 and CO groups reduced IL-12β level (~0.6 fold and ~0.2 fold respectively. All the three types of EVs significantly reduced iNOS level (~0.4-0.6 fold). BDNF was significantly decreased (~0.4 fold) by the CO group. TREM2 was inhibited by all three types of EVs (~0.5-0.7 fold). CASS4 was inhibited (~0.5 fold) by the M0 and CO groups. In the neural degeneration model, he EVs from the healthy MGC and the co-culture showed regulation of genes involved in pro-inflammation, Aβ and Tau pathology, microglial activation, and carbon metabolism, which provide multiple targets for potential AD therapies. This study paves the way for understanding the role of microglia and brain organoids in modeling neural degeneration and the development of EV-based therapeutic interventions.

Acknowledgements and/or References (Optional):: Research reported in this publication was supported by the National Institutes of Health (USA) under Award Number R01NS125016 (to YL) and R21EB033495 (to XW and YL)