Developing Human Pluripotent Stem Cell-Based Cerebral Organoids with a Controllable Microglia Ratio for Modeling Brain Development and Pathology

Abstract

Microglia, as the brain-resident macrophages, play critical roles in brain development, homeostasis, and disease. Microglia in animal models cannot accurately model the properties of human microglia, because there are due to notable transcriptomic and functional differences between human and other animal microglia at transcriptomic and functional levels. Efficient generation of microglia from human pluripotent stem cells (hPSCs) provides unprecedented opportunities to study the function and behavior of human microglia. Particularly, incorporating hPSCs-derived microglia into brain organoids facilitates their development in a 3-dimensional context, mimicking the brain environment. However, an optimized method that integrates an appropriate amount of microglia into brain organoids at a proper time point, similar to what is seen in vivo, is still needed. Here, we report the development of a new brain region-specific, microglia-containing organoid model by co-culturing hPSCs-derived primitive neural progenitor cells (pNPCs) and primitive macrophage progenitors (PMPs). In these organoids, hPSCs-derived pNPCs and PMPs interact with each other and develop into functional neurons, astroglia, and microglia, respectively. Importantly, the numbers of human microglia population in the organoids can be controlled, resulting in a cell type at a ratio similar to that seen in the human brain. Importantly, uUsing super-resolution microscopy, we demonstrate that these human microglia are able to phagocytize neural progenitor cells (NPCs) and dead cells, as well as to prune synapses at different developmental stages of the organoids. Furthermore, these human microglia respond to Zika virus infection in of the organoids, as indicated by exhibiting amoeboid-like morphology, an increased expression of gene transcripts encoding inflammatory cytokines, and excessive pruning of synaptic materials. ThusTogether, our findings establish a new microglia-containing brain organoid model that will serve to study human microglial function in a variety of neurological disorders.

Type I Interferon Signaling Drives Microglial Dysfunction and Senescence in Human iPSC Models of Down Syndrome and Alzheimer’s Disease

“…However, in the cerebral organoids, microglia extend fewer processes and do not show morphology as highly ramified as they do in vivo (Benito-Kwiecinski and Lancaster, 2020; Ormel et al, 2018; Xu et al, 2021). Consequently, we did not observe any significant morphological differences between DS vs. Cont microglia in cell size, as indicated by the quantification of microglia cell volume (Fig.…”

“…We recently developed a novel microglia-containing cerebral organoid model that provides a convenient tool to study human microglial functions in neurodevelopmental disorders (Xu et al, 2021). As shown in Fig.…”

“…2A, here we used PMPs derived from the three pairs of Cont and DS hiPSCs and primitive neural progenitor cells (pNPCs) derived from Cont hiPSC cells (Xu et al, 2019). Then, hiPSC-derived pNPCs and PMPs were mixed to develop organoids, as described in our recent study (Xu et al, 2021). By week 4, both Cont and DS organoids contained cells that expressed CD45, which is a marker for all nucleated cells of the hemopoietic lineage (Carson et al, 1998) and labels microglia in the organoids (Fig.…”

“…Each brain organoid was generated from a total of 10,000 cells (7,000 pNPCs and 3,000 PMPs), in each well of ultra-low-attachment 96-well plates in the presence of ROCK inhibitor Y-27632 (10 mM) (Xu et al, 2021). The culture medium was composed of a 1:1 mixture of PMP medium and NPC medium (1:1 mixture of Neurobasal and DMEM/F12, supplemented with 1x N2, 1x B27-RA, and FGF2 (20 ng/ml, Peprotech) for three days (day 3).…”

“…Recent advances in stem cell technology have led to the efficient generation of microglia from human induced pluripotent stem cells (hiPSCs) (Abud et al, 2017; Brownjohn et al, 2018; Douvaras et al, 2017; Haenseler et al, 2017; Jiang et al, 2020; Muffat et al, 2016; Pandya et al, 2017), providing an unlimited source of human microglia to study their pathophysiology. Very recently, we have developed new hiPSC-based microglia-containing cerebral organoid and chimeric mouse brain models (Jiang et al, 2020; Xu et al, 2021; Xu et al, 2020), in which hiPSC-derived microglia undergo maturation and develop appropriate functions. With DS hiPSC established in our previous studies (Chen et al, 2014; Xu et al, 2019), these new models provide unprecedented opportunities to investigate the role of human microglia in DS brain development and DSAD.…”