Human iPSC-derived mature microglia retain their identity and functionally integrate in the chimeric mouse brain

Abstract

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. Here, we develop a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage progenitors into neonatal mouse brains. Single-cell RNAsequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a tool to study the role of human microglia in brain development and degeneration.

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

“…The identity of hPSCs-derived pNPCs was confirmed by staining with Nestin, a marker for NPCs (Wiese et al, 2004) ( Figure 1B). To induce microglia differentiation and obtain PMPs, we followed a published protocol (Haenseler et al, 2017;Xu et al, 2020). At day 5, yolk sac embryoid bodies (YS-EB) were plated on dishes, and PMPs were released from attached YS-EB into the supernatant starting around day 20; PMPs were continuously produced for more than three months, with a cumulative yield about 40-fold higher than the number of the input hPSCs.…”

“…The hiPSC lines were generated from healthy adult-derived fibroblasts using the "Yamanaka" reprogramming factors in our previous study (Chen et al, 2014). The hPSC line has been fully characterized, including karyotyping, teratoma assay, Pluritest (www.PluriTest.org), DNA fingerprinting STR (short tandem repeat) analysis and gene expression profiling (Xu et al, 2019;Xu et al, 2020). The hPSCs, from passage number 15-30, were cultured on dishes coated with hESC-qualified Matrigel Human pNPCs were generated from hPSCs as previously reported (Chen et al, 2016;Xu et al, 2019).…”

“…The PMPs were derived from hPSCs, as previously reported (Haenseler et al, 2017;Xu et al, 2020), and produced in a Myb-independent manner to recapitulate primitive hematopoiesis (Buchrieser et al, 2017;Haenseler et al, 2017). Briefly, yolk sac embryoid bodies (YS-EBs) were generated by treating EBs with bone morphogenetic protein 4 (BMP4, 50 ng/ml, Peprotech; to induce mesoderm), stem cell factor (SCF, 20 ng/ml, Peprotech), and vascular endothelial growth factor (VEGF, 50 ng/ml, Peprotech) in mTeSR1 medium (STEM CELL Technologies).…”

“…Zeiss Airyscan super-resolution microscope at 63X with 0.2 mm z-steps was used to acquire super-resolution images for visualizing synaptic puncta pruning. Imaris software (Bitplane 9.5) was used to process super-resolution images and generate 3D-surface rendered images as previously reported (Xu et al, 2020). To visualize the engulfed puncta within microglia, the mask function of Imaris was used to subtract any fluorescence that was outside of the microglia, as previously reported (Schafer et al, 2014).…”

Dendrimers as Modulators of Brain Cells

“…Such models are particularly suitable for investigations of the mechanisms implicated in microglia regulation upon exposure to different neuroactive agents, toxins and nanostructures including dendrimers. Aside from rodent microglia, human stem cells are valuable sources for microglia, astrocytes, oligodendrocytes, neurons and other cell types [ 49 , 50 , 51 , 52 ]. Astrocytes are essential for physiological neuronal functions, but if they acquire genetic mutations, they become highly proliferative and give rise to astrocytoma.…”