Chimeric brain models to study human glial-neuronal and macroglial-microglial interactions [scRNA-seq]

Human-mouse chimeric brain models, generated by transplanting human induced pluripotent stem cell (hiPSC)-derived neural cells, are valuable for studying the development and function of human neural cells in vivo. Understanding glial-glial and glial-neuronal interactions is essential for unraveling the complexities of brain function and developing treatments for neurological disorders. To explore these interactions between human neural cells in vivo, we co-engrafted hiPSC-derived neural progenitor cells together with primitive macrophage progenitors into the neonatal mouse brain. This approach creates human-mouse chimeric brains containing human microglia, macroglia (astroglia and oligodendroglia), and neurons. Using super-resolution imaging and 3D reconstruction techniques, we examine the dynamics between human neurons and glia, and observe human microglia pruning synapses of human neurons, and often engulfing neurons themselves, as well as the interactions between human oligodendrocytes and neurons. Single-cell RNA sequencing analysis of the chimeric brain uncovers a close recapitulation of the human glial progenitor cell population, along with a dynamic stage in astroglial development that mirrors the processes found in the human brain. Furthermore, cell-cell communication analysis highlights significant neuronal-glial and macroglial-microglial interactions, especially the interaction between adhesion molecules neurexins and neuroligins. This innovative co-transplantation model opens up new avenues for exploring the complex pathophysiological mechanisms underlying human neurological diseases. It holds particular promise for studying disorders where glial-neuronal interactions and non-cell-autonomous effects play crucial roles. Overall design: Two hMAN (human microglia-astrocyte-neuron co-transplanted) Rag2-/- IL2r?-/- hCSF1KI chimeric mice were anesthetized and perfused with cold 1x Dulbecco's Phosphate-Buffered Saline (DPBS). Brains were then dissected and dissociated using the Adult Brain Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Human cells were isolated using the Mouse Cell Depletion Kit (Miltenyi Biotec), following the manufacturer's protocols. The isolated cell suspension from the two mice were subsequently loaded into the Chromium Controller respectively for single-cell RNA sequencing (scRNA-seq) library preparation, utilizing the Chromium Next GEM Single Cell 3' Kit v3.1 and Dual Index Kit TT Set A, in accordance with the manufacturer's guidelines. Quality control of the scRNA-seq libraries was performed using an Agilent 2100 Bioanalyzer, and the libraries were sequenced on a NovaSeq 6000 system using S4 flow cells.