Humanized Chimeric Mouse Models to Study Human Neural Development and Pathogenesis of Brain Diseases

Abstract

The development of humanized neural chimeric mouse models consists in grafting human neuronal progenitor cells (NPC) or differentiated neurons derived from induced pluripotent stem cells (iPSC) into the mouse brain at different timepoints. As the main features of cortical networks and their alterations during brain development cannot be reproduced in vitro, these in vivo models have been used to investigate the mechanisms by which reprogrammed human neurons and non-neuronal cells integrate and migrate into the mouse brain at early stages of brain development and display functional activities several months after their grafting. Here, we describe the neonatal grafting technique of human NPC which we use in our laboratory.We also present a new method based on the grafting of human NPC into the brain of mouse embryos, in utero. A third method consists in the stereotaxic grafting of human NPC into selected brain regions of adult mice. The iPSC technology combined with the use of chimeric mouse models offers numerous possibilities to study human neural development within wellcontrolled and defined temporal windows and to model neuropathological disorders.

Pathogenic variants associated with speech/cognitive delay and seizures affect genes with expression biases in excitatory neurons and microglia in developing human cortex

“…Finally, although large scale transcriptomic analyses like this one highlight new hypotheses and areas of focus, they do not obviate the need for detailed functional molecular studies of individual genes that determine how pathogenic variants affect protein function, and how protein dysfunction in turn leads to cellular pathology, alters developmental mechanics, disrupts neuronal microcircuitry, and ultimately generates neurodevelopmental symptomatology. To this end, the union between the increased availability of high-depth clinical genetic testing, 48 the increasing scale of multimodal single cell profiling, 31,49 and novel methods for testing the impact of pathogenic variants on human cortical development 43 via organotypic slice culture, 50 cerebral organoids, [51][52][53] and humanized animal models, 54 will together help unlock the complexity of human NDDs. 2), demonstrated that Microglia Broad genes were significantly enriched for gene trend G10 (adjusted pval=0.03), while Microglia Narrow genes did not show significant enrichment after correction for multiple comparisons but…”

Developmental Changes of Human Neural Progenitor Cells Grafted into the Ventricular System and Prefrontal Cortex of Mouse Brain in Utero

“…The NPCs were transduced with 99.5 ng of lentivirus (p24) in a 6-well culture plate filled with 500 μL of fresh culture medium for 3 h. The volume was completed to 2 mL, incubated for the next 48 h, and then rinsed with the culture medium. All media and products are described in Thiberge, Llach Pou et al [ 31 ] and are summarized in Table S1 .…”

“…Before their transplantation, the cells were kept in a water bath at 37 °C. The injection solution also contained 20 mM EGTA to avoid cell aggregation upon transplantation [ 14 , 31 , 32 ]. The transplantation protocol has been previously detailed [ 31 ].…”

“…The injection solution also contained 20 mM EGTA to avoid cell aggregation upon transplantation [ 14 , 31 , 32 ]. The transplantation protocol has been previously detailed [ 31 ]. Briefly, 2 μL containing 2 × 10 5 cells were injected within 1 lateral ventricle or within 1 side of the PFC of E17.5 embryos.…”

“…Immunofluorescence analysis on brain sections was performed at 1 and 3 MPT, according to our previously described protocol with slight modifications [ 9 , 31 ]. Animals received a lethal dose of a mixture of Ketamine (Imalgen ® , 100 mg/kg, Merial) and Xylazine (Rompun ® , 10 mg/kg, Bayer), and were transcardially perfused with 15 mL of DPBS, followed by 40 mL of ice-cold paraformaldehyde (PFA) (4%) in 0.1 M phosphate buffer (pH 7.4).…”