Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

Abstract

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apicobasal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits

“…To circumvent this technical hurdle, we developed a transplantation procedure that increases the rate of integration of human cortical pyramidal neurons into the neonatal cortex. Specifically we took advantage of the observation that EGTA, added together with the cells during intraventricular injection, can promote integration and migration of mouse and human neurons into the mouse host cortex (Nagashima et al, 2014) ( Figure 1A, S1A). In these conditions, transplanted human ESC-derived cortical pyramidal neurons, examined 2-8 weeks post-transplantation, were found within the cortical gray matter, from deep (5/6) to superficial (2/3) cortical layers, displaying a radial orientation ( Figure 1B, S1B) and expressing markers ( Figure 1C-D, S1C-D) typical of cortical pyramidal neurons (Greig et al, 2013).…”

“…The observation of structural dynamics, together with an increase in the rate of incoming spontaneous synaptic events over the course of several months ( Figure 2H), raised the possibility that transplanted human neurons might display synaptic plasticity. To assess this, we applied a long-term potentiation (LTP) protocol (Nevian and Sakmann, 2006) to xenotransplanted neurons in ex vivo slice preparations ( Figure 5A). We found that human cells aged 3 to 6…”

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain

“…The immunostaining protocol for fixed brain slices and dissociated cells is described elsewhere (Nagashima et al, 2014). The primary antibodies used were as follows: rabbit anti-Pax6 (1:500; PRB-278P, Covance), mouse anti-Tuj1 (1:500; MMS-435P, Covance), and rat anti-Eomes eFluor 660 (1:1000; 14-4875-80, eBioscience).…”

Stem Cells, Genome Editing, and the Path to Translational Medicine

“…Importantly, genetic depletion of the host melanoblast lineage significantly increased integration of human cells, suggesting that an “empty host niche” gives donor cells a selective advantage to efficiently contribute to interspecies chimeras. Similarly, transplanted human neural precursor cells into E14.5 embryos or terminally differentiated cortical neurons into neonatal mice robustly functionally integrate into mouse brain (Espuny-Camacho et al, 2013; Nagashima et al, 2014). …”

“…In both experimental paradigms, the overall contribution of human cells is rather limited. This could be caused by technical challenges related to transplantation (Cohen et al, 2016; Nagashima et al, 2014), imperfect adjustment of developmental timing between donor and host cells at the time of transplantation (Cohen et al, 2018) or species-specific differences.…”