Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex.

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".

Xenotransplanted Human Cortical Neurons Reveal Species-Specific Development and Functional Integration into Mouse Visual Circuits.

How neural circuits develop in the human brain has remained almost impossible to study at the neuronal level. Here, we investigate human cortical neuron development, plasticity, and function using a mouse/human chimera model in which xenotransplanted human cortical pyramidal neurons integrate as single cells into the mouse cortex. Combined neuronal tracing, electrophysiology, and in vivo structural and functional imaging of the transplanted cells reveal a coordinated developmental roadmap recapitulating key milestones of human cortical neuron development. The human neurons display a prolonged developmental timeline, indicating the neuron-intrinsic retention of juvenile properties as an important component of human brain neoteny. Following maturation, human neurons in the visual cortex display tuned, decorrelated responses to visual stimuli, like mouse neurons, demonstrating their capacity for physiological synaptic integration in host cortical circuits. These findings provide new insights into human neuronal development and open novel avenues for the study of human neuronal function and disease. VIDEO ABSTRACT.

Repair of the injured adult hippocampus through graft-mediated modulation of the plasticity of the dentate gyrus in a rat model of temporal lobe epilepsy.

Intracerebroventricular kainate administration in rat, a model of temporal lobe epilepsy (TLE), causes degeneration of the hippocampal CA3 pyramidal and dentate hilar neurons. This leads to a robust but aberrant sprouting of the granule cell axons (mossy fibers) into the dentate supragranular layer and the CA3 stratum oriens. Because this plasticity is linked to an increased seizure susceptibility in TLE, strategies that restrain the aberrant mossy fiber sprouting (MFS) are perceived to be important for preventing the TLE development after the hippocampal injury. We ascertained the efficacy of fetal hippocampal CA3 or CA1 cell grafting into the kainate-lesioned CA3 region of the adult rat hippocampus at early post-kainic acid injury for providing a lasting inhibition of the aberrant MFS. Analyses at 12 months after grafting revealed that host mossy fibers project vigorously into CA3 cell grafts but avoid CA1 cell grafts. Consequently, in animals receiving CA3 cell grafts, the extent of aberrant MFS was minimal, in comparison with the robust MFS observed in both "lesion-only" animals and animals receiving CA1 cell grafts. Analyses of the graft axon growth revealed strong graft efferent projections into the dentate supragranular layer with CA3 cell grafting but not with CA1 cell grafting. Thus, the formation of reciprocal circuitry between the dentate granule cells and the grafted CA3 pyramidal neurons is likely the basis of inhibition of the aberrant MFS by CA3 cell grafts. The results also underscore that grafting of cells capable of differentiating into CA3 pyramidal neurons is highly efficacious for a lasting inhibition of the abnormal mossy fiber circuitry development in the injured hippocampus.

Fetal hippocampal CA3 cell grafts transplanted to lesioned CA3 region of the adult hippocampus exhibit long-term survival in a rat model of temporal lobe epilepsy.

Intracerebroventricular administration of kainic acid in the adult rat, a widely used model for studying human temporal lobe epilepsy, results in widespread degeneration of CA3-pyramidal neurons. Transplantation of specific fetal hippocampal CA3 cell grafts into the lesioned CA3-region at a prolonged post lesion delay of 45-day leads to 31% graft cell survival at 1 month postgrafting and significantly facilitates appropriate recovery of the lesioned host hippocampus. However, the capability of hippocampal CA3 cell grafts for enduring survival in this model is unknown. We hypothesize that a significant fraction of fetal CA3 cells grafted into the lesioned CA3 region of the adult hippocampus at 45-days postlesion exhibit long-term survival. We measured the extent of cell survival within 5'-bromodeoxyuridine-labeled CA3 cell grafts at 1 year postgrafting, following their transplantation at 45 days postlesion into the lesioned CA3-region. Quantification of absolute graft cell survival using BrdU immunostaining and the optical fractionator counting method revealed survival of 36% of grafted cells at 1 year postgrafting. Thus, over a third of fetal hippocampal CA3 cells transplanted to the lesioned CA3-region at 45 days postlesion exhibit long-term survival. Further, the extent of cell survival in these grafts is highly analogous to the degree of cell survival in CA3 grafts analyzed earlier at 1 month postgrafting, suggesting that specific fetal cells that survive the first month of grafting into the lesioned CNS area are capable of exhibiting enduring survival.

Fetal cell grafts provide long-term protection against scrapie induced neuronal loss.

We have transplanted fetal neurons to prolong hippocampal pyramidal cell survival in a mouse scrapie model in which 50% of CA1 pyramidal cells have died by day 180 of the 250-day incubation period. Cells prepared from embryonic PrP deficient mice were intracerebrally injected into infected mice on day 150 and groups killed on day 171 and with terminal disease. Neuron counts and CA1 depth measurements were made on semi-serial sections using an image analysis system. Both grafted groups retained more CA1 neurons than controls injected with medium alone, and showed greater depth of CA1 than controls. This new approach may have potential as a late-stage therapy for TSEs for which there are currently no available treatments.

Morphometric analysis of hippocampal pyramidal neurons in situ and in grafts developing in the anterior eye chambers of young and aged Wistar rats.

We performed a morphometric analysis of the somatic and nuclear areas in the pyramidal neurons of the hippocampal fields CA1 and CA3 in situ and in grafts developing for six weeks in the anterior eye chambers of young (3-to-9 wk.) and of aged (18-to-19.5 mos.) Wistar rats. The mean areas of the CA1 pyramidal somata and nuclei were significantly decreased in the aged animals in situ. The mean parameters of the CA3 pyramidal neurons were not changed, although their distribution was different (bimodal versus unimodal in the young animals). In both groups of recipients, the areas of CA1 neurons and of their nuclei were significantly larger in the grafted tissue than those found in situ. The areas of CA3 neurons did not show any difference in aged recipients and demonstrated only slight hypertrophy in young recipients. We concluded that the area sizes of the pyramidal cell bodies and nuclei in CA1 neurons are more sensitive than those of CA3 neurons to both aging and transplantation. The age of recipients did not significantly influence the growth and development of grafted pyramidal cells.

Morphological alterations of hippocampal pyramidal neurons heterotopically transplanted into the somatosensory cortex of adult rats: a quantitative Golgi study.

A characteristic feature of hippocampal organization is the laminated termination of extrinsic and intrinsic afferents. At present, it is not known to what extent these layer-specific fiber projections modulate the development and final shape of the dendritic arbor of hippocampal target neurons. In the present study, pieces of late embryonic (E18) rat hippocampus were transplanted heterotopically into a cavity in the somatosensory cortex of 6-8 week-old recipient rats. Here, the transplanted neurons differentiated and survived up to several months in the absence of their specific extrinsic afferents. Moreover, tracing of transplant connections with the carbocyanine dye DiI revealed only a limited projection between the transplant and the host neocortex. Golgi-impregnated transplants were used to analyze the postsynaptic structures (dendrites and spines) of hippocampal pyramidal cells quantitatively. Compared with controls, the transplanted pyramidal neurons showed a significant reduction of apical primary dendrites and basal dendritic branches, i.e. of peripheral dendritic portions that originate farther from the soma. In contrast, the number of basal primary dendrites originating directly from the perikaryon was enhanced. Spine density on the main apical dendritic shaft was significantly lower in all peripheral dendritic segments in transplanted neurons. We conclude from our results that the absence of layer-specific extrinsic afferents that normally terminate on peripheral parts of the dendritic arbor of hippocampal pyramidal neurons caused a reduction of these peripheral dendrites and spines. In contrast, the increase of dendrites and spines near the cell body might be induced by intrinsic fibers that normally terminate on these proximal dendritic portions and are known to sprout under transplant conditions.

Intracortical dentate fascia grafts: mossy fiber synapses in the host neocortex.

Embryonic dentate fascia was grafted into a cavity in the area of the adult rat neocortex which represents the vibrissae (barrel field). We wished to test the possibility of development of connections between the two brain areas which do not have synaptic or tissue contacts in situ. The unique characteristics of the giant synaptic boutons of the dentate mossy fibers were used for detection of the dentate synaptic contacts with neocortical neurons at the electron microscopic level. Ultrastructural analysis nine months postgrafting has shown that the bundles of mossy axons enter the host neocortex and develop multiple terminal and en passant contacts with typical characteristics. Neuronal perikarya, large dendritic trunks and fine caliber terminal dendritic branches were used by the mossy fibers as postsynaptic targets, as well as spines of various complexity and configurations. The subsynaptic dendrites seemed to be modified by synapsing giant boutons. Accumulation of cytoplasmic organelles was observed at these sites. Various bumps and protuberances were formed by the subsynaptic dendrite surface. The contents of these appendages were variable; some of them contained organelles typical of dendroplasm, while others were more spine-like, often with inclusion of ribosomes. It is concluded that mossy fibers growing into the host neocortex can develop typical contacts with inappropriate targets with all the ultrastructural features of functional synapses.

Ultrastructural signs of regenerative-degenerative processes in long-term dentate fascia grafts.

An ultrastructural investigation of embryonic (E20) dentate fascia grafts transplanted into an acute cavity in the somatosensory neocortex of adult rats revealed a continuous dynamic state of the tissue nine months postgrafting. The grafts consisted mainly of typical granular cells with some admixture of hippocampal pyramidal neurons and polymorph hilar cells with a normal, mature ultrastructure. Many features of the transplanted tissue suggested continuing development and growth. Dendritic branches with growth tips, axonal growth cones, synaptic boutons with growth vesicles, immature myelin sheaths and myelin-producing cells were observed. In contrast, ultrastructural signs of degeneration were present in some axons, and, less often, in dendrites. These processes, as well as some of the terminal synapses, contained various amounts of lysosomes and lipofuscine granules. In many such terminals the signs of degenerative change were combined with the presence of multiple mitochondria, polymorph vesicles and tubular reticulum, indicating simultaneous reparative processes. It is suggested that continuous recycling of neuronal processes occurs in long-term dentate grafts. This morphological instability may depend on the paucity of synaptic targets within the dentate tissue transplanted with a minimal quantity of hippocampal pyramidal cells and on the limitation of the afferent input. However, the observed features of the grafted dentate tissue are not qualitatively different from those observed in normal dentate with its protracted development and active compensatory reorganization.