Embryonic Cortical Tissue Research Articles

Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice.

Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic mice to evaluate the reconstruction of neuronal network ( Falkner et al., 2016 ) and the repopulation of a subset of cortical cells. By using this approach, we have shown that infiltrating cells from host brain can restore the microglial population in the graft tissue ( Wang et al., 2016 ). This protocol describes the detailed procedure of the transplantation surgery in mice, including establishing a lesion model in the host brain, preparing the embryonic cortical graft, and transplanting the embryonic cortical graft to adult brain.

Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft.

Is the adult mouse striatum a hostile host for neural transplant survival?

Human donor cells, including neurally directed embryonic stem cells and induced pluripotent stem cells with the potential to be used for neural transplantation in a range of neurodegenerative disorders, must first be tested preclinically in rodent models of disease to demonstrate safety and efficacy. One strategy for circumventing the rejection of xenotransplanted human cells is to desensitize the host animal to human cells in the early neonatal period so that a subsequent transplant in adulthood is not immunorejected. This method has been robustly validated in the rat, but currently not in the mouse in which most transgenic models of neurodegeneration have been generated. Thus, we set out to determine whether this could be achieved through modification of the existing rat protocol. Mice were inoculated in the neonatal period with a suspension of human embryonic cortical tissue of varying cell numbers, and received a subsequent human embryonic cortical tissue cell transplant in adulthood. Graft survival was compared with those in mice immunosuppressed with cyclosporine A and those receiving allografts of mouse whole ganglionic eminence tissue. Poor survival was found across all groups, suggesting a general problem with the use of mouse hosts for testing human donor cells.

Reestablishment of damaged adult motor pathways by grafted embryonic cortical neurons

Damage to the adult motor cortex leads to severe and frequently irreversible deficits in motor function. Transplantation of embryonic cortical neurons into the damaged adult motor cortex was previously shown to induce partial recovery, but reports on graft efferents have varied from no efferent projections to sparse innervation. Here, we grafted embryonic cortical tissue from transgenic mice overexpressing a green fluorescent protein into the damaged motor cortex of adult mice. Grafted neurons developed efferent projections to appropriate cortical and subcortical host targets, including the thalamus and spinal cord. These projections were not a result of cell fusion between the transplant and the host neurons. Host and transplanted neurons formed synaptic contacts and numerous graft efferents were myelinated. These findings demonstrate that there is substantial anatomical reestablishment of cortical circuitry following embryonic cortex grafting into the adult brain. They suggest that there is an unsuspected potential for neural cell transplantation to promote reconstruction after brain injury.

Neuronal and glial differentiation following culture of the human embryonic cortical stem cells.

To set up long-term in vitro culture system of the human neural stem cells (hNSC) and to study their biological properties. Human fetuses aged about 20 weeks following spontaneous abortion were adopted. A serum-free medium containing basic fibroblast growth factor and epidermal growth factor was used to make the hNSCs divide continuously in the culture. The growth curve of continually passaged cells was examined. The effects of long-term culture on the cell cycle, cell differentiation were analyzed. The cell cycles of these cells were analyzed using flow cytometry. The cells from the human embryonic cortical tissue could be maintained and propagated in the presence of growth factors. Neurospheres were generated continually. Only one month after the primary culture, the precursors could be effectively discarded. The cells could be cultured for ten months. The cells had an exponential, consistent growth. The cell cycle analysis indicated that the hNSCs maintained remarkable proliferation. Upon differentiation, the hNSCs gave rise to mature cells. The multi-lineage potential of differentiation after different passages remained unchanged. The hNSCs isolated from the human embryonic tissues retained their biological features after long-term culture in vitro.

Transplants of fetal frontal cortex grafted into the occipital cortex of newborn rats receive a substantial thalamic input from nuclei normally projecting to the frontal cortex

A number of molecular and hodological experiments have provided evidence that there is an early commitment of neocortical neurons to express features unique to a certain cortical area. However, the findings of several transplantation experiments have indicated that late embryonic cortical tissue heterotopically grafted into the neocortex of newborn rats receives a set of thalamic projections appropriate for the host cortical locus within which it develops. To provide further information on the extent to which neocortical neurons are predetermined to develop area-specific systems of connections, in this study we have compared the pattern of thalamic afferents to grafts of embryonic day 16 occipital or frontal neocortex transplanted into the occipital cortex of newborn rats. Two months after grafting, a retrograde neurotracer (cholera toxin, subunit b) was injected into the grafts to precisely assess the number of cells in the visual- and/or motor-related nuclei of the host thalamus projecting to each category of transplants (occipital-to-occipital or frontal-to-occipital). Transplants of embryonic occipital cortex received significant input from several visual-related thalamic nuclei, i.e. the lateral posterior and lateral dorsal nuclei, and no input from motor-related thalamic nuclei. However, only few labeled cells were found in the dorsal lateral geniculate nucleus, which was systematically affected by a severe atrophy, probably in response to the lesion of the occipital cortex performed prior to the transplantation. By comparison, transplants of frontal origin received a substantial input from the ventrolateral and ventromedial thalamic nuclei, which normally project to the frontal cortex, but received a weak input from the lateral posterior and lateral dorsal nuclei. Neocortical neurons grafted heterotopically into the neocortex of newborn hosts are not only able to contact cortical and subcortical targets appropriate for their embryonic site of origin, but are also susceptible to derive thalamic inputs closely related to their embryonic origin.

Neocortical grafting to newborn and adult rats: developmental, anatomical and functional aspects.

This report presents the results of neural transplantation experiments that were designed either to study neural developmental phenomena or to appraise the possible restorative capacity of grafts. The first part of the report deals with the findings of transplantation studies that were performed in newborn recipient rats in an attempt to determine the importance of extrinsic or intrinsic factors in the process of areal cortical differentiation. More precisely, we examined the extent to which extrinsic signals drive the pattern of efferent connections of neurons residing in different cortical areas. In the first experiment, we examined the general distribution pattern of efferents arising from homotopic as compared to heterotopic transplants of embryonic cortical tissue placed into the frontal cortex of newborn rats. Our findings indicated that embryonic occipital neurons transplanted heterotopically into the sensorimotor cortex: (a) only rarely contacted normal targets of the motor cortex, (b) systematically projected towards normal targets of the visual cortex, and (c) distributed fibers to structures normally receiving fibers from both the motor and visual cortex, either exclusively into the visual corticorecipient zone of the structure or into both the visual and motor corticorecipient zones. In the second experiment, we attempted to assess the densities of the spinal projections arising from homotopic or heterotopic transplants implanted into the frontal or occipital cortex of newborn rats. We provided evidence that transplants of embryonic neocortical neurons of frontal origin developed and maintained a spinal cord projection whatever their rostrocaudal position within the host neocortex, whereas transplants of occipital origin did not maintain a significant spinal cord projection in adulthood. Finally, in the third experiment, we analyzed the laminar and tangential distribution of the tectal projections developed by transplants of embryonic occipital cortex placed into the primary or secondary subdivisions of the occipital cortex of newborn rats. Abnormalities in the laminar and/or tangential organization of the tectal distribution of the transplant efferents were systematically found. Using these different transplantation paradigms, we provided increasing evidence that, in their heterotopic location, the embryonic neurons retain some of the developmental characteristics corresponding to their embryonic cortical site of origin. Our findings strongly suggested, therefore, that there is an early specification of neocortical neurons to develop area-specific efferents. In conclusion, converging lines of evidence suggest that regional differences in the neuroepithelium are predetermined early in development, thus leading to cerebral cortex parcellation, as hypothesized by Rakic (1988). In the second part of the report, we describe the results of a series of experiments dealing with the consequences of grafting embryonic cortex into the damaged cortex of adult rats. These effects were examined from an anatomical, metabolic, behavioral, and electrophysiological point of views. The experiments were conducted using either the frontal or the visual cortex as models to appraise the functional integration of the grafts into the motor or visual circuits, respectively. The first study was undertaken to examine the capacity of transplants of embryonic frontal neocortical tissue placed into the frontal cortex of an adult recipient to develop efferents into the host CNS. The results indicated that transplants of fetal neocortex placed into adult CNS had the capacity to develop efferents which seemed to grow over significant distances within the host corpus callosum but, in most cases, failed to penetrate deeply into the gray matter. The density of the efferent projections was, however, far weaker than that seen in newborn hosts. In the second study, the 2-deoxyglucose (2-DG) technique was used to examine the functional integration of hom

Transplants of embryonic cortical tissue placed in the previously damaged frontal cortex of adult rats: local cerebral glucose utilization following execution of forelimb movements.

Transplantation of fetal cortical tissue into the motor cortex of adult rats was used as an experimental model to examine the functional integration of homotopic fetal neocortical grafts into the motor pathways of adult host brain. We have employed the [14C]2-deoxy-D-glucose method to analyse the metabolic activity of the transplant and host sensorimotor cortex: (i) in animals solicited to perform specific lever-pressing movements with the limb contralateral to the transplant (experimental group); and (ii) in non-solicited animals or in animals using the limb ipsilateral to the transplant (control group). Grafts in the control group displayed homogeneous uptake of 2-deoxy-D-glucose throughout the rostrocaudal extent of the transplant. The local cerebral glucose utilization levels were low as compared to those of the surrounding cortex but were at least two-times higher than in the corpus callosum. Increase in 2-deoxy-D-glucose uptake by the transplant cells was found only in the experimental group. In this group, 2-deoxy-D-glucose uptake was higher in the caudal (AP: +3.0 to +1.7 mm, relative to Bregma) than in the rostral sectors of the transplants suggesting the existence of a topographic organization within the transplant. In addition, except in the rostral part, glucose utilization was higher in the transplant of the experimental group than in the sensorimotor areas of the non-activated cortex in the control group. Moreover, glucose utilization of the transplant cells was systematically higher in the experimental than in the control group. The transplants appear to display a certain level of metabolic integration with the host sensorimotor cortex since, in the experimental group, there was no significant differences in local cerebral glucose utilization values in the caudal sector of the transplant and in the surrounding sensorimotor cortical areas of the host. The 2-deoxy-D-glucose uptake was even higher in the caudal sector of the transplant than in some of the subfields of the contralateral sensorimotor cortex. The present findings indicate for the first time that motor activation of the contralateral forelimb produces an increase in metabolic activity in distinct transplant sectors, the topographic distribution of which matches the normal topographic organization of the forelimb somatomotor map. This suggests that transplants of embryonic frontal neocortex placed in the frontal cortex of adult hosts become functionally integrated with the host motor system.(ABSTRACT TRUNCATED AT 400 WORDS)

Skilled forelimb use in the rat: amelioration of functional deficits resulting from neonatal damage to the frontal cortex by neonatal transplantation of fetal cortical tissue

Limb preference and dexterity of right and left forelimbs were studied in a food reaching task in normal rats (control group), in rats that sustained a neonatal lesion of the left frontal cortex (lesion group) and in animals that received a transplant obtained from the frontal cortex of 16-day-old embryos immediately after the lesion (graft group). In addition, an anatomical study of host-transplant interconnections was performed in several transplanted animals. The results indicate that the lesion slightly increased the preference for the limb ipsilateral to the lesion and transplantation of fetal cortical tissue did not restore the preference for the contralateral limb. Furthermore, lesion of the motor cortex induced a deficit in the dexterity of limb use in the food-reaching task. This motor deficit was more pronounced when the limb contralateral to the lesion was used. Transplantation of embryonic cortical tissue led to a reduction of the motor deficit. Compared to the lesion group, the graft group had a higher success rate and a lower percentage of motor abnormalities, whichever forelimb was used, ipsi- or contralateral to the transplant. Nevertheless, larger improvement was noted with contralateral forelimb usage. Functional recovery was not complete since the control group still performed significantly better than the graft group, although almost complete sparing in skilled reaching was noted when the limb ipsilateral to the transplant was used. Analysis of host-transplant interconnections indicates that the transplants sent fibers to the host spinal cord, caudate-putamen, thalamus and homotopic contralateral cortex and received projections from the host thalamus and contralateral cortex. It is therefore suggested that neonatal transplantation of fetal cortical tissue promotes functional recovery from damage to the motor cortex occurring at birth.

Replacement of damaged cortical projections by homotypic transplants of entorhinal cortex.

The extent to which transplants of embryonic cortical tissue can be used to replace damaged cortical projections has been examined. Embryonic entorhinal cortex was implanted into the entorhinal region of young adult rats that had previously received a lesion through the angular bundle. Projections between transplant and host were examined by using WGA-HRP and the fluorescent dye Fast Blue. Implants selectively innervated areas of the host hippocampus and amygdala which normally receive entorhinal afferents. Implants were innervated by cells in the host diagonal band and, in one case, by cells in the contralateral entorhinal and/or presubicular cortex. In most cases, host fibers were differentially distributed within transplants, possibly reflecting an ability of host fibers to recognize and selectively innervate their appropriate targets even though the cellular organization of the implant is different from that present during normal development. These data suggest that homotypic implants of embryonic entorhinal cortex can, in some ways, replace severed cortical projections and may eventually be able to reconstitute normal cortical circuitry.

Transplantation into the mammalian adult spinal cord.

Embryonic cerebral cortical tissue obtained from rat embryos of 15-day gestation was transplanted into the cervical spinal cord of adult rats. The cortical transplants survived, grew, and established connections with the host animal's spinal cord. In other animals, knife lesions were first made in the host's spinal cord, and then embryonic cortical tissue was transplanted into the site of the lesion. The cortical transplants in these animals were observed to become an integral part of the host animal's spinal cord.