Microglia regulate the number of neural precursor cells in the developing cerebral cortex.

Neurogenesis must be properly regulated to ensure that cell production does not exceed the requirements of the growing cerebral cortex, yet our understanding of mechanisms that restrain neuron production remains incomplete. We investigated the function of microglial cells in the developing cerebral cortex of prenatal and postnatal macaques and rats and show that microglia limit the production of cortical neurons by phagocytosing neural precursor cells. We show that microglia selectively colonize the cortical proliferative zones and phagocytose neural precursor cells as neurogenesis nears completion. We found that deactivating microglia in utero with tetracyclines or eliminating microglia from the fetal cerebral cortex with liposomal clodronate significantly increased the number of neural precursor cells, while activating microglia in utero through maternal immune activation significantly decreased the number of neural precursor cells. These data demonstrate that microglia play a fundamental role in regulating the size of the precursor cell pool in the developing cerebral cortex, expanding our understanding of the mechanisms that regulate cortical development. Furthermore, our data suggest that any factor that alters the number or activation state of microglia in utero can profoundly affect neural development and affect behavioral outcomes.

Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery.

Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.

Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system.

BACKGROUND: While it is clear that inbred strains of mice have variations in immunological responsiveness, the influence of genetic background following tissue damage in the central nervous system is not fully understood. A cortical explant system was employed as a model for injury to determine whether the immediate transcriptional response to tissue resection revealed differences among three mouse strains. METHODS: Immunological mRNAs were measured in cerebral cortex from SJL/J, C57BL/6J, and BALB/cJ mice using real time RT-PCR. Freshly isolated cortical tissue and cortical sections incubated in explant medium were examined. Levels of mRNA, normalized to ß-actin, were compared using one way analysis of variance with pooled samples from each mouse strain. RESULTS: In freshly isolated cerebral cortex, transcript levels of many pro-inflammatory mediators were not significantly different among the strains or too low for comparison. Constitutive, baseline amounts of CD74 and antisecretory factor (ASF) mRNAs, however, were higher in SJL/J and C57BL/6J, respectively. When sections of cortical tissue were incubated in explant medium, increased message for a number of pro-inflammatory cytokines and chemokines occurred within five hours. Message for chemokines, IL-1α, and COX-2 transcripts were higher in C57BL/6J cortical explants relative to SJL/J and BALB/cJ. IL-1ß, IL-12/23 p40, and TNF-α were lower in BALB/cJ explants relative to SJL/J and C57BL/6J. Similar to observations in freshly isolated cortex, CD74 mRNA remained higher in SJL/J explants. The ASF mRNA in SJL/J explants, however, was now lower than levels in both C57BL/6J and BALB/cJ explants. CONCLUSIONS: The short-term cortical explant model employed in this study provides a basic approach to evaluate an early transcriptional response to neurological damage, and can identify expression differences in genes that are influenced by genetic background.

The influence of the environment on Cajal-Retzius cell migration.

During cerebral cortex development, different cell populations migrate tangentially through the preplate, traveling from their site of origin toward their final positions. One of the earliest populations formed, the Cajal-Retzius (C-R) cells, is mainly generated in different cortical hem (CH) domains, and they migrate along established and parallel routes to cover the whole cortical mantle. In this study, we present evidence that the phenotype of -Retzius cells, as well as some of their migratory characteristics, is specified in the area where the cells are generated. Nevertheless, when implanted ectopically, these cells can follow new migratory routes, indicating that locally provided genetic cues along the migratory path nonautonomously influence the position of these cells emanating from different portions of the CH. This was witnessed by performing CH implants of tissue expressing fluorescent tracers in live whole embryos. In the same way, tracer injections into the hem of Small eye mutant mice were particularly informative since the lack of Pax6 affects some guidance factors in the migratory environment. As a result, in these animals, the C-R cell population is disorganized, and it forms 1 day late, showing certain differences in gene expression that might help explain these disruptions.

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

BACKGROUND: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively. RESULTS: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. CONCLUSION: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

Cell biology of synaptic plasticity.

The nervous system of mammals retains throughout the animals' life-span the ability to modify the number, nature, and level of activity of its synapses. Synaptic plasticity is most evident after injury to the nervous system, and the cellular and molecular mechanisms that make it possible are beginning to be understood. Transplantation of brain tissue provides a powerful approach for studying mechanisms of synaptic plasticity. In turn, understanding the response of the central nervous system to injury can be used to optimize transplant survival and integration with the host brain.

Fetal brain transplant: reduction of cognitive deficits in rats with frontal cortex lesions.

Frontal cortex and cerebellar tissue from fetal rats was implanted into the damaged frontal cortex of adults. Cognitive deficits in spatial alternation learning that follow bilateral destruction of medial frontal cortex were reduced in rats with frontal cortex implants but not in those with implants of cerebellum. Histological evaluation showed that connections were made between the frontal cortex implants and host brain tissue.

Neocortical transplants in the mammalian brain lack a blood-brain barrier to macromolecules.

In order to determine whether the blood-brain barrier was present in transplants of central nervous tissue, fetal neocortex, which already possesses blood-brain and blood-cerebrospinal fluid barriers to protein, was grafted into the undamaged fourth ventricle or directly into the neocortex of recipient rats. Horseradish peroxidase or a conjugated human immunoglobulin G-peroxidase molecule was systemically administered into the host. These proteins were detected within the cortical transplants within 2 minutes regardless of the age of the donor or postoperative time. At later times these compounds, which normally do not cross the blood-brain barrier, inundated the grafts and adjacent host brain and also entered the cerebrospinal fluid. Endogenous serum albumin detected immunocytochemically in untreated hosts had a comparable although less extensive distribution. Thus, transplants of fetal central nervous tissue have permanent barrier dysfunction, probably due to microvascular changes, and are not integrated physiologically within the host. Blood-borne compounds, either systemically administered or naturally occurring, which should never contact normal brain tissue, have direct access to these transplants and might affect neuronal function.

Transplanted human embryonic neural stem cells survive, migrate, differentiate and increase endogenous nestin expression in adult rat cortical peri-infarction zone.

Transplantation of stem cells is a potential therapeutic strategy for stroke damage. The survival, migration, and differentiation of transplanted human embryonic neural stem cells in the acute post-ischemic environment were characterized and endogenous nestin expression after transplantation was investigated. Human embryonic neural stem cells obtained from the temporal lobe cortex were cultured and labeled with fluorescent 1,1'-dioctadecy-6,6'-di (4-sulfopheyl)-3,3,3',3'-tetramethylindocarbocyanin (DiI) in vitro. Labeled cells were transplanted into cortical peri-infarction zones of adult rats 24 h after permanent middle cerebral artery occlusion. Survival, migration, and differentiation of grafted cells were quantified in immunofluorescence-stained sections from rats sacrificed at 7, 14, and 28 days after transplantation. Endogenous nestin-positive cells in the cortical peri-infarction zone were counted at serial time points. The cells transplanted into the cortical peri-infarction zone displayed the morphology of living cells and became widely located around the ischemic area. Moreover, some of the transplanted cells expressed nestin, GFAP, or NeuN in the peri-infarction zone. Furthermore, compared with the control group, endogenous nestin-positive cells in the peri-infarction zone had increased significantly 7 days after cell transplantation. These results confirm the survival, migration, and differentiation of transplanted cells in the acute post-ischemic environment and enhanced endogenous nestin expression within a brief time window. These findings indicate that transplantation of neural stem cells into the peri-infarction zone may be performed as early as 24 h after ischemia.

Directed fiber outgrowth from transplanted embryonic cortex-derived neurospheres in the adult mouse brain.

Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.

Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex.

Neural stem cells (NSCs) hold great promise for neural repair in cases of CNS injury and neurodegeneration; however, conventional cell-based transplant methods face the challenges of poor survival and inadequate neuronal differentiation. Here, we report an alternative, tissue-based transplantation strategy whereby cerebral organoids derived from human pluripotent stem cells (PSCs) were grafted into lesioned mouse cortex. Cerebral organoid transplants exhibited enhanced survival and robust vascularization from host brain as compared to transplants of dissociated neural progenitor cells (NPCs). Engrafted cerebral organoids harbored a large NSC pool and displayed multilineage neurodifferentiation at two and four weeks after grafting. Cerebral organoids therefore represent a promising alternative source to NSCs or fetal tissues for transplantation, as they contain a large set of neuroprogenitors and differentiated neurons in a structured organization. Engrafted cerebral organoids may also offer a unique experimental paradigm for modeling human neurodevelopment and CNS diseases in the context of vascularized cortical tissue.

Cholinergic grafts, memory and ageing.

Neural grafts rich in cholinergic neurones can survive transplantation to the neocortex or hippocampus in rats. Such grafts have the capacity to ameliorate a variety of functional deficits associated both with explicit lesions that deafferent the neocortex or hippocampus and with natural ageing. The transplantation technique enhances our understanding of the involvement of forebrain cholinergic systems in normal cognitive functions (including memory) and of the role of cholinergic degeneration in the dysfunctions associated with ageing. It is unlikely, however, that these observations will extend to a therapeutic strategy for dementia using neural transplantation, because the human diseases (at least in the case of Alzheimer's disease and multi-infarct dementia) involve widespread degeneration of other populations of cortical neurones that are not so amenable to functional transplantation as the diffuse forebrain cholinergic systems.

Where, oh where, have my stem cells gone?

During the summer of 2001, Americans were treated to high political drama courtesy of the debate over embryonic and adult stem cell research. The popular press was flush with predictions about how neural stem cells would reverse, almost by magic, the devastation caused by diseases such as Alzheimer's, Parkinson's, stroke or spinal cord injury. Unfortunately, this promise remains unfulfilled because we have such a poor understanding of how stem cells function. With regard to adult stem cells, we are not even completely sure where they are, or how or when they got there. A provocative study by Ourednik et al. published in Science suggests that in primates, adult neural stem cells are allocated during early corticogenesis. The study also provides evidence for the existence of stem cells dispersed throughout the frontal cortex and striatum.

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.

Astrocytes from cerebral cortex or striatum attract adult host serotoninergic axons into intrastriatal ventral mesencephalic co-grafts.

The identification of axon growth inhibitory molecules offers new hopes for repair of the injured CNS. However, the navigational ability of adult CNS axons and the guidance cues they can recognize are still essentially unknown. Astrocytes may express guidance molecules and are known to have different regional phenotypes. To evaluate their influence on the affinity of adult serotoninergic (5-HT) axons for a projection target, we co-implanted astrocytes from the neonatal striatum, cortex, or ventral mesencephalon together with fetal ventral mesencephalic tissue into the striatum of adult rats. Two months after surgery, quantification after in vitro 5-[1,2-(3)H]serotonin ([(3)H]5-HT) uptake and autoradiography showed that ventral mesencephalic grafts with co-grafted cortical or striatal astrocytes were four times and three times, respectively, more densely innervated by host 5-HT axons than control ventral mesencephalic grafts with or without co-grafted ventral mesencephalic astrocytes. Immunohistochemistry for glial fibrillary acidic protein, vimentin, or chondroitin-sulfate proteoglycans revealed no qualitative or quantitative differences in host astroglial scar or production of inhibitory molecules that could explain these differences in 5-HT innervation. These results demonstrate that astrocytes grown in culture from different brain regions have the potential to influence the growth and maintenance of adult 5-HT axons in a graft of neural tissue from another brain region. It should now be feasible to identify the molecules expressed by cultured cortical or striatal, but not by ventral mesencephalic, astrocytes that have these tropic actions on 5-HT axons of the neostriatum.

Initial studies of embryonic transplants of human hippocampus and cerebral cortex derived from schizophrenic women.

Human fetal brain tissue was obtained from first-trimester elective abortions of two women who also had schizophrenia. Portions of the embryonic hippocampus or cerebral cortex were transplanted into the anterior eye chamber of immunologically compromised athymic nude rats. In this environment, embryonic brain tissue derived from normal women generally continues organotypic growth and development for many months. Although initial survival after transplantation was normal, the tissue derived from schizophrenic women manifested less robust growth. However, cells in the transplants showed typical neuronal differentiation, with development of different neuronal types, such as pyramidal cells, granule cells, and gamma-aminobutyric acid (GABA)-containing interneurons. Rhythmic electrical activity was also observed, indicative of some local synaptic organization. The presence of messenger RNA (mRNA) for brain-derived neuronotrophic factor (BDNF) was observed using in situ hybridization. The reason for the decreased rate of growth of these transplants remains unknown and the significance of the finding cannot be assessed from only two fetuses. However, these preliminary findings suggest that fetal transplants may be a useful model system for the detection of developmental pathogenic processes in the expression and transmission of schizophrenia.

Time course of neocortical graft innervation by AChE-positive fibers.

Acetylcholinesterase (AChE)-containing axons are the only extrinsic fibers projecting to the adult cortex that readily innervate embryonic cortical grafts up to normal densities without prior manipulation of the host brain. In the present paper we compare the time course of AChE-positive fiber innervation in the normal mouse cortex with that seen in neocortical grafts by using AChE histochemistry as a marker for presumed cholinergic fibers. Donor tissue was taken at two different stages of gestation; before (embryonic days 12-14, or E12-14) and after (E17-19) the cortical plate is formed. Three features are analyzed: 1) the distribution and density of AChE-containing fibers, 2) the presence of AChE-positive cells, and 3) the distribution of butyrylcholinesterase (BuChE)-positive elements. The modification of Koelle's method used for AChE localization showed AChE-positive fibers in developing parietal neocortex as early as E18-19. The distribution of AChE-labeled fibers in the normal cortex achieves the mature pattern by the end of the third postnatal week. The rate of innervation of transplants takes longer and depends on the age of the donor tissue. Tissue from both donor ages first showed AChE-positive fibers crossing the host-transplant interface by 7 days postsurgery. E17-19 tissue approaches the density of AChE-positive fibers in the normal adult cortex by 15 weeks after grafting, whereas the E12-14 donor tissue does not approach normal innervation densities until after 20 weeks. While the degree of innervation in the E12-14 donor tissue never equalled the surrounding adult cortex within our range of survival times, a few of the E17-19 transplants did develop densities equal to that of the host cortex. AChE-positive cells are first detectable in the normal parietal cortex on the day of birth, peak by the end of the first postnatal week, and then decline in number to the low levels of the mature cortex after the second postnatal week. Grafted cells in E12-14 tissue stain lightly for AChE by 7 days postsurgery, achieve maximal densities by 3 weeks, and become markedly reduced in number and density by 10 weeks. Cells in E17-19 tissue are lightly reactive by 7 days postsurgery, reach maximal numbers by 2 weeks postsurgery, and become similar in number and density to those seen in the mature cortex after 4 weeks. The appearance of BuChE-reactive blood vessels, neurons, and glia in both normal development and in the transplants is described and discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Fiber outgrowth from anterior hypothalamic and cortical xenografts in the third ventricle.

Fetal grafts of the anterior hypothalamus (SCN/AH) containing the suprachiasmatic nucleus (SCN) restore circadian rhythms to SCN-lesioned host hamsters and rats following implantation into the third ventricle. Previous studies suggest that intraventricular SCN/AH grafts are variable in their attachment sites, the extent of their outgrowth, and the precise targets innervated in the host brain. However, the use of different methods to analyze graft outgrowth in this model has previously led to inconsistent results. We have reevaluated the outgrowth of fetal rat SCN/AH grafts implanted in the third ventricle of hamsters by using two methods: the carbocyanine dye, 1,1'dioctadecyl-3,3'-tetramethylindocarbocyanine percholate (DiI), was placed directly onto grafted tissue; and a donor-specific neurofilament marker was used in conjunction with xenografts. We examined the specificity of outgrowth by comparing SCN/AH xenografts with that of control cortical (CTX) xenografts. To evaluate whether SCN/AH graft efferents arise from the donor SCN, we used micropunch grafts that contained minimal extra-SCN tissue. The results show that the use of a donor-specific neurofilament marker reveals more extensive SCN/AH graft outgrowth than DiI. SCN/AH graft efferents project into areas normally innervated by the intact SCN. However, this outgrowth is variable among graft recipients, is not specific to SCN/AH tissue, and does not necessarily derive from the donor SCN. The precise functional role of neural efferents arising from SCN/AH grafts in the restoration of circadian clock function and the extent of SCN-derived efferents remain to be determined.

Fetal tectal or cortical tissue transplanted into brachial lesion cavities in rats: influence on the regrowth of host retinal axons.

Fetal neural tissue was transplanted into suction lesions of the left brachium and pretectal region in young rats. Tectal tissue was grafted into 6-18-day-old rats and cortical tissue was transplanted into 17-20-day-old animals. The aim was to determine whether grafts could potentiate the regrowth of damaged retinal axons and, as a consequence, stimulate the axons to reenter their host target, the superior colliculus (SC). Fifteen to 581 days after transplantation, host retinal projections were traced by injecting the right eye with horseradish peroxidase (HRP). Parallel series of frozen brain sections were stained for HRP histochemistry, acetylcholinesterase, Nissl, or neurofibrils. At all ages studied, grafts survived and grew within the wound cavity; survival was better in the older animals. Most cortical grafts and a small number of tectal grafts filled the wound cavity and formed complete tissue bridges across the lesion. The majority of tectal grafts were attached to one or the other side of the lesion and were connected to the opposite lesion face by glial and connective tissue membranes that formed over the lesion site. In many animals that received tectal transplants, host retinal axons were traced growing into the grafts. Regenerating axons innervated specific, localized areas within the grafts, and it appeared that the axons retained the ability to recognize their appropriate target cells within the graft neuropil. Comparable ingrowth into cortical grafts was not observed. Optic axons were occasionally seen reentering the superficial layers of the host SC; however, compared to fetal tectal grafts, the density of host SC innervation was sparse. The implications of these data are discussed with regard to the possible use of fetal neural tissue grafts as reconstructive tissue bridges in the mammalian central nervous system.

Patterns of angiogenesis in neural transplant models: II. Fetal neocortical transplants.

Vascular integration between transplanted fetal CNS tissues and host brain is essential for long-term transplant survival. This study compares the time course and mechanism of vascularization in allografts of fetal cerebral cortex inserted either into the fourth ventricle or directly into the parietal cortex or hippocampus of perinatal rats. Recipient animals were administered 3H-thymidine after various postoperative time periods. The tissues were processed for light microscopic autoradiography to determine the temporal pattern of endothelial proliferation at the graft sites. Correlative electron microscopy depicted the morphological changes in transplant vasculature. Some recipients were prelabelled with 3H-thymidine prior to transplantation to determine if host vessels invaded the grafts; conversely, some donor tissues were prelabelled in utero to ascertain if the intrinsic vascular anlagen survived. Intraventricular transplants contained patent vessels, probably originating from the host pia mater, as early as 24 hours postoperative. Intraparenchymal transplants had patent vessels by 72 hours and a more complete network by 5 days. Prelabelling experiments and ultrastructural observations demonstrated that adjacent host pial vessels became incorporated into the perimeter of the intraventricular transplants and later grew centrally into the grafts. Intraparenchymal transplants also contained host vessels that exhibited a similar growth pattern. Intrinsic graft vessels remained viable and continued their development, and presumably anastomosed with the ingrowing host vasculature. Temporal labelling studies revealed that both vessel populations attained their highest proliferative rates within 72 hours after transplantation. This study demonstrates that the vasculature which develops within both intraventricular and intraparenchymal fetal CNS transplants is chimeric, consisting of intrinsic fetal vasculature and proliferating host vessels. The mechanism of transplant vascularization may be significant with regard to astrocytic, immunological, or blood-brain-barrier characteristics at these transplantation sites.