Xeno-free culture for generation of forebrain oligodendrocyte precursor cells from human pluripotent stem cells.

Oligodendrocytes (OLs) show heterogeneous properties that depend on their location in the central nervous system (CNS). In this regard, the investigation of oligodendrocyte precursor cells (OPCs) derived from human pluripotent stem cells (hPSCs) should be reconsidered, particularly in cases of brain-predominant disorders for which brain-derived OPCs are more appropriate than spinal cord-derived OPCs. Furthermore, animal-derived components are responsible for culture variability in the derivation and complicate clinical translation. In the present study, we established a xeno-free system to induce forebrain OPCs from hPSCs. We induced human forebrain neural stem cells (NSCs) on Laminin 511-E8 and directed the differentiation to the developmental pathway for forebrain OLs with SHH and FGF signaling. OPCs were characterized by the expression of OLIG2, NKX2.2, SOX10, and PDGFRA, and subsequent maturation into O4+ cells. In vitro characterization showed that >85% of the forebrain OPCs (O4+ ) underwent maturation into OLs (MBP+ ) 3 weeks after mitogen removal. Upon intracranial transplantation, the OPCs survived, dispersed in the corpus callosum, and matured into (GSTπ+ ) OLs in the host brains 3 months after transplantation. These findings suggest our xeno-free induction of forebrain OPCs from hPSCs could accelerate clinical translation for brain-specific disorders.

Muscarinic Receptor M3R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells.

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases, such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination, but the receptor subtypes that mediate these receptors are unclear. In this study, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.

Interneuron progenitors attenuate the power of acute focal ictal discharges.

Interneuron progenitors from the embryonic medial ganglionic eminence (MGE) can migrate, differentiate, and enhance local inhibition after transplantation into the postnatal cortex. Whether grafted MGE cells can reduce ictal activity in adult neocortex is unknown. We transplanted live MGE or killed cells (control) from pan green fluorescent protein expressing mice into adult mouse sensorimotor cortex. One week, 2 and 1/2 weeks, or 6 to 8 weeks after transplant, acute focal ictal epileptiform discharges were induced by injection of 4-aminopyridine (4-AP) 2 mm away from the site of transplantation. The local field potential of the events was recorded with 2 electrodes, 1 located in the 4-AP focus and the other 1 in the transplantation site. In all control groups and in the 1-week live cell transplant, 4-AP ictal discharges revealed no attenuation in power and duration from the onset site to the site of transplantation. However, 2.5 or 6 ~ 8 weeks after MGE transplants, there was a dramatic decrease in local field potential power at the MGE transplanted site with little decrease in ictal duration. Surprisingly, there was no relationship between grafted cell distribution or density and the degree of attenuation. As remarkably low graft densities still significantly reduced discharge power, these data provide further support for the therapeutic potential of interneuron precursor transplants in the treatment of neocortical epilepsy.

Extracerebellar progenitors grafted to the neurogenic milieu of the postnatal rat cerebellum adapt to the host environment but fail to acquire cerebellar identities.

Stem or progenitor cells acquire specific regional identities during early ontogenesis. Nonetheless, there is evidence that cells heterotopically transplanted to neurogenic regions of the developing or mature central nervous system may switch their fate to adopt host-specific phenotypes. Here, we isolated progenitor cells from different germinative sites along the neuraxis where GABAergic interneurons are produced (telencephalic subventricular zone, medial ganglionic eminence, ventral mesencephalon and dorsal spinal cord), and grafted them to the prospective white matter of the postnatal rat cerebellum, at the time when local interneurons are generated. The phenotype acquired by transplanted cells was assessed by different criteria, including expression of region-specific transcription factors, acquisition of morphological and neurochemical traits, and integration in the cerebellar cytoarchitecture. Regardless of their origin, all the different types of donor cells engrafted in the cerebellar parenchyma and developed mature neurons that shared some morphological and neurochemical features with local inhibitory interneurons, particularly in the deep nuclei. Nevertheless, transplanted cells failed to activate cerebellar-specific regulatory genes. In addition, their major structural features, the expression profiles of type-specific markers and the laminar placement in the recipient cortex did not match those of endogenous interneurons generated during the same developmental period. Therefore, although exogenous cells are influenced by the cerebellar milieu and show remarkable capabilities for adapting to the foreign environment, they essentially fail to switch their fate, integrate in the host neurogenic mechanisms and adopt clear-cut cerebellar identities.

Long-term survival, robust neuronal differentiation, and extensive migration of human forebrain stem/progenitor cells transplanted to the adult rat dorsal root ganglion cavity.

Neurons in dorsal root ganglia (DRGs) transmit sensory information from peripheral tissues to the spinal cord. This pathway can be interrupted, for example, as the result of physical violence, traffic accidents, or complications during child delivery. As a consequence, the patient permanently loses sensation and often develops intractable neuropathic pain in the denervated area. Here we investigate whether human neural stem/progenitor cells (hNSPCs) transplanted to the DRG cavity can serve as a source for repairing lost peripheral sensory connections. We found that hNSPCs robustly differentiate to neurons, which survive long-term transplantation. The neuronal population in the transplants was tightly surrounded by astrocytes, suggesting their active role in neuron survival. Furthermore, 3 months after grafting hNSPCs were found in the dorsal root transitional zone (DRTZ) and within the spinal cord. The level of differentiation of transplanted cells was high in the core of the transplants whereas cells that migrated to the DRTZ and spinal cord were undifferentiated, nestin-expressing precursors. These data indicate that peripherally transplanted hNPSCs can be used for repair of dorsal root avulsion or spinal cord injuries; however, additional factors are required to guide their differentiation to the desired type of neurons. Furthermore, hNPSCs that migrate from the DRG cavity graft site to the DRTZ and spinal cord may provide trophic support for regenerating dorsal root axons, thereby allowing them to reenter the host spinal cord.

Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres.

There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (>350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue>9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord.

Generation of distinct types of periglomerular olfactory bulb interneurons during development and in adult mice: implication for intrinsic properties of the subventricular zone progenitor population.

The subventricular zone (SVZ) of the lateral ventricle develops from residual progenitors of the embryonic lateral ganglionic eminence (LGE) and maintains neurogenic activity throughout life. Precursors from LGE/SVZ migrate to the olfactory bulb (OB) where they differentiate into local interneurons, principally in the granule layer and glomerular layer (GL). By in situ dye labeling, we show that neonatal and adult SVZ progenitors differentially contribute to neurochemically distinct types of periglomerular interneurons in the GL. Namely, calbindin-positive periglomerular cells are preferentially generated during early life, whereas calretinin- and tyrosine hydroxylase-expressing neurons are mainly produced at later ages. Furthermore, homochronic/heterochronic transplantation demonstrates that progenitor cells isolated from the LGE or SVZ at different stages (embryonic day 15 and postnatal days 2 and 30) engraft into the SVZ of neonatal or adult mice, migrate to the OB, and differentiate into local interneurons, including granule and periglomerular cells as well as other types of interneurons. The total number of integrated cells and the relative proportion of granule or periglomerular neurons change, according to the donor age, whereas they are weakly influenced by the recipient age. Analysis of the neurochemical phenotypes acquired by transplanted cells in the GL shows that donor cells of different ages also differentiate according to their origin, regardless of the host age. This suggests that progenitor cells at different ontogenetic stages are intrinsically directed toward specific lineages. Neurogenic processes occurring during development and in adult OB are not equivalent and produce different types of periglomerular interneurons as a consequence of intrinsic properties of the SVZ progenitors.

A roof plate-dependent enhancer controls the expression of Homeodomain only protein in the developing cerebral cortex.

The smallest known homeodomain protein, Homeodomain only protein (Hop), was identified and described here as a temporally and spatially restricted gene in the neurogenic regions of the developing murine CNS including the cerebral cortex. Furthermore, an evolutionarily conserved 418 base pair upstream cis-regulatory DNA sequence was found to confine the Hop expression to the CNS of transgenic mice, but not to the heart which is the second major Hop expressing organ Chen, F., Kook, H., Milewski, R., Gitler, A.D., Lu, M.M., Li, J., Nazarian, R., Schnepp, R., Jen, K., Biben, C., Runke, G., Mackay, J.P., Novotny, J., Schwartz, R.J., Harvey, R.P., Mullins, M.C., Epstein, J.A., 2002. Hop is an unusual homeobox gene that modulates cardiac development. Cell 110, 713-723; Shin, C.H., Liu, Z.P., Passier, R., Zhang, C.L., Wang, D.Z., Harris, T.M., Yamagishi, H., Richardson, J.A., Childs, G., Olson, E.N., 2002. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110, 725-735. The forebrain enhancer activity was successfully reproduced in vitro utilizing a combination of the electroporation and the organotypic brain culture method. Using this approach, the minimal requirement for the forebrain-specific enhancer sequence was delineated down to 200 base pairs. We further demonstrate that the Hop enhancer activity is inducible ectopically in a transgenic tissue by wild-type roof plate transplantation in vitro. Thus Hop is regulated in the forebrain by a so far unidentified paracrine signaling factor from the roof plate. Furthermore, the identified enhancer sequence provides an important tool for the targeted expression of transgenes in the medial cortex and the cortical hem.

Comparison between fetal spinal-cord- and forebrain-derived neural stem/progenitor cells as a source of transplantation for spinal cord injury.

Recently, we have shown that the transplantation of spinal-cord-derived neural stem/progenitor cells (NSPCs) can contribute to the repair of injured spinal cords in adult rats, which may correspond to a behavioral recovery. To apply these results to clinical practice, a system for supplying human NSPCs on a large scale must be established. However, human spinal-cord-derived NSPCs are known to have a low proliferation rate, compared with forebrain-derived NSPCs. This low proliferative potency limits the feasibility of large-scale spinal cord-derived NSPC use. Thus, forebrain-derived NSPCs should be examined as an alternative to spinal-cord-derived NSPCs for the treatment of spinal cord injuries. In this study, we compared spinal-cord- and forebrain-derived NSPCs transplanted into injured spinal cords with respect to their fates in vivo as well as the animals' functional recovery. Both spinal-cord- and forebrain-derived NSPCs promoted functional recovery in rats with spinal cord injuries. While both spinal-cord- and forebrain-derived NSPCs survived, migrated and differentiated into neurons, astrocytes and oligodendrocytes in response to the microenvironment within the injured spinal cord after transplantation, forebrain-derived NSPCs differentiated into more neurons and fewer oligodendrocytes, compared to spinal-cord-derived NSPCs. Neurons that had differentiated from the transplanted forebrain-derived NSPCs were shown to be positive for neurotransmitters like GABA, glutamate and glycine, although authentic glycinergic neurons are not normally present within the forebrain. Thus, at least a subpopulation of the transplanted forebrain-derived NSPCs differentiated into spinal-cord-type neurons. In conclusion, forebrain-derived NSPCs could be used as an alternative to spinal-cord-derived NSPCs as a potential therapeutic agent for spinal cord injuries.

Neuroprotection by human neural progenitor cells after experimental contusion in rats.

Neural progenitor/stem cells (HNPC) have been suggested to contribute essential trophic factors and promote survival of degenerating neurons after traumatic brain injury. For these reasons we hypothesize that the addition of HNPC to a post-injury region could possibly protect injured neurons. Experimental brain contusions were carried out in 18 rats. Immediately post-injury, rats were injected with 0.1 ml of medium (n=8), dead cells (n=4), or live cells (n=6) in the medial border of the lesion. The rats were sacrificed 6 days post-surgery and evaluated by immunohistochemistry using a human nuclear marker (huN), hematoxylin and Fluoro-Jade (FJ). Human neural stem cells showed engraftment detectable by positive huN staining in 5/6 animals. The non-grafted animal was excluded from further analyses. Those given dead HNPC or medium showed no detectable huN immunoreactivity. A statistical comparison between the numbers of FJ positive degenerating endogenous neurons was made between rats receiving vehicle and dead cells to evaluate whether the presence of human cells would increase neuronal degeneration in comparison to vehicle alone. The rats receiving vehicle had a median of 117.5 FJ positive cells and dead progenitor cell recipients 175.0 per counted section (P<0.05, Mann-Whitney). Consequently, the animals receiving dead human cells were chosen as controls to the animals receiving live progenitor cells. The rats that received live HNPC demonstrated significantly fewer FJ positive cells per counted section than controls (58.0 vs. 175.0, P<0.01, Mann-Whitney). The post-traumatic perilesional environment allowed for the engraftment of live HNPC. The stepwise analysis indicated that host neuronal degeneration was higher in animals transplanted with non-viable human neuronal progenitor cells than in those receiving vehicle, indicating a bystander effect from introducing foreign antigen. In contrast, transplantation of viable progenitor cells attenuated neuronal degeneration, indicating that a potentially beneficial effect in progenitor cell transplantation is not limited to restoration by transplanted cells, but also improving survival of host cells.

A transplantation study of expanded human embryonic forebrain precursors: evidence for selection of a specific progenitor population.

Neural stem and progenitor cells can be expanded under growth factor stimulation in vitro. It is likely that different mitogens and different culturing techniques selectively expand specific subclasses of cells, but this selection has not been well studied. We have expanded human cells isolated from the lateral ganglionic eminence (LGE) of a 10-week-old embryo in the presence of serum and epidermal growth factor. We provide evidence that culturing in this manner favors expansion of cells with characteristics similar to a subpopulation of LGE cells, the olfactory bulb progenitors, as revealed by their expression of Er81 in vitro. After transplantation into neonatal rats, the cells displayed similar properties to endogenous olfactory bulb progenitors when exposed to local cues present in the subventricular zone (SVZ) and rostral migratory stream (RMS). However, the human LGE cells do not migrate or undergo region-specific differentiation when placed outside the SVZ and RMS.

Layer specification of transplanted interneurons in developing mouse neocortex.

The six-layered neocortex is composed of excitatory projection neurons and inhibitory interneurons. Recent studies have established separate embryological origins for these two cellular populations. However, it remains uncertain how interneurons arising from the subcortical ganglionic eminences are able to participate in the orderly stratification of the cortical layers. A related question concerns whether or not early and late interneuron progenitors have equivalent developmental potentials. To address these issues, we performed transplantation experiments to test the fates of early-versus late-born interneuron populations using cells labeled with a genetic marker. Our results indicate that transplanted interneurons from the medial ganglionic eminence give rise to specific layers of the neocortex in an inside-out order. To test the potency of interneurons born at different ages, heterochronic transplantations were also performed. Both early- and late-born progenitors were able to switch their fates in the new environment, and, similar to projection neurons, fate-switching was dependent on progenitor receptivity to environmental cues during their last round of cell division. Our data also demonstrate, for the first time, that interneuron-layering cues are present within the medial ganglionic eminence, suggesting that, before the commencement of long-distance tangential migration, interneurons are already specified with respect to their future layer addresses. So, although the generation of diverse neuronal phenotypes in separate locations is an effective strategy to pursue separate developmental programs, our results indicate that excitatory and inhibitory neurons share similar mechanisms for integrating sequentially born neurons from two places into a single layered structure.

Restorative potential of cholinergic rich transplants in cholchicine induced lesioned rats: a comparative study of single and multiple micro-transplantation approach.

Restorative potential of fetal neural transplantation in colchicine induced neurodegeneration was studied in rats; where colchicine (2.5mg per site) was administered bilaterally into the hippocampus followed by bilateral infusions of fetal neural cell suspension rich in cholinergic neurons as single macro- or multiple micro-transplants in the hippocampal region 3 weeks post-colchicine (2.5mg per site) lesion. Animals were studied for neuro behavioural and neurochemical recovery at 4 and 24 weeks post-transplantation and electrophysiological (single cell recording) and immunohistochemical parameters, choline acetyl transferase (ChAT) expression was studied in hippocampus at 24 weeks post-transplantation. Colchicine lesioned rats receiving single macro- or multiple micro-transplants exhibited significant restoration in cognitive dysfunction caused by colchicine after 4 weeks of transplantation which remain persistent in multiple micro-transplanted group upto 24 weeks post-transplantation, whereas, single macro-transplanted animals did not exhibit any significant recovery. Neurochemical studies revealed significant restoration in acetylcholine esterase activity and cholinergic (muscarinic) receptor binding after 24 weeks post-transplantation as compared to 4 weeks post-transplantation in multiple micro-transplanted group. Single cell recording studied at 24 weeks post-transplantation exhibited significant restoration in firing rates when compared with lesioned group. The viability of cholinergic fibre at transplanted sites has further been confirmed by increase in ChAT immuno positivity in hippocampal region using monoclonal antibody and quantified using image analyser Leica Qwin 500 software. The results suggest that intra-hippocampal multiple site cholinergic rich transplants provide better and long term restoration in the cholinergic deficits induced by colchicine lesion as compared to single site macro-transplantation.

Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions.

In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes. In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons. Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse. Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats. Cell suspensions of primary LGE tissue were prepared and grafted in parallel. Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes. Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons. Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb. MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus. The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.

Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor AB (PDGF AB) promote adult SVZ-derived oligodendrogenesis in vivo.

The capacity of multipotential progenitor cells of the adult mammalian forebrain to generate myelin-forming oligodendrocytes was tested by grafting fragments of different regions of the subventricular zone (SVZ) of the lateral ventricle and the striatum of 6-month-old wild-type mice into the brain of neonate shiverer and wild-type mice. Without growth factor treatment, only few cells of the rostral SVZ survived and formed myelin after engraftment. Treating donors prior to transplantation with a single intraperitoneal injection of epidermal growth factor, basic fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor AB (PDGF(AB)) vigorously promoted the survival, migration, and differentiation of the grafted SVZ cells into myelin-forming oligodendrocytes. In situ, both growth factors expanded the constitutively proliferative PSA-NCAM+ population and favored their differentiation toward the neuronal and oligodendroglial cell fate. The adult central nervous system thus harbors a focal reservoir of FGF-2 and PDGF(AB)-responsive cells which are able to generate substantial amounts of myelin-forming oligodendrocytes in vivo, opening a new prospective area for therapy in demyelinating diseases.

Fate map of the chicken neural plate at stage 4.

A detailed fate map was obtained for the early chick neural plate (stages 3d/4). Numerous overlapping plug grafts were performed upon New-cultured chick embryos, using fixable carboxyfluorescein diacetate succinimidyl ester to label donor chick tissue. The specimens were harvested 24 hours after grafting and reached in most cases stages 9-11 (early neural tube). The label was detected immunocytochemically in wholemounts, and cross-sections were later obtained. The positions of the graft-derived cells were classified first into sets of purely neural, purely non-neural and mixed grafts. Comparisons between these sets established the neural plate boundary at stages 3d/4. Further analysis categorized graft contributions to anteroposterior and dorsoventral subdivisions of the early neural tube, including data on the floor plate and the eye field. The rostral boundary of the neural plate was contained within the earliest expression domain of the Ganf gene, and the overall shape of the neural plate was contrasted and discussed with regard to the expression patterns of the genes Plato, Sox2, Otx2 and Dlx5 (and others reported in the literature) at stages 3d/4.

Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections.

Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1-3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.

Correlation between tectum formation and expression of two PAX family genes, PAX7 and PAX6, in avian brains.

Heterotopic transplantation of brain vesicles between chick and quail were performed, and the correlation between tectum formation and the expression of two PAX family genes, PAX7 and PAX6, analyzed. Reciprocal transplantation between the prosencephalon and mesencephalon showed that formation of the tectum always coincided with induction/maintenance of PAX7 and suppression of PAX6, indicating that switch-on or -off of these two PAX family genes in region specific manners are responsible for the differentiation of brain vesicles into the tectum. On the other hand, transplantation of the mesencephalic floor plate into the dorsal mesencephalon suppressed PAX7 expression in the dorsal mesencephalon and changed its fate from the tectum to the tegmentum, indicating that factors in the mesencephalic floor plate suppress PAX7 and limit tectum territory to the dorsal part of the mesencephalon.

Calbindin D28K and parvalbumin gene expression in rat embryonic ventral forebrain grafts.

The present study characterizes expression of calbindin D28K (CB-D28K) and parvalbumin (PV) in ventral forebrain (VFB) grafts placed in the neocortex of adult rats bearing quisqualic acid lesions to the nucleus basalis magnocellularis. Three to nine months after transplantation surgery, rats were killed for in situ hybridization with probes to CB-D28K or PV and for immunohistochemistry with antibodies to CB-D28K or PV. In addition, an antibody to choline acetyltransferase (ChAT) was used to characterize the cholinergic component in the graft and an antibody to tyrosine hydroxylase (TH) to explore catecholaminergic innervation of the graft. Quantitative analysis of CB-D28K and PV messenger ribonucleic acid (mRNA) was based on counts of silver grains generated by emulsion autoradiography. Cells expressing CB-D28K mRNA were significantly larger than such cells in the adult VFB and the mean number of silver grains per cell was significantly greater than to such cells in the adult VFB. The level of CB-D28K mRNA expression as calculated by ratio of silver grains per unit area was also significantly increased. Quantification of PV mRNA showed no significant differences between the cells in the graft and in the adult VFB. In order to begin to interpret these findings, a comparison was made with such cells in the VFB of developing rats. Brain sections were sampled from embryonic day 17 and postnatal days 1, 5, 12, 19 and adult (6-12 months of age). Cells expressing CB-D28K mRNA were detected in ventral forebrain from postnatal day 5 and cells expressing PV mRNA were detected in ventral forebrain from postnatal day 19. In the course of normal development of the ventral forebrain, no CB-D28K cells were found that were as large or expressed such high levels of CB-D28K mRNA as observed in the grafts. We conclude that changes in grafted cells expressing CB-D28K do not reflect an arrest of developmental processes. TH immunohistochemistry revealed lack of catecholaminergic innervation of the graft, whereas adult mediolateral septal cells that express CB-D28K receive such innervation in addition to other neurotransmitter inputs. Imbalance in neurotransmitter inputs to grafted cells expressing CB-D28K is discussed as a possible factor in their increased size and gene expression.