Human Fetal Central Nervous System Research Articles

Deciphering Oligodendrocyte Lineages in the Human Fetal Central Nervous System Using Single-Cell RNA Sequencing.

Oligodendrocytes form myelin sheaths and wrap axons of neurons to facilitate various crucial neurological functions. Oligodendrocyte progenitor cells (OPCs) persist in the embryonic, postnatal, and adult central nervous system (CNS). OPCs and mature oligodendrocytes are involved in a variety of biological processes such as memory, learning, and diseases. How oligodendrocytes are specified in different regions in the CNS, in particular in humans, remains obscure. We here explored oligodendrocyte development in three CNS regions, subpallium, brainstem, and spinal cord, in human fetuses from gestational week 8 (GW8) to GW12 using single-cell RNA sequencing. We detected multiple lineages of OPCs and illustrated distinct developmental trajectories of oligodendrocyte differentiation in three CNS regions. We also identified major genes, particularly transcription factors, which maintain status of OPC proliferation and promote generation of mature oligodendrocytes. Moreover, we discovered new marker genes that might be crucial for oligodendrocyte specification in humans, and detected common and distinct genes expressed in oligodendrocyte lineages in three CNS regions. Our study has demonstrated molecular heterogeneity of oligodendrocyte lineages in different CNS regions and provided references for further investigation of roles of important genes in oligodendrocyte development in humans.

A Novel Multicellular Placental Barrier Model to Investigate the Effect of Maternal Aflatoxin B1 Exposure on Fetal-Side Neural Stem Cells.

Ingestion of food toxins such as aflatoxin B1 (AFB1) during pregnancy may impair fetal neurodevelopment. However, animal model results may not be accurate due to the species' differences, and testing on humans is ethically impermissible. Here, we developed an in vitro human maternal-fetal multicellular model composed of a human hepatic compartment, a bilayer placental barrier, and a human fetal central nervous system compartment using neural stem cells (NSCs) to investigate the effect of AFB1 on fetal-side NSCs. AFB1 passed through the HepG2 hepatocellular carcinoma cells to mimic the maternal metabolic effects. Importantly, even at the limited concentration (0.0641 ± 0.0046 μM) of AFB1, close to the national safety level standard of China (GB-2761-2011), the mixture of AFB1 crossing the placental barrier induced NSC apoptosis. The level of reactive oxygen species in NSCs was significantly elevated and the cell membrane was damaged, causing the release of intracellular lactate dehydrogenase (p < 0.05). The comet experiment and γ-H2AX immunofluorescence assay showed that AFB1 caused significant DNA damage to NSCs (p < 0.05). This study provided a new model for the toxicological evaluation of the effect of food mycotoxin exposure during pregnancy on fetal neurodevelopment.

Culturing and Expansion of "Clinical Grade" Neural Stem Cells from the Fetal Human Central Nervous System.

NSCs have been demonstrated to be very useful in grafts into the mammalian central nervous system to investigate the exploitation of NSC for the therapy of neurodegenerative disorders in animal models of neurodegenerative diseases. To push cell therapy in CNS on stage of clinical application, it is necessary to establish a continuous and standardized, clinical grade (i.e., produced following the good manufacturing practice guidelines) human neural stem cell lines.In this chapter we will illustrate some of the protocols for the production and characterization routinely used into our GMP "cell factory" for the production of "clinical grade" human neural stem cell lines already in use in clinical trials on neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS- Clinicaltrials.gov number NCT01640067) and secondary progressive multiple sclerosis (SPMS- Clinicaltrials.gov number NCT03282760).

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133+/CD24lo), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Human neural progenitor cells in central nervous system lesions

Various immature cells can be isolated from human embryonic and fetal central nervous system (CNS) residual tissue and potentially be used in cell therapy for a number of neurological diseases and CNS insults. Transplantation of neural stem and progenitor cells is essential for replacing lost cells, particularly in the CNS with very limited endogenous regenerative capacity. However, while dopamine released from transplanted cells can substitute the lost dopamine neurons in the experimental models of Parkinson's disease, stem and progenitor cells primarily have a neuroprotective effect, probably through the release of trophic factors. Understanding the therapeutic effects of transplanted cells is crucial to determine the design of clinical trials. During the last few years, a number of clinical trials for CNS diseases and insults such as amyotrophic lateral sclerosis (ALS), stroke, and spinal cord trauma using neural progenitor cells have been initiated. Data from these early studies will provide vital information on the safety of transplanting these cells, which still is a major concern. That the beneficial results observed in experimental models also can be repeated in the clinical setting is highly hoped for.

Maternal Thyroid and Glucocorticoid Hormone Interactions in Larval Fish Development, and Their Applications in Aquaculture

Thyroid hormones (THs) have long been known to have regulatory roles in the differentiation and maturation of vertebrate embryos, beginning with the knowledge that hormones of maternal origin are essential for human fetal central nervous and respiratory system development. Precise measurements of circulating THs led to insights into their critically important actions throughout vertebrate growth and development, initially with amphibian metamorphosis and including embryogenesis in fishes. Thyroid cues for larval fish differentiation are enhanced by glucocorticoid hormones, which promote deiodinase activity and thereby increase the generation of triiodothyronine (T3) from the less bioactive thyroxin (T4). Glucocorticoids also induce the expression of thyroid hormone receptors in some vertebrates. Maternally derived thyroid hormones and cortisol are deposited in fish egg yolk and accelerate larval organ system differentiation until larvae become capable of endogenous endocrine function. Increases in the T3/T4 ratio during larval development may reflect the regulatory importance of maternal thyroid hormones. Experimental applications of individual hormones have produced mixed results, but treatments with combinations of thyroid and corticoid hormones consistently promote larval fish development and improve survival rates. The developmental and survival benefits of maternal endocrine provisioning are increased in viviparous fishes, in which maternal/larval chemical contact is prolonged. Treatments with exogenous thyroid and corticoid hormones consistently promote development and reduce mortality rates in larval fishes, with potential hatchery-scale applications in aquaculture.

Dual effects of daily FTY720 on human astrocytes in vitro: relevance for neuroinflammation

BackgroundFTY720 (fingolimod, Gilenya™) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1β.MethodsAstrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1β-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA.ResultsWe observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1β. Recurrent FTY720 treatments did not inhibit serum- or IL-1β-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1β were unaffected by FTY720 treatment(s).ConclusionOur results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.

Cross-hemispheric functional connectivity in the human fetal brain.

Compelling evidence indicates that psychiatric and developmental disorders are generally caused by disruptions in the functional connectivity (FC) of brain networks. Events occurring during development, and in particular during fetal life, have been implicated in the genesis of such disorders. However, the developmental timetable for the emergence of neural FC during human fetal life is unknown. We present the results of resting-state functional magnetic resonance imaging performed in 25 healthy human fetuses in the second and third trimesters of pregnancy (24 to 38 weeks of gestation). We report the presence of bilateral fetal brain FC and regional and age-related variation in FC. Significant bilateral connectivity was evident in half of the 42 areas tested, and the strength of FC between homologous cortical brain regions increased with advancing gestational age. We also observed medial to lateral gradients in fetal functional brain connectivity. These findings improve understanding of human fetal central nervous system development and provide a basis for examining the role of insults during fetal life in the subsequent development of disorders in neural FC.

The Tryptophan Metabolite 3-Hydroxyanthranilic Acid Plays Anti-Inflammatory and Neuroprotective Roles During Inflammation: Role of Hemeoxygenase-1

Tryptophan metabolism by the kynurenine pathway (KP) is important to the pathogenesis of inflammatory, infectious, and degenerative diseases. The 3-hydroxykynurenine (3-HK) branch of the KP is activated in macrophages and microglia, leading to the generation of 3-HK, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid, which are considered neurotoxic owing to their free radical-generating and N-methyl-d-aspartic acid receptor agonist activities. We investigated the role of 3-HAA in inflammatory and antioxidant gene expression and neurotoxicity in primary human fetal central nervous system cultures treated with cytokines (IL-1 with or without interferon-γ) or with Toll-like receptor ligands mimicking the proinflammatory central nervous system environment. Results were analyzed by microarray, Western blot, immunostain, enzyme-linked immunosorbent assay, and neurotoxicity assays. 3-HAA suppressed glial cytokine and chemokine expression and reduced cytokine-induced neuronal death. 3-HK also suppressed cytokine-induced neuronal death. Unexpectedly, 3-HAA was highly effective in inducing in astrocytes the expression of hemeoxygenase-1 (HO-1), an antioxidant enzyme with anti-inflammatory and cytoprotective properties. Optimal induction of HO-1 required 3-HAA and cytokines. In human microglia, 3-HAA weakly induced HO-1 and lipopolysaccharide suppressed microglial HO-1 expression. 3-HAA-mediated HO-1 expression was confirmed in cultured adult human astrocytes and in vivo after 3-HAA injection to mouse brains. Together, our results demonstrate the novel neuroprotective activity of the tryptophan metabolite 3-HAA and have implications for future therapeutic approaches for neuroinflammatory disorders.

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells ( Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8–9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133 +CD24 −/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca 2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Markers of Pluripotency and Differentiation in Human Neural Precursor Cells Derived from Embryonic Stem Cells and CNS Tissue

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

222 Ofatumumab binds to a membrane-proximal epitope which comprises amino acids in the small and large loops of CD20

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005, Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8–9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133+CD24−/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Assessment of ghrelin, GHS-R, GH, and neurohormones in human fetal pituitary glands and central nervous system: an immunohistochemical study.

The aim of this work was evaluation of expression of ghrelin and GHS-R1a receptor in somatotrops and in neuronal cells of brain tissue in the process of human fetal ontogenesis. Relations were also looked for between GHRH and SS in the pituitary and in the CNS neurones of the studied fetuses. The study was based on 8 pituitaries and 8 brains from fetuses in different periods of intrauterine life. The immunocytochemical technique was used. The presence of ghrelin, GHS-R was shown in the glandular part of the pituitary and CNS during the whole period of intrauterine life. Neurohormones in the stalk of the pituitary were found in fetuses from the 32nd week of pregnancy whereas in the CNS neurones these hormones could be detected throughout the whole period of intrauterine life. The results obtained suggest that stimulation of GH secretion by ghrelin is independent of the feedback concentration and these two hormones act like signals of metabolic balance. GH release by ghrelin in fetal life is independent of somatostatin. The hypothalamic-pituitary axis which regulates pulsatile GH release from the pituitary matures functionally in the third trimester of pregnancy independent of the previous anatomical differentiation.

Clade‐specific differences in neurotoxicity of human immunodeficiency virus‐1 B and C Tat of human neurons: significance of dicysteine C30C31 motif

Human immunodeficiency virus-1 (HIV-1) causes mild to severe cognitive impairment and dementia. The transactivator viral protein, Tat, is implicated in neuronal death responsible for neurological deficits. Several clades of HIV-1 are unequally distributed globally, of which HIV-1 B and C together account for the majority of the viral infections. HIV-1-related neurological deficits appear to be most common in clade B, but not clade C prevalent areas. Whether clade-specific differences translate to varied neuropathogenesis is not known, and this uncertainty warrants an immediate investigation into neurotoxicity on human neurons of Tat derived from different viral clades We used human fetal central nervous system progenitor cell-derived astrocytes and neurons to investigate effects of B- and C-Tat on neuronal cell death, chemokine secretion, oxidative stress, and mitochondrial membrane depolarization by direct and indirect damage to human neurons. We used isogenic variants of Tat to gain insights into the role of the dicysteine motif (C30C31) for neurotoxic potential of Tat Our results suggest clade-specific functional differences in Tat-induced apoptosis in primary human neurons. This study demonstrates that C-Tat is relatively less neurotoxic compared with B-Tat, probably as a result of alteration in the dicysteine motif within the neurotoxic region of B-Tat This study provides important insights into differential neurotoxic properties of B- and C-Tat, and offers a basis for distinct differences in degree of HIV-1-associated neurological deficits observed in patients in India. Additional studies with patient samples are necessary to validate these findings.

FTY720 modulates human oligodendrocyte progenitor process extension and survival

FTY720, a sphingosine-1-phosphate (S1P) receptor agonist that crosses the blood-brain barrier, is a potential immuno-therapy for multiple sclerosis. Our objective was to assess the effect of FTY720 on process extension, differentiation, and survival of human oligodendrocyte progenitor cells (OPCs), and link the functional effects with S1P receptor expression and signaling. Functional assays and receptor expression studies were conducted on A2B5+ OPCs derived from the human fetal central nervous system. Cells were treated with physiologically relevant concentrations of the active phosphorylated form of FTY720. S1P receptor/signaling modulators were used to elucidate the basis of the FTY720-induced functional responses. Short-term (1 day) FTY720 treatment caused initial process retraction that was reversed by uncoupling S1P3 and 5 from their G protein using suramin, and with a Rho-kinase inhibitor H1152. Retraction was associated with RhoA-mediated cytoskeletal signaling and with inhibition of OPC differentiation into more mature phenotypes. Continued FTY720 treatment (2 days) induced process extension and enhanced cell survival associated with increased extracellular signal-regulated kinases 1 and 2 phosphorylation, mimicked with the S1P1-specific agonist SEW2871, but not reversed with suramin. Quantitative real-time polymerase chain reaction showed that FTY720 induced reciprocal and cyclic modulation of S1P1 and S1P5 messenger RNA levels. The observed initial downregulation of S1P5 and subsequently of S1P1 messenger RNA supports functional responses being mediated sequentially by S1P5- and later S1P1-associated signaling. FTY720 induces time-dependent modulation of S1P receptors on human OPCs with consequent functional responses that are directly relevant for the remyelination process.

BARHL1 homeogene, the human ortholog of the mouse Barhl1 involved in cerebellum development, shows regional and cellular specificities in restricted domains of developing human central nervous system

The mouse homeobox gene Barhl1 plays a central role in cerebellum development and its expression is activated by the transcription factor Math1 which is involved in bone morphogenetic protein response pathways. We studied the human ortholog BARHL1 and we found that human, mouse, monkey, rat, and zebrafish orthologs were highly conserved and are members of the BarH homeogene family, containing Drosophila BarH1 and BarH2. The N-terminus of BARHL1 protein presents two FIL domains and an acidic domain rich in serine/threonine and proline, while the C-terminus contains a canonical proline-rich domain. Secondary structure analysis showed that outside the three helixes of the homeodomain, BARHL1 protein has essentially random coil structure. We isolated BARHL1 and defined its expression pattern in human embryonic and fetal central nervous system (CNS) and compared it to the mouse Barhl1 transcription. BARHL1 mRNA was found exclusively in the CNS restricted to p1–p4 prosomeres of the diencephalon, to the dorsal cells of the mesencephalon, to the dorsal dl1 sensory neurons of the spinal cord, and to the rhombic lips yielding the cerebellar anlage. Detailed analysis of BARHL1 expression in fetal cerebellar cell layers using our new optic microscopy technology showed BARHL1 expression in external and internal granular cells and also in mouse adult granular cells, in agreement to Barhl1 null mouse phenotype affecting the differentiation and migration of granular cells. These findings indicate that the regional and cellular specificities of BARHL1 transcriptional control well correspond to the mouse Barhl1 transcription and suggest a potential role of this gene in the differentiation of BARHL1-expressing neuronal progenitors involved in the pattern formation of human cerebral and cerebellar structures.

Studies on protocadherin-2 expression in the human fetal central nervous system.

Protocadherin (Pcad) is a group of molecules obtained by polymerase chain reaction (PCR) utilizing the sequence that is well preserved in the extracellular domain of cadherin. Sano et al. analyzed Pcad (PC42,43) that had been cloned from rats, and found that it basically had homology to cadherin, but contained more than six cadherin repeats with a completely different intracellular domains (Sano et al. 1993). In the present study, of the Pcad (Pcad-1,2) cloned from a human cDNA library, as-yet-unspecified Pcad-2 was analyzed for expression in the human fetal central nervous system (CNS). Northern blot analysis of adult human tissue showed that Pcad-2 was expressed in the brain and the placenta, and that Pcad-2 mRNA was expressed in actively dividing neural tumor cell lines. Monoclonal antibodies against Pcad-2 were then made, and the CNS of fetuses were immunohistochemically stained. The expression was hardly visible at the 6th week of pregnancy, and began to become visible along the nerve fiber in the brain stem at the 8th week, and spread over the entire brain at the 11th week. At the 18th week, however, expression in the nerve fascicles, which had been visible by that time, was no longer visible or had decreased. These results suggest that Pcad-2 appears relatively early in the critical stage of development of the fetal CNS, and is involved in the induction, fasciculation, and extension of axons.

Staging and preparation of human fetal striatal tissue for neural transplantation in Huntington's disease.

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.

Expression of β‐chemokines and chemokine receptors in human fetal astrocyte and microglial co‐cultures: Potential role of chemokines in the developing CNS

Chemokines play specific roles in directing the recruitment of leukocyte subsets into inflammatory foci within the central nervous system (CNS). The involvement of these cytokines as mediators of inflammation is widely accepted. Recently, it has become evident that cells of the CNS (astrocytes, microglia, and neurons) not only synthesize, but also respond functionally or chemotactically to chemokines. We previously reported developmental events associated with colonization of the human fetal CNS by mononuclear phagocytes (microglial precursors), which essentially takes place within the first two trimesters of life. As part of the array of signals driving colonization, we noted specific anatomical distribution of chemokines and chemokine receptors expressed during this period. In order to further characterize expression of these molecules, we have isolated and cultured material from human fetal CNS. We demonstrate that unstimulated subconfluent human fetal glial cultures express high levels of CCR2 and CXCR4 receptors in cytoplasmic vesicles. Type I astrocytes, and associated ameboid microglia in particular, express high levels of surface and cytoplasmic CXCR4. Of the chemokines tested (MIP-1alpha, MIP-1beta, MCP-1, MCP-3, RANTES, SDF-1, IL-8, IP-10), only MIP-1alpha, detected specifically on microglia, was expressed both constitutively and consistently. Low variable levels of MCP-1, MIP-1alpha, and RANTES were also noted in unstimulated glial cultures. Recombinant human chemokines rhMCP-1 and rhMIP-1alpha also displayed proliferative effects on glial cultures at [10 ng/ml], but displayed variable effects on CCR2 levels on these cells. rhMCP-1 specifically upregulated CCR2 expression on cultured glia at [50 ng/ml]. It is gradually becoming evident that chemokines are important in embryonic development. The observation that human fetal glial cells and their progenitors express specific receptors for chemokines and can be stimulated to produce MCP-1, as well as proliferate in response to chemokines, supports a role for these cytokines as regulatory factors during development.

Erythropoietin gene expression in different areas of the developing human central nervous system

Evidence from cell culture and animal experiments suggests a neuroprotective and neurotrophic function of erythropoietin (EPO). We have quantitated the distribution of EPO mRNA expression in the developing human central nervous system (CNS). Patients and Methods: Up to seven biopsies from different areas of the CNS of four preterm fetuses (gestational age 23–37 weeks) were obtained at routine postmortem examinations. EPO mRNA was quantitated by competitive PCR in samples from the CNS, the kidneys, and the liver where the EPO gene is predominantly expressed at this gestational age. Results: EPO mRNA was most abundant in one sample from the cerebellum (0.29 amol/μg total RNA [amol=10 −18mol]) and two from the pituitary gland (0.23 amol/μg total RNA), but levels varied considerably. EPO mRNA in the cortex cerebri (median 0.12 amol/μg total RNA; n=4) dominated over the expression in the corpora amygdala (median 0.05 amol/μg total RNA; n=4), the hippocampus (median 0.03 amol/μg total RNA; n=4), or the basal ganglia (median 0.01 amol/μg total RNA; n=3). Only little EPO mRNA (<0.01 and 0.06 amol/μg total RNA) was found in the spinal cord. EPO mRNA levels in the cerebellum, pituitary gland, or the cerebral cortex were within the same range as in the liver (0.03–1.67 amol/μg total RNA; n=4), or the kidneys (0.06–0.79 amol/μg total RNA; n=4). Conclusion: We found the EPO gene expressed throughout the fetal human CNS. Our data provide the basis to discuss a function for EPO in the brain of humans as well.