Human Fetal Cerebral Cortex Research Articles

Separation and identification of neural stem cells from human fetal cerebral cortex in vitro

目的 探讨人脑皮质中分离培养神经干细胞并鉴定其增殖及分化潜能.方法采用包含碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的无血清培养和单细胞克隆技术,从30周自愿水囊引产人胎脑皮质中分离出神经干细胞, 并进行培养、传代、分化观察,应用免疫组织化学染色对培养的细胞及其分化的细胞进行鉴定.结果从30周人胎脑皮质中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈悬浮状态生长,形成神经球,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原(Nestin);在含血清培养时诱导神经干细胞分化,分化后的细胞表达神经元细胞和胶质细胞的特异性抗原.结论神经干细胞的存活和分裂有赖于EGF和bFGF的共同作用;30周人胎脑皮质仍能培养出具有自我复制和分化潜能的神经干细胞。

Early expression of thyroid hormone deiodinases and receptors in human fetal cerebral cortex

Thyroid hormones are known to be important for optimal development of the human central nervous system. Classically, maternal thyroid hormones have not been thought to have a major role in defining central nervous system development. However, recent epidemiological evidence has indicated that subtle deficiencies in circulating maternal thyroid hormones in the first trimester of pregnancy are associated with adverse neurodevelopment. We have used real time PCR to quantitate the expression of mRNAs encoding the thyroid receptor isoforms (TR α1, α2, β1 and β2) and thyronine deiodinase subtypes (5′-DI, 5′-DII and 5-DIII) in human fetal cerebral cortex from the first and second trimesters of pregnancy. Deiodinase subtype activities have also been determined in these tissues and compared to ‘normal’ adult human cerebral cortex. Iodothyronine deiodinase mRNAs were expressed in human fetal cerebral cortex from 7 to 8 weeks of gestation. The expression of 5′-DI mRNA was variable in fetal life but increased relative to adult cortex ( P<0.05), whereas the activity of this enzyme was below the level of assay detection. 5′-DII mRNA and activity in fetal cerebral cortex was detectable from as early as 7–8 weeks but not significantly different from that in adult life except at 15–16 weeks when mRNA expression increased ( P<0.05). Fetal cortex 5-DIII mRNA expression was present from the early first trimester but less abundant than in adult tissue ( P<0.01) and 5-DIII activity appeared greateter in fetal cortex ( P<0.01) as compared to adults. Only TR α1 mRNA was more abundantly expressed in fetal cortex than adult tissues ( P<0.01). In contrast, the TR isoforms (α2 and β1) were expressed significantly less than in adult tissues ( P<0.05). Only 26% of fetal cerebral cortex samples expressed TRβ1. There is evidence that the developing fetal brain, as early as the first trimester, expresses TRs and exhibits the mechanisms of pre-receptor control of thyroid hormone supply.

Expression of thyroid receptor isoforms in the human fetal central nervous system and the effects of intrauterine growth restriction.

Congenital hypothyroidism is known to be associated with mental retardation which, if recognized promptly, is largely prevented by thyroid hormone replacement. Intrauterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity, and is also associated with neurodevelopmental delay. Fetuses with IUGR have reduced circulating concentrations of free thyroxine (T4) and free triiodothyronine (T3), leading to the hypothesis that a reduction in the tissue effects of thyroid hormones in the central nervous system (CNS) may contribute to neurodevelopmental morbidity. Since thyroid hormone effects are mediated through binding to specific nuclear thyroid hormone receptors (TRs), we have defined the pattern of TR isoform expression in the CNS throughout normal human development and have compared TR expression in the CNS of normal fetuses with those affected by IUGR. Samples of normal human fetal brain from first and second trimesters were obtained at surgical termination of pregnancy. Appropriately grown and third trimester fetuses affected by Intrauterine growth restriction (IUGR) were also investigated after unexplained stillbirth at post mortem examination. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of TR isoform mRNAs in frozen cerebral cortex from 10 to 16 weeks gestation. TR protein expression in human fetal brains (both cerebral hemispheres and cerebellum) was also examined in formalin fixed sections and expression of TR alpha1, alpha2, beta1 and beta2 isoforms being defined using semiquantitative immunocytochemistry. RT-PCR revealed the presence of mRNAs encoding TR alpha1, beta1 and beta2 isoforms and the nonfunctional TRalpha2 variant in the fetal cerebral cortex from week 10 of human pregnancy. Immunostaining of the fetal brain revealed TR alpha1 and alpha2 protein from week 11 of human gestation. Expression of all TR isoform proteins was largely confined to the pyramidal neurones of the cerebral cortex and the Purkinje cells of the cerebellum with increasing receptor expression evident with gestational age. Semiquantitative observer scoring showed that by the second trimester, there was a marked increase in the proportion of pyramidal and Purkinje cells expressing TR isoforms, while by the third trimester, all these cells immunostained. Comparison of TR immunostaining in the cerebral cortex and cerebellum from IUGR fetuses (n = 18) matched for gestational age to normal fetuses revealed a lower intensity of expression of all the TR isoforms confirmed by observer scoring and quantification using TR protein immunofluoresence (P<0.01). Our findings indicate both pre- and post-translational expression of TR alpha and beta isoforms in the cerebral cortex of first trimester fetuses. These findings support the view that the transplacental passage of thyroid hormone in early gestation may be critical to neurological development. Our finding that in severe IUGR the expression of TR isoforms in the human fetal cerebral cortex and cerebellum was significantly reduced, in association with reduced circulating thyroid hormone concentrations indicate that changes in free ligand and receptors may affect CNS development. These findings should prompt further investigation of the potential therapeutic role of peripartum thyroid hormone treatment.

Differential expression of S100B and S100A6 1 in the human fetal and aged cerebral cortex

S100B and S100A6 (calcylin) are two members of the S100 Ca 2+-binding protein family and have been localized in the mammalian nervous system. However, information on their distribution in the human nervous system, especially in the developing human fetal brain, is scarce. In the present study, an immunocytochemical method was used to examine the spatio-temporal protein expression patterns of S100B and S100A6 in normal human fetal hippocampus, entorhinal cortex and occipital cortex. Normal aged adult human brain specimens were also included for comparison. From week 15 onwards, an increase with advancing gestation age in both the number and staining intensity of S100B positive, astrocyte-like cells was found in the pyramidal layer of the hippocampus, while both the molecular and polymorphic layers showed similar S100B immunoreactivities at all stages examined. A decrease in the immunoreactivities was found in the molecular layer of the aged adult hippocampus while other layers exhibited immunoreactivities similar to those of the late fetus. At week 15, the molecular, pyramidal and ganglionic/multiform layers of the entorhinal cortex also showed positive S100B immunoreactivities which were maintained throughout the rest of the gestation and in adult specimens. In the occipital cortex, the numbers of positive cells for all layers were about twofold higher than those found in the hippocampus and entorhinal cortex, and immunoreactivities detected in the granular layer increased from week 21, reaching a plateau at around week 27. S100B positive fibers were also found at week 30 but were not observed in aged adult specimens. S100A6 positive cells were on the whole fewer in number than those of S100B in the brain regions examined. The S100A6 immunoreactivities which were localized in some pyramidal neuron-like and some glial-like cells of the pyramidal and molecular layers of the hippocampus increased by midgestation and became weak in the late fetus and in aged adult specimens. Weakly stained S100A6 positive cells were also observed in the entorhinal cortex throughout the gestation and in aged adult cortex. S100A6 immunoreactivities were weak in the fetal occipital cortex. They were also localized in the glial-like cells of the aged adult occipital cortex. The differential spatio-temporal expression of S100B and S100A6 proteins suggests that the proteins play different roles in different brain regions during development and in adulthood.

Full length article

Microglia represent the primary immune effector cells of the adult central nervous system (CNS). The origin of these cells has been a subject of intense debate over the last century. However, immunohistochemical and chimera developmental studies in rodents support the hypothesis that microglia are monocytic in origin. There have been relatively few studies to date on microglia in human fetal development, and the mechanisms by which microglial precursors enter the developing CNS are as yet unknown. It is possible that microglial precursors use combinations of adhesion molecules on cerebral endothelium to gain entry into the developing CNS. In the present study, we have shown the distribution of microglia within human fetal cerebral cortex between 16 and 22 weeks of gestation using RCA-1 lectin histochemistry. We have also demonstrated dual anti-macrophage antibody labelling of these cells in conjunction with adhesion molecules ICAM-1, ICAM-2 and PECAM on cerebral endothelium throughout this period. We conclude that fetal microglia usually occur at highly vascularised sites within the developing human fetal brain and are more specifically associated with the expression of ICAM-2 on cerebral endothelium.

NMDA induces NO release from primary cell cultures of human fetal cerebral cortex

We and others have previously reported that N-methyl- d-aspartate (NMDA) induces nitric oxide (NO) release from the rat cerebral cortex in vivo. It is crucial to determine if this phenomenon also exists in human brain tissue. In this study, we investigated the interactions of NMDA and NO in human primary neocortical cell cultures obtained from elective abortions. Extracellular NO concentration was monitored through Nafion- and porphyrine-coated carbon fiber electrodes, which have previously been demonstrated sensitive and selective responses to NO. We found that local application of NMDA induced NO release from neocortical neuron-enriched cultures but not from glial cultures. Pretreatment with N G-nitro- l-arginine methyl ester hydrochloride ( l-NAME), a nitric oxide synthase inhibitor, or MK801 significantly attenuated NMDA-induced NO overflow. This is the first time, to our knowledge, that extracellular NO concentration evoked by exogenous NMDA has been directly measured from the fetal human cortical neurons.

The properties of cultured fetal human and rat brain tissue and its use as grafts for the relief of the parkinsonian syndrome.

Primary cultures were derived from human fetal ventral mesencephalon and cerebral cortex at 7-11 weeks gestation, and from fetal rat mesencephalon and cortex at embryonic day 14-15. Immunohistochemical analysis of the mesencephalic cultures using antibodies to tyrosine hydroxylase (TH) showed between 0.1-0.5% of human cells to be TH positive and 0.1-1% of rat cells to be TH positive. HPLC analysis of extracts from the cultures showed that they had the ability to synthesise and store dopamine. Implantation of the cultured human and rat mesencephalic tissue into a 6-hydroxydopamine rat model of Parkinson's disease produced marked recovery from amphetamine induced rotational asymmetry in the recipient rats, but no such recovery was observed following implantation of cortical cultures. Histological examination demonstrated the presence of surviving human mesencephalic and cortical grafts at least 6 months after implantation. Implants of cultured fetal rat tissue were less obviously but still significantly effective in these experiments. These rat tissue grafts were detectable for periods of at least 6-8 weeks by histological staining.

Chapter 76 Electrophysiological studies of human cerebral and cerebellar cortical tissue grafted to the anterior eye chamber of athymic rodents

Publisher Summary Syngeneic grafting of brain tissue has emerged as a valuable approach in studying the development and regeneration of neural connections in the central nervous system (CNS) of mammals. This chapter describes the electrophysiological studies of human fetal cerebellar and cortical brain tissue grafted to the anterior chamber of the eye of athymic nude rats. The results demonstrate that human fetal cerebellar and cerebral cortex grafts survive in athymic nude rat recipients, and continue their development in the anterior chamber of the eye. It has also been shown that several different human fetal central nervous system areas can survive and develop, when grafted to the anterior chamber of the eye of cyclosporin A-treated host rats. However, in spite of rigorous immunosuppression, only some of the grafted material survives intraocularly. Thus, the athymic nude rat offers an alternative model for xenogeneic brain tissue grafting with several advantages. The chapter concludes that this technique uniquely permits the study of human brain development, connectivity, and pharmacological properties in an otherwise immunologically incompatible host species.

Studies on the development, antigenic phenotype and function of human glial cells in tissue culture.

Cell type-specific neural markers and indirect immunofluorescence were used to study the antigenic phenotype, development and function of astrocytes and oligodendrocytes in dissociated cell cultures of human fetal optic nerve and cerebral cortex. It was found that (1) two populations of GFAP+ astrocytes could be distinguished in optic nerve cultures using the A2B5 monoclonal antibody; (2) the proportion of A2B5+ astrocytes and of oligodendrocytes which developed in optic nerve cultures was dependent on the tissue culture medium; (3) A2B5+ cells were required for the development of galactocerebroside+ oligodendrocytes and at least some GFAP+A2B5+ astrocytes; (4) astrocytes purified from cerebral cortex were A2B5- and NCAM+; and (5) astrocytes supported the unfasciculated growth of rat CNS neurons. These results demonstrate close similarities between cultured rat and human neural cells. Such observations are likely to be of significance in future studies of the developing human nervous system and of a variety of neurological diseases.

THE ONTOGENESIS OF SOMATOSTATIN IN THE FETAL RAT AND SHEEP PANCREAS

The localization of somatostatin (GIF) in pancreatic D cells and in the gastrointestinal tract led to the concept that SRIF may have a physiologic role at these sites as well as in the CNS. Its role in the fetus is not known. Previously we demonstrated GIF in the human fetal hypothalamus and cerebral cortex. The present study examines the ontogeny of GIF In rat and sheep fetal pancreas using a homologous radioimmunoassay with tyrosyl 11-GIF as label and standard, with a sensitivity of 5 pg cyclic GIF. In the rat, GIF is detected at 14 days gestation in pooled extracted pancreases at .0016 ng/pancreas. GIF increases proportional to DNA until day 19, with a subsequent rapid rise to 3.8 ng/pancreas (72 pg/μg DNA) at term. Two days postnatally GIF Increases to 8.3 ng/pancreas (135 pg/μg DNA). In the adult rat 71.3 ng GIF/pancreas is detected (54 pg/μg DNA). In the fetal sheep, GIF is present by 58 days at 1.6 ng/pancreas, rises to 5.3 ng at 65 days and to 1900 ng/pancreas at term. The content in the adult sheep is 7000 ng GIF/pancreas. GIF is present by mid-gestation in the fetal rat and sheep pancreas with a sharp 1000-fold increase by term. The biphasic developmental pattern of somatostatin in the fetus is similar to that of insulin but differs from that for glucagon.

The fine structure of the axons and growth cones of the human fetal cerebral cortex

The fine structure of axon cylinders and growth cones has been examined in serial sections through the marginal zone of the human cerebral cortex from 10 to 22 weeks of gestation. Neurotubules, mitochondria, agranular reticulum, coated vesicles, and synaptic vesicles both of the translucent and dense core variety are found throughout the axon cylinders. At periodic intervals along the axons, bulged-out regions display accumulations of either synaptic vesicles or transversely oriented neurotubules. Prominent, growth cone varicosities are encountered at the distal segment of the axon cylinders. In these, mitochondria, channels of both tubular and dilated agranular reticulum, dense core vesicles, and multivesicular bodies lie in a finely filamentous matrix. Processes characterized by their content of either agranular reticulum and synaptic vesicles or a finely filamentous meshwork are infrequently continuous with these varicosities. Dense membrane thickenings are associated with some varicosities and their processes. The ultrastructural features of the human fetal growth cones have been compared to growth cones previously described in other mammalian and submammalian species.

Autoradiographic studies of migrating neurons and astrocytes of human fetal cerebral cortex in vitro

Autoradiographic studies with tritiated thymidine were carried out utilizing organotypic cultures established from full-thickness tissue blocks taken from primordial posterior frontal-anterior parietal human fetal cerebral neocortex. The specimens cultured came from fetuses of 8 wk to 20 wk gestation and were grown in vitro for one to 126 days. The principal cellular components in cultures were neurons and astrocytes as determined by special stains and electronmicroscopy. In all specimens studied, there was marked labeling of astrocytes, but no labeling of neurons could be found either in outgrowth zones or within primary explant areas. The findings were identical in 24 hr pulse-labeled and in cumulatively-labeled cultures. It is suggested that the final DNA synthesis of neurons destined for the posterior frontal-anterior parietal region of cerebral cortex in the human fetus may take place near the end of the first trimester. Inherent in these findings are implications concerning the vulnerability of developing human brain to various types of insults at different gestational ages.

A Correlated Light and Electron Microscopic Study of the Early Phase of Growthin Vitroof Human Fetal Cerebellar and Cerebral Cortex

Cerebellar and cerebral cortex from human fetuses of 12–20 weeks' gestation removed for the purpose of terminating pregnancy were studied in vitro and in tissue blocks by light and electron microscopy. In cultures, the morphologic features characterising the first three weeks of growth were examined. By use of multiple criteria, immature neurons and astrocytes and their processes could be identified with reasonable certainty. In cerebellar cultures, migrating immature neurons were similar to cells of the external granular layer observed in tissue blocks; whereas migrating young neurons in cerebral cultures were similar to cells of the cortical plate. Immature astrocytes developed abundant 100 A filaments in vitro . Similar cells with abundant filaments were not observed in tissue blocks of either cerebellar or cerebral cortex. This study demonstrated not only the capacity of immature neurons of both human cerebellar and cerebral cortex to migrate in vitro , but also a consistent pattern of outgrowth of cells from explants during the first three weeks of growth in culture. Immature astrocytes first emerged from an explant followed by neuritic processes and subsequently by cell bodies of immature neurons from which the neurites arose. In addition to making contacts with other young neurons, intimate relationships were observed by light microscopy (i) between endings of neurites and the cytoplasm of astrocytes, and (ii) between cell bodies of migrating immature neurons and migrating immature astrocytes. The close relationship between migrating neurons and astrocytes was a particularly striking characteristic of the early outgrowth.