Human Embryonic Forebrain Research Articles

Transcriptomic analysis of left-right differences in human embryonic forebrain and midbrain

Left-right asymmetry is subtle but pervasive in the human central nervous system. This asymmetry is initiated early during development, but its mechanisms are poorly known. Forebrains and midbrains were dissected from six human embryos at Carnegie stages 15 or 16, one of which was female. The structures were divided into left and right sides, and RNA was isolated. RNA was sequenced with 100 base-pair paired ends using Illumina Hiseq 4000. After quality control, five paired brain sides were available for midbrain and forebrain. A paired analysis between left- and right sides of a given brain structure across the embryos identified left-right differences. The dataset, consisting of Fastq files and a read count table, can be further used to study early development of the human brain.

Zic-associated holoprosencephaly: zebrafish Zic1 controls midline formation and forebrain patterning by regulating Nodal, Hedgehog, and retinoic acid signaling

Holoprosencephaly (HPE) is the most frequently observed human embryonic forebrain defect. Recent evidence indicates that the two major forms of HPE, classic HPE and midline interhemispheric (MIH) HPE, are elicited by two different mechanisms. The only gene known to be associated with both forms of HPE is Zic2. We used the zebrafish Danio rerio as a model system to study Zic knockdown during midline formation by looking at the close homolog Zic1, which is expressed in an overlapping fashion with Zic2. Zic1 knockdown in zebrafish leads to a strong midline defect including partial cyclopia due to attenuated Nodal and Hedgehog signaling in the anterior ventral diencephalon. Strikingly, we were able to show that Zic1 is also required for maintaining early forebrain expression of the retinoic acid (RA)-degrading enzyme cyp26a1. Zic1 LOF leads to increased RA levels in the forebrain, subsequent ventralization of the optic vesicle and down-regulation of genes involved in dorsal BMP signaling. Repression of BMP signaling in dorsal forebrain has been implicated in causing MIH HPE. This work provides a mechanistical explanation at the molecular level of why Zic factors are associated with both major forms of HPE.

In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133(+)/SSEA4(+) and CD133(+)/CD15(+) isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.

The first neurons of the human cerebral cortex

We describe a distinctive, widespread population of neurons situated beneath the pial surface of the human embryonic forebrain even before complete closure of the neural tube. These 'predecessor' cells include the first neurons seen in the primordium of the cerebral cortex, before the onset of local neurogenesis. Morphological analysis, combined with the study of centrosome location, regional transcription factors and patterns of mitosis and neurogenesis, indicates that predecessor cells invade the cortical primordium by tangential migration from the subpallium. These neurons, described here for the first time, precede all other known cell types of the developing cortex.

Tangential Networks of Precocious Neurons and Early Axonal Outgrowth in the Embryonic Human Forebrain

We used a combination of immunohistochemistry and carbocyanine dye tracing to study neurons and their processes in the human embryonic forebrain, 4-7 weeks after conception, before the onset of synaptogenesis. We discovered a widespread network of precocious MAP2 (microtubule-associated protein 2)-immunoreactive cells, with long, nonaxonal processes, before the appearance of the cortical plate and the establishment of thalamocortical connectivity. Dye tracing revealed that the processes of these precocious cells form tangential links between intermediate zones of the thalamus, ganglionic eminence, hypothalamus, and cortical preplate. The spatiotemporal distribution and morphology of the precocious neurons in the cortical preplate suggest that they are generated outside the cerebral wall rather than in the local ventricular zone. The first thalamocortical axons and axons of preplate cells extend across diencephalo-telencephalic and striatocortical boundaries before the arrival of the first cortical plate neurons. Precocious cells may provide initial communication between subdivisions of the embryonic brain as well as guidance cues for navigation of growing axons and/or transverse neuronal migration.

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.

In Vitro Expansion of a Multipotent Population of Human Neural Progenitor Cells

The isolation and expansion of human neural progenitor cells have important potential clinical applications, because these cells may be used as graft material in cell therapies to regenerate tissue and/or function in patients with central nervous system (CNS) disorders. This paper describes a continuously dividing multipotent population of progenitor cells in the human embryonic forebrain that can be propagated in vitro. These cells can be maintained and expanded using a serum-free defined medium containing basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF). Using these three factors, the cell cultures expand and remain multipotent for at least 1 year in vitro. This period of expansion results in a 107-fold increase of this heterogeneous population of cells. Upon differentiation, they form neurons, astrocytes, and oligodendrocytes, the three main phenotypes in the CNS. Moreover, GABA-immunoreactive and tyrosine hydroxylase-immunoreactive neurons can be identified. These results demonstrate the feasibility of long-term in vitro expansion of human neural progenitor cells. The advantages of such a population of neural precursors for allogeneic transplantation include the ability to provide an expandable, well-characterized, defined cell source which can form specific neuronal or glial subtypes.

HUMAN FIRST TRIMESTER FOREBRAIN CELLS EXPRESS GENES FOR INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES

Using in situ hybridization and immunohistochemistry the induction of potential cytokines [interleukin 1β (IL-1β), IL-4, IL-6, IL-10, tumour necrosis factor α (TNF-α), interferon γ (IFN-γ) and transforming growth factor β (TGF-β)] in human embryonic forebrain cells at weeks 5, 7 and 10 of gestation was studied. The aims were to investigate the capacity of these cells to express cytokines, to determine the kinetics of induction and to display the type of cytokine expressing cells in the developing brain. Constitutive cytokine gene expression was recorded from week 5, augmented by about 50% at week 7 and by more than 100% at week 19 (except TGF-β at week 10). Among other cytokines, IL-1β exhibited the highest expression at week 10. TGF-β showed maximum expression at week 7 and declined at week 10. Combined techniques revealed that glial cells are a major source of cytokines. The study show and present for the first time the kinetics of spontaneous cytokine expression in human embryonic forebrain cells. The increased mRNA expressions with age suggest an important role for cytokines in promotion of brain development. The capacity of inducing these cytokines may, however, be implicated in the immunopathogenesis of several brain diseases. The distinctive TGF-β profile suggests a further role for TGF-β on modulation of cytokine responses during development.

Tyrosine kinases are required for IFN-gamma-induced growth of human embryonic forebrain astrocytes.

The cytokine IFN-gamma was shown to regulate growth and differentiation of human embryonic forebrain astrocytes. This work investigated a signalling pathway used by IFN-gamma during the process of growth regulation of human fetal astrocytes obtained from first trimester embryos. IFN-gamma induced significantly higher cell survival compared to that of unexposed cultures, and this survival could be suppressed by incubation with the tyrosine protein kinase (TPK) specific inhibitor tyrphostin A47 at the non-toxic concentration of 10(-6) M. The signal transducer and activator of transcription (STAT)-1 was translocated into the nucleus upon IFN-gamma stimulation, which was also blocked by incubation with A47. Our data demonstrate that TPKs are actively involved in growth regulation of the developing brain astrocytes induced by IFN-gamma.

Interactions between human embryonic forebrain cells and the cytokines interferon-gamma and interleukin-4.

In the present study we investigated the capacity of human first trimester forebrain cells at different gestational ages to produce IFN-gamma and IL-4. We also studied the effects of IFN-gamma on their proliferation, survival and expression of MHC antigens. IFN-gamma but not IL-4 was spontaneously produced after 24 h cultures. Furthermore, IFN-gamma exhibited anti-proliferative effects and induced MHC expression on these cells. However, the IFN-gamma exposed cultures showed significantly higher cell survival compared to un-exposed cultures. Co-culture with IL-4 blocked the IFN-gamma production and reversed its anti-proliferative effects. These interactions suggest important roles for cytokines in the survival, proliferation and differentiation of human embryonic and fetal forebrain cells.