The Subventricular Zone: A Key Player in Human Neocortical Development.

One of the main characteristics of the developing brain is that all neurons and the majority of macroglia originate first in the ventricular zone (VZ), next to the lumen of the cerebral ventricles, and later on in a secondary germinal area above the VZ, the subventricular zone (SVZ). The SVZ is a transient compartment mitotically active in humans for several gestational months. It serves as a major source of cortical projection neurons as well as an additional source of glial cells and potentially some interneuron subpopulations. The SVZ is subdivided into the smaller inner (iSVZ) and the expanded outer SVZ (oSVZ). The enlargement of the SVZ and, in particular, the emergence of the oSVZ are evolutionary adaptations that were critical to the expansion and unique cellular composition of the primate cerebral cortex. In this review, we discuss the cell types and organization of the human SVZ during the first half of the 40 weeks of gestation that comprise intrauterine development. We focus on this period as it is when the bulk of neurogenesis in the human cerebral cortex takes place. We consider how the survival and fate of SVZ cells depend on environmental influences, by analyzing the results from in vitro experiments with human cortical progenitor cells. This in vitro model is a powerful tool to better understand human neocortex formation and the etiology of neurodevelopmental disorders, which in turn will facilitate the design of targeted preventive and/or therapeutic strategies.

Slice Culture Method for Studying Migration of Neuronal Progenitor Cells Derived from Human Embryonic Stem Cells (hESC).

In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low-adhesion plates to generate transgenic human neurospheres (t-hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t-hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue.

Increasing doublecortin expression promotes migration of human embryonic stem cell-derived neurons.

Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs.

Oligodendrocyte development and the onset of myelination in the human fetal brain.

Oligodendrocytes are cells that myelinate axons, providing saltatory conduction of action potentials and proper function of the central nervous system. Myelination begins prenatally in the human, and the sequence of oligodendrocyte development and the onset of myelination are not thoroughly investigated. This knowledge is important to better understand human diseases, such as periventricular leukomalacia, one of the leading causes of motor deficit in premature babies, and demyelinating disorders such as multiple sclerosis (MS). In this review we discuss the spatial and temporal progression of oligodendrocyte lineage characterized by the expression of specific markers and transcription factors in the human fetal brain from the early embryonic period (5 gestational weeks, gw) until midgestation (24 gw). Our in vitro evidence indicated that a subpopulation of human oligodendrocytes may have dorsal origin, from cortical radial glia cells, in addition to their ventral telencephalic origin. Furthermore, we demonstrated that the regulation of myelination in the human fetal brain includes positive and negative regulators. Chemokines, such as CXCL1, abundant in proliferative zones during brain development and in regions of remyelination in adult, are discussed in the view of their potential roles in stimulating oligodendrocyte development. Other signals are inhibitory and may include, but are not limited to, polysialic acid modification of the neural cell adhesion molecule on axons. Overall, important differences in temporal and spatial distribution and regulatory signals for oligodendrocyte differentiation exist between human and rodent brains. Those differences may underlie the unique susceptibility of humans to demyelinating diseases, such as MS.

Electrical excitability of early neurons in the human cerebral cortex during the second trimester of gestation.

Information about development of the human cerebral cortex (proliferation, migration, and differentiation of neurons) is largely based on postmortem histology. Physiological properties of developing human cortical neurons are difficult to access experimentally and therefore remain largely unexplored. Animal studies have shown that information about the arousal of electrical activity in individual cells within fundamental cortical zones (subventricular zone [SVZ], intermediate zone, subplate [SP], and cortical plate [CP]) is necessary for understanding normal brain development. Here we ask where, in what cortical zone, and when, in what gestational week (gw), human neurons acquire the ability to generate nerve impulses (action potentials [APs]). We performed electrical recordings from individual cells in acute brain slices harvested postmortem from the human fetal cerebral cortex (16-22 gw). Tetrodotoxin-sensitive Na(+) current occurs more frequently among CP cells and with significantly greater peak amplitudes than in SVZ. As early as 16 gw, a relatively small population of CP neurons (27%) was able to generate sodium APs upon direct current injection. Neurons located in the SP exhibited the highest level of cellular differentiation, as judged by their ability to fire repetitive APs. At 19 gw, a fraction of human CP and SP neurons possess beta IV spectrin-positive axon initial segments populated with voltage-gated sodium channels (PanNav). These results yield the first physiological characterization of developing human fetal cortical neurons with preserved morphologies in intact surrounding brain tissue.

Radial glia cells in the developing human brain.

Human radial glia (RG) share many of the features described in rodents, but also have a number of characteristics unique to the human brain. Results obtained from different mammalian species including human and non-human primates reveal differences in the involvement of RG in neurogenesis and oligodendrogenesis and in the timing of the initial expression of typical RG immunomarkers. A common problem in studying the human brain is that experimental procedures using modern molecular and genetic methods, such as in vivo transduction with retroviruses or creation of knockout or transgenic mutants, are not possible. Nevertheless, abundant and valuable information about the development of the human brain has been revealed using postmortem human material. Additionally, a combination and spectrum of in vitro techniques are used to gain knowledge about normal developmental processes in the human brain, including better understanding of RG as progenitor cells. Molecular and functional characterization of multipotent progenitors, such as RG, is important for future cell replacement therapies in neurological and psychiatric disorders, which are often resistant to conventional treatments. The protracted time of development and larger size of the human brain could provide insight into processes that may go unnoticed in the much smaller rodent cortex, which develops over a much shorter period. With that in mind, we summarize results on the role of RG in the human fetal brain.

Neuroprotective role of minocycline in co-cultures of human fetal neurons and microglia.

Bacterial infections during pregnancy often result in premature birth and neonatal white matter damage. During these infections, microglia, the resident immune cells of the CNS, undergo activation and contribute to further neuronal damage of the CNS. Minocycline, a second-generation tetracycline antibiotic, inhibits microglial activation and protects neurons in rodents but data about its effects on human cells are limited. We studied the mechanism of the neuroprotective effect of minocycline in either purified cell cultures or co-cultures of microglia and neurons from human fetal brain during inflammation induced by lipopolysaccharide (LPS). In neuron/microglial co-cultures, minocycline treatment prevented activation and proliferation of microglia and protected neurons as demonstrated by decreased neuronal cell death and a shift of Bcl-2 family proteins toward anti-apoptotic ratio. Notably, neither minocycline nor LPS had an effect on neurons in purified neuronal cultures. The ability of minocycline to regulate activation of human fetal microglia might be relevant in therapies used towards treating neonatal CNS infections.

The effect of CXCL1 on human fetal oligodendrocyte progenitor cells.

Chemokine CXCL1 is abundantly present in proliferative zones during brain development and in regions of remyelination, suggesting that it influences development of oligodendrocyte progenitors (OPC) in these regions. We studied in vitro the effects and possible mechanisms by which CXCL1 acts on human fetal OPC. In organotypic slice cultures of human fetal cortical ventricular/subventricular (VZ/SVZ) zones, blocking of CXCL1 signaling reduced significantly the proliferation of OPC. Moreover, exogenously added CXCL1 induced increase of OPC proliferation. Treatments of purified OPC cultures and cell depletion experiments demonstrated that this effect of CXCL1 was mainly indirect, mediated through astrocytes. We identified that CXCL1 acted through the extracellular signal regulated kinase (ERK1/2) pathway, activated primarily in astrocytes. In vitro, astrocytes stimulated with CXCL1 released several cytokines, but only the release of interleukin-6 (IL-6) was completely blocked by inhibition of ERK1/2 pathway. When released IL-6 was neutralized in slices, a decrease in OPC proliferation was demonstrated, while addition of IL-6 was able to return OPC proliferation in astrocyte-depleted slices to the control level. These results suggest that in the human fetal brain CXCL1 promotes proliferation of early OPC, acting in part through an ERK1/2-dependent pathway and release of IL-6 from astrocytes.

Human cortical neurons originate from radial glia and neuron-restricted progenitors.

Understanding the molecular and physiological determinants of cortical neuronal progenitor cells is essential for understanding the development of the human brain in health and in disease. We used surface marker fucose N-acetyl lactosamine (LeX) (also known as CD15) to isolate progenitor cells from the cortical ventricular/subventricular zone of human fetal brain at the second trimester of gestation and to study their progeny in vitro. LeX+ cells had typical bipolar morphology, radial orientation, and antigen profiles, characterizing them as a subtype of radial glia (RG) cells. Four complementary experimental techniques (clonal analysis, immunofluorescence, transfection experiments, and patch-clamp recordings) indicated that this subtype of RG generates mainly astrocytes but also a small number of cortical neurons. The neurogenic capabilities of RGs were both region and stage dependent. Present results provide the first direct evidence that RGs in the human cerebral cortex serve as neuronal progenitors. Simultaneously, another progenitor subtype was identified as proliferating cells labeled with neuronal (beta-III-tubulin and doublecortin) but not RG markers [GFAP, vimentin, and BLBP (brain lipid-binding protein)]. Proliferative and antigenic characteristics of these cells suggested their neuron-restricted progenitor status. In summary, our in vitro study suggests that diverse populations of cortical progenitor cells, including multipotent RGs and neuron-restricted progenitors, contribute differentially to cortical neurogenesis at the second trimester of gestation in human cerebral cortex.

Interaction between microglia and oligodendrocyte cell progenitors involves Golli proteins.

Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease characterized by plaques, areas of destroyed myelin sheaths in the CNS, which results in multiple disabilities for patients. In addition to demyelinated plaques, pathophysiological studies have shown "shadow plaques" that represent areas of partial remyelination. New myelin can be made by oligodendrocytes (OLs) generated from oligodendrocyte progenitor cells (OPCs) that pre-exist in the demyelinated area or recruited from surrounding areas. To successfully repopulate the demyelinated area, OPCs have to proliferate, migrate, and differentiate into mature OLs capable of forming myelin. Identifying factors that influence remyelination is a current topic in developmental neurobiology. Previously, we showed that Golli proteins, which have a broad distribution in the nervous and immune systems, are present both in OPCs and activated microglia around MS lesions. We hypothesized that in response to inflammation, Golli proteins may promote proliferation of OPCs through microglial cells. To test this, we established neonatal mouse brain slice and cell cultures and used lipopolysaccharide (LPS) to induce inflammation. In LPS-treated brain slices, Golli proteins displayed increased expression in the cortical subventricular zone. Furthermore, Golli proteins were demonstrated only in the conditioned medium from LPS-treated microglial cell cultures (LPS-MCM), and were absent in either conditioned medium from LPS-treated astrocytes or control media. Finally, proliferation of purified OPCs was promoted with LPS-MCM or Golli proteins, but not with LPS alone. In summary, these results demonstrate that activated microglia are beneficial for proliferation of OPCs and suggest possible involvement of Golli proteins as one of mediators in this process.

Downregulation of glial scarring after brain injury: the effect of purine nucleoside analogue ribavirin.

The weak regenerative capacity of self-repair after injury to the adult brain is caused by the formation of glial scar due to reactive astrogliosis. In the present study the beginning of reactive astrogliosis in the adult, as shown immunocytochemically by upregulation of glial fibrillary acidic protein (GFAP) and vimentin, was seen two days after the left sensorimotor cortex lesion, being maximal during the first two weeks and declining by 30 days after the lesion. This was accompanied by intensive glial scarring. Conversely, after the neonatal lesion a lack of gliotic scar was seen until 30 days postsurgery, although the pattern of GFAP and vimentin expression during recovery period was the same. The aim of the study was to define an appropriate therapeutic intervention that could modulate astrocyte proliferation and diminish glial scar formation after adult brain lesion. For this purpose the effects of an antiproliferative agent, the purine nucleoside analogue ribavirin was examined. It was shown that daily injection of ribavirin for 5 and 10 days considerably decreased the number of reactive astrocytes, while slight GFAP labeling was restricted to the lesion site. Obtained results show that ribavirin treatment downregulates the process of reactive astrogliosis after adult brain injury, and thus may be a useful approach for improving neurological recovery from brain damage.

Contributions of cortical subventricular zone to the development of the human cerebral cortex.

The cortical subventricular zone (SVZ), a proliferative compartment in the forebrain, has a uniquely important role during the second half of intrauterine development in human. This is best observed in numerous neonatal pathologies that result from prenatal SVZ damage. These conditions highlight a need to better understand the contribution of the SVZ to the development of the human cerebral cortex. With this goal in mind, we analyzed histological organization, cell proliferation, and molecular diversity in the human fetal SVZ from 7-27 gestational weeks (gw) using light and electron microscopy, immunohistochemistry, and in vitro methods. Complex histological organization distinguishes human cortical SVZ from that of other mammals. In vitro quantification showed that approximately 50% of cells in the VZ/SVZ region are neurons, 30% are astroglia, 15% are nestin+ cells, with other cell types representing smaller fractions. Immunolabeling with BrdU showed that a considerable number of cells ( approximately 10%) are generated in the human cortical SVZ during midgestation (18-24 gw) under in vitro conditions. Immunofluorescence with cell type-specific markers and BrdU revealed that all major cell types, neural precursors (nestin+), astroglia including radial glia (GFAP+, vimentin+), and oligodendrocyte progenitors (PDGFR-alpha+) were proliferating. An increase in the ratio of the size of the SVZ to VZ, protracted period of cell proliferation, as well as cellular and histological complexity of the human fetal SVZ are directly related to the evolutionary expansion of the human cerebral cortex.

Lipopolysaccharide affects Golli expression and promotes proliferation of oligodendrocyte progenitors.

Proliferation of oligodendrocyte progenitor cells (OPCs) is important for initial myelination as well as for remyelination in demyelinating diseases. Previously, we showed that numerous OPCs and activated microglia, are present around multiple sclerosis lesions, and that they accumulate Golli proteins. Golli proteins, present in both neuronal and immune cells, might have a role in the immune processes, as well as in development of neurons and oligodendrocytes. We hypothesize that Golli proteins, generated by microglia in response to inflammation, promote proliferation of OPCs. To test this hypothesis, we induced inflammation in neonatal mouse brain slice culture with bacterial endotoxin lipopolysaccharide (LPS). Treated slices showed an increase in the number of OPCs. Several results support the notion that this effect of LPS is conveyed through activation of microglia and upregulation of Golli proteins. First, LPS-treated brain slices have increased expression of Golli proteins observed by immunofluorescence and Western blot analysis. Second, Golli proteins were demonstrated only in the conditioned medium from LPS-treated microglial cell cultures (LPS-MCM), and were absent in either the conditioned media from LPS-treated astrocytes or the control media. Third, proliferation of purified OPCs was promoted with LPS-MCM or Golli proteins, but not with LPS alone. Taken together, these results demonstrate that microglia and/or microglia secreted factors, are necessary for the LPS-promoted proliferation of OPCs and suggest possible involvement of Golli proteins as one of mediators in this process.