Monique Dubois-Dalcq

Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats

Limited experimental access to the central nervous system (CNS) is a key problem in the study of human neural development, disease, and regeneration. We have addressed this problem by generating neural chimeras composed of human and rodent cells. Fetal human brain cells implanted into the cerebral ventricles of embryonic rats incorporate individually into all major compartments of the brain, generating widespread CNS chimerism. The human cells differentiate into neurons, astrocytes, and oligodendrocytes, which populate the host fore-, mid-, and hindbrain. These chimeras provide a unique model to study human neural cell migration and differentiation in a functional nervous system.

Role of the α-chemokine stromal cell-derived factor (SDF-1) in the developing and mature central nervous system

α‐chemokines, which control the activation and directed migration of leukocytes, participate in the inflammatory processes in host defense response. One of the α‐chemokines, CXCL12 or stromal cell‐derived factor 1 (SDF‐1), not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development as we discuss here. SDF‐1 indeed activates the CXCR4 receptor expressed in a variety of neural cells, and this signaling results in diverse biological effects. It enhances migration and proliferation of cerebellar granule cells, chemoattracts microglia, and stimulates cytokine production and glutamate release by astrocytes. Moreover, it elicits postsynaptic currents in Purkinje cells, triggers migration of cortical neuron progenitors, and produces pain by directly exciting nociceptive neurons. By modulating cell signaling and survival during neuroinflammation, SDF‐1 may also play a role in the pathogenesis of brain tumors, experimental allergic encephalitis, and the nervous system dysfunction associated with acquired immunodeficiency syndrome. GLIA 42:139–148, 2003.

Growth and Fate of PSA-NCAM+ Precursors of the Postnatal Brain

Oligodendrocyte-type 2 astrocyte (O-2A) lineage cells are derived from multipotential stem cells of the developing CNS. Precursors of O-2A progenitors express the polysialylated (PSA) form of the neural cell adhesion molecule (NCAM) and are detected in neonatal rat brain glial cultures. It is unclear how such PSA-NCAM+ “pre-progenitors” are related to neural stem cells and whether they still have the potential to differentiate along several neural lineages. Here we isolated PSA-NCAM+ pre-progenitor cells from glial cultures by immunopanning and found that most of these cells expressed nestin and PDGF-receptor-α but not O-2A antigens. PSA-NCAM+ cells synthesized transcripts for fibroblast growth factor (FGF) receptors 1, 2, and 3 and responded to FGF2 by survival and proliferation, growing into large clusters resembling neural spheres. FGF2-induced proliferation of PSA-NCAM+ pre-progenitors was significantly enhanced by thyroid hormone (T3), which on its own did not increase cell survival or mitosis. After adhesion and withdrawal of the mitogen, spheres generated mostly oligodendrocytes and astrocytes but very rarely neurons. PSA-NCAM immunopanned cells grown in epidermal growth factor (EGF) also adopted a mostly glial fate after differentiation. In contrast, PSA-NCAM-negative cells and striatal neonatal stem cells, grown in EGF or FGF2, generated the three CNS cell types. Like neural stem cells, PSA-negative cells generated more oligodendrocytes and fewer neurons when expanded in FGF2 and T3. Thus emergence of PSA-NCAM at the surface of neonatal brain precursors coincides with their restriction to a glial fate. T3 modulates these events by enhancing PSA-NCAM+ pre-progenitor growth in FGF2 and favoring an oligodendrocyte fate.

Differential signalling of the chemokine receptor CXCR4 by stromal cell-derived factor 1 and the HIV glycoprotein in rat neurons and astrocytes.

CXCR4 is the Gi protein‐linked seven‐transmembrane receptor for the alpha chemokine stromal cell‐derived factor 1 (SDF‐1), a chemoattractant for lymphocytes. This receptor is highly conserved between human and rodent. CXCR4 is also a coreceptor for entry of human immunodeficiency virus (HIV) in T cells and is expressed in the CNS. To investigate how these CXCR4 ligands influence CNS development and/or function, we have examined the expression and signalling of this chemokine receptor in rat neurons and astrocytes in vitro. CXCR4 transcripts and protein are synthesized by both cell types and in E15 brain neuronal progenitors. In these progenitors, SDF‐1, but not gp120 (the HIV glycoprotein), induced activation of extracellular signal regulated kinases (ERKs) 1/2 and a dose‐dependent chemotactic response. This chemotaxis was inhibited by Pertussis toxin, which uncouples Gi proteins and the bicyclam AMD3100, a highly selective CXCR4 antagonist, as well as by an inhibitor of the MAP kinase pathway. In differentiated neurons, both SDF‐1 and the glycoprotein of HIV, gp120, triggered activation of ERKs with similar kinetics. These effects were significantly inhibited by Pertussis toxin and the CXCR4 antagonist. Rat astrocytes also responded to SDF‐1 signalling by phosphorylation of ERKs but, in contrast to cortical neurons, no kinase activation was induced by gp120. Thus neurons and astrocytes can respond differently to signalling by SDF‐1 and/or gp120. As SDF‐1 triggers directed migration of neuronal progenitors, this alpha chemokine may play a role in cortex development. In differentiated neurons, both natural and viral ligands of CXCR4 activate ERKs and may therefore influence neuronal function.

From Neural Stem Cells to Myelinating Oligodendrocytes

The potential to generate oligodendrocytes progenitors (OP) from neural stem cells (NSCs) exists throughout the developing CNS. Yet, in the embryonic spinal cord, the oligodendrocyte phenotype is induced by sonic hedgehog in a restricted anterior region. In addition, neuregulins are emerging as potent regulators of early and late OP development. The ability to isolate and grow NSCs as well as glial-restricted progenitors has revealed that FGF2 and thyroid hormone favor an oligodendrocyte fate. Analysis of genetically modified mice showed that PDGF controls the migration and production of oligodendrocytes in vivo. Interplay between mitogens, thyroid hormone, and neurotransmitters may maintain the undifferentiated stage or result in OP growth arrest. Notch signaling by axons inhibits oligodendrocyte differentiation until neuronal signals--linked to electrical activity-trigger initiation of myelination. To repair myelin in adult CNS, multipotential neural precursors, rather than slowly cycling OP, appear the cells of choice to rapidly generate myelin-forming cells.

Developmental pattern of expression of the alpha chemokine stromal cell‐derived factor 1 in the rat central nervous system

Stromal cell‐derived factor 1 (SDF‐1) is an alpha‐chemokine that stimulates migration of haematopoietic progenitor cells and development of the immune system. SDF‐1 is also abundantly and selectively expressed in the developing and mature CNS, as we show here. At embryonic day 15, SDF‐1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF‐1 in situ hybridization signal that decreased progressively within the next 2 weeks. In other regions such as cortex, thalamus and hippocampus, SDF‐1 transcripts detected at birth progressively increased in abundance during the postnatal period. SDF‐1 protein was identified by immunoblot and/or immunocytochemistry in most brain regions where these transcripts were detected. SDF‐1 was selectively localized in some thalamic nuclei and neurons of the fifth cortical layer as well as in pontine and brainstem nuclei which relay the nociceptive response. The presence of SDF‐1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF1 mRNA signal increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life. The selective and regulated expression of SDF‐1 in these regions suggests a role in precursor migration, neurogenesis and, possibly, synaptogenesis. Thus this alpha chemokine may be as essential to nervous system function as it is to the immune system.

The neurobiology of X-linked adrenoleukodystrophy, a demyelinating peroxisomal disorder

Adrenoleukodystrophy (ALD) is caused by mutations in an ATP-binding-cassette transporter located in the peroxisomal membrane, which result in a fatal demyelinating disease in boys and a milder phenotype in men and some heterozygous women. There is no molecular signature to indicate a particular clinical course. The underlying molecular mechanisms of this disease have yet to be targeted clinically. Is the increase in very-long-chain fatty acids (VLCFA) the disease trigger? Why is there no phenotype in ALD null mice that show this increase? Do VLCFA destabilize human myelin, once formed, and lead to the inflammation seen in this genetic disease? Bone-marrow transplantation might save a child by providing normal brain macrophages and allowing myelin regeneration early in disease. The processes that underlie ALD challenge neuroscientists to elucidate peroxisomal transporter functions in the nervous system and to pursue the gene-transfer strategies leading to remyelination until a preventive therapy emerges.

Emergence of oligodendrocytes from human neural spheres

To study the development of human oligodendrocyte precursors (OP), we expanded human embryonic brain‐derived neural precursors into spheres with basic fibroblast growth factor (FGF2). Over 90% of the cells in the expanded spheres were precursors coexpressing nestin and the polysialylated (PSA) form of NCAM. The remaining cells were mostly astrocytes and neuronal cells located at the periphery of the floating spheres. When spheres were allowed to adhere on fibronectin‐coated substrate in the absence of FGF2, neural precursors migrated in the outgrowth and often formed chains of cells expressing high levels of PSA‐NCAM. Many migrating cells also expressed beta‐3 tubulin while only scattered elongated cells radiating from the spheres were GFAP+ astrocytes. Spindle‐shaped cells not associated with the chains were labeled for the PDGF‐alpha receptor and often coexpressed MAP2 neuronal isoforms. Neuronal cells in the outgrowth rapidly established a rich neuritic network where OP expressing O4 and DM20/proteolipid antigens appeared. T3 treatment of neural spheres increased the rate of OP formation and the complexity of their shape. Thus, the generation of human oligodendrocytes from neural precursors is tightly correlated with growth of neuronal processes and enhanced by hormonal signals. J. Neurosci. Res. 50:146–156, 1997.

Origin of Oligodendrocytes within the Human Spinal Cord

To determine the time and site of origin of the oligodendrocyte lineage in the developing human spinal cord, we have examined tissues from 45 to 83 d postconception (dpc) using sets of probes and antibodies recognizing oligodendrocyte-specific glycolipids, transcripts, and proteins. We found that two clusters of oligodendrocyte precursors appear on or before 45 dpc on each side of the cord ventral ependyma above the floor plate. These precursors express glycolipids recognized by the O4 and Rmab antibodies, platelet-derived growth factor α-receptor, myelin basic protein (MBP), and 2′, 3′-cyclic nucleotide 3′ phosphodiesterase as well as MBP and proteolipid transcripts. Expression of the morphogen sonic hedgehog was detected in the floor plate at 45 dpc and decreased at 58 dpc. During this period, oligodendrocyte precursors emerged in the ventral and lateral region of the forming white matter, a process occurring first in cervical and later in lumbar cord. The majority of O4+ cells express the proliferating cell nuclear antigen (PCNA), and their pattern of dispersion suggests that these cells progressively populate the lateral and dorsal cord regions. Oligodendrocytes expressing galactocerebroside appeared at 53 dpc and did not express PCNA. Oligodendrocyte precursors were detected in dorsal cord regions at 74 dpc and at 83 dpc when myelination started in the ventral roots. Thus, oligodendrocyte precursors expressing myelin transcripts and proteins emerge in the ventral region of the embryonic cord several weeks before myelination.

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro

Schwann cells, the myelin‐forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl‐transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase‐N stripping of PSA. PSA‐positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX‐transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co‐cultures. Thus, migratory properties of PNS myelin‐forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.