Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy

The classical recessive mouse mutant, "the twitcher," is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.     

Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.     

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.     

Commingling and segregation analysis of blood pressure in a French-Canadian population.

Using genetic linkage we have localized the gene coding for galactocerebrosidase (GALC) to human chromosome 14. Patients with Krabbe disease and their family members were assayed for GALC activity in leukocytes or fibroblasts and were classified as affected, carrier, noncarrier, or unknown. Polymorphic DNA markers from chromosome 14 demonstrated a multipoint LOD score of 3.40 with GALC located 13 cM centromere distal to CRI-C70 (D14S24). This finding is consistent with the location of the mouse twitcher mutation (a model of human GALC deficiency) on chromosome 12, which has substantial homology to human chromosome 14. Our data do not support a previous report's localization of GALC to chromosome 17.     

Neural transplantation of human MSC and NT2 cells in the twitcher mouse model

BACKGROUND: Accumulating evidence has demonstrated that the NT2 embryonal carcinoma cell line and multipotential stem cells found in BM, mesenchymal stromal cells (MSC), have the ability to differentiate into a wide variety of cell types. This study was designed to explore the efficacy of these two human stem cell types as a graft source for the treatment of demyelinating disorders such as Krabbe's disease and multiple sclerosis (MS). METHODS: We examined the engraftment and in vivo differentiation of adult MSC and NT2 cells after transplantation into two demyelinating environments, the neonatal and postnatal twitcher mouse brain. RESULTS: Both types of xenografts led to anatomical integration, without tumor formation, and remained viable in the normal and twitcher mouse brain, showing differentiation into neurons, astrocytes and oligodendrocytes. DISCUSSION: This study represents a platform for further stem cell transplantation studies in the twitcher model and potentially has important therapeutic implications.     

Biosynthesis of galactosylsphingosine (Psychosine) in the twitcher mouse

In attempts to elucidate the origin of accumulated galactosylsphingosine in the twitcher mouse, a murine model of human globoid cell leukodystrophy (Krabbe's disease), UDP-galactose: sphingosine galactosyltransferase activity was assayed in tissues from normal and twitcher mice. Among several tissues from normal, 20 day postnatal mice, the highest galactosyltransferase activity was found in the brainstem and spinal cord, followed by cerebrum, kidney and liver, in that order. Chronologically, the enzyme activity in the central nervous tissue increased with age, reached a maximum at 25 postnatal days, and declined thereafter. In the kidney and liver, however, the activity remained much the same during development. In the twitcher mouse, developmental change in the enzyme activity was similar to that seen in control mouse, but the decrease in activity in the central nervous tissue after the 25 postnatal days was more rapid. The galactosyltransferase activity and the accumulation of galactosylsphingosine in the tissue of the twitcher mouse were closely related; where and when the enzyme activity was higher, the greater was the accumulation of galactosylsphingosine in the tissue of the twitcher mouse. These results strongly suggest that the accumulated galactosylsphingosine in the twitcher mouse is synthesized mainly by UDP-galactose: sphingosine galactosyltransferase.     

Cloning of the Canine GALC cDNA and Identification of the Mutation Causing Globoid Cell Leukodystrophy in West Highland White and Cairn Terriers

Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3′-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A to C change at 473 is the disease-causing mutation. A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectively and will permit the establishment of a colony of dogs for use in therapy trials.     

Murine, canine and non-human primate models of Krabbe disease

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessively inherited neurological disease caused by mutations in the gene coding for the lysosomal enzyme galacto-cerebrosidase (GALC). GALC is responsible for the degradation of specific galactolipids, including several that are important in the production of compact, stable myelin. A failure to adequately degrade galactosylceramide and psychosine (galactosylsphingosine) results in the characteristic pathological findings observed in tissue from humans and animals affected with GLD. These galactosphingolipids are normally synthesized during active myelination, and psychosine accumulates in individuals with very low GALC activity. Psychosine is highly toxic to the myelin-forming oligodendrocytes, causing their death and the paucity of myelin found on autopsy. While most human patients present with symptoms before six months of age and die before 18 months of age, older children and adults can also be diagnosed with GLD[1,2]. The cloning of both the human GALC cDNA and the GALC gene opened the way for the identification of mutations causing GLD in humans and animals and the development of novel strategies to treat this severe and fatal disease[3]. The pheno-typic differences between human patients result from the wide range of mutations identified, as well as additional unknown factors. Treatment of late-onset patients and pre-symptomatic individuals (identified either because prenatal testing was not requested or a fetus predicted to be affected was not aborted) by hemato-poietic stem cell transplantation (HSCT) resulted in a less severe phenotype than was predicted and, in some cases, a significant delay in the onset of symptoms[4]. Attempts to treat this disorder by in utero HSCT have not been successful[5].GLD in dogs     

Application of neuronal cell culture in human degenerative brain disorders

Neuronal cell culture system has been used for the study of pathochemical evaluations in human degenerative brain disorders, particularly for Krabbe's disease and neuronal ceroid lipofuscinosis. To understand the pathochemistry of Krabbe's disease, we added psychosine into neuronal cell cultures and psychosine treated cells showed the destruction of cytoskeleton and pathy intracellular changes. Electron microscopic finding showed the swelling of the mitochondria. Oligodendrocytes and Schwann cells were isolated from the brains and sciatic nerve of twitcher mouse as an authentic murine model of globoid cell leukodystrophy. Oligodendroglial cells cultured for 22 days were stained by anti-galactocerebroside antibodies. In twitcher oligodendrocyte processes were wirelike and progressively degenerated and there were few membranous expansion. Schwann cells from twitcher could not elongated their processes. These data suggest that psychosine might be important factor to result in these pathological conditions. Furthermore, we studied the effect of protease inhibitors, E-64 on dissociated primary cultures from fetal rat brain. After treated with E-64 in a concentration from 0.1-50 micrograms/ml, numerous cytoplasmic accumulations appeared in neuronal cells. These morphological pictures resemble with those of neuronal ceroid lipofuscinosis, Batten disease. We will discuss the relationship between the deficiency of catepsin H in Batten disease and inclusion bodies found in E-64.     

Optic nerve tissue sections derived from myelin-deficient mutant mice as culture substrata for fibroblast adhesion and spreading

The substrate properties were compared between normal and myelin-deficient central nervous system (CNS) tissues by an in vitro assay of cell attachment and spreading. Fibroblasts (3T3) were plated onto culture substrata consisting of optic nerve tissue sections cut from normal or two myelin-deficient mutant mice, Shiverer and Quaking. Optic nerve sections from either of the mutant animals supported more 3T3 fibroblast spreading and adhesion than sections derived from animals with normal myelin. These results demonstrate that CNS myelin influences the ability of cells to attach and spread and that it is the actual presence of myelin which is inhibitory rather than the presence of optic nerve axons or oligodendrocytes.     

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells

Abstract In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His→Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.     

Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: evidence from a mouse model of Krabbe leukodystrophy

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.     

Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse,a Model of Krabbe's Disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.     

NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration

The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.     

Steroid synthesis in rat brain cell cultures

Primary cultures derived from neonatal rat forebrains were established and cultured for several weeks. They grow entirely as glial cultures composed of oligodendrocytes and astrocytes. Glial cells undergo maturation and differentiation in culture. This was shown by measuring the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a specific marker for expression of oligodendrocyte differentiation. CNPase activity increased from days 10-21 of culture. Both cell types were characterized by indirect immunofluorescence staining using monoclonal antibodies to galactocerebroside (Gal C) and myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) for astrocytes. Using the above criteria, we measured about 60% oligodendrocytes and 40% astrocytes after 3 weeks of culture. Oligodendrocytes, expressing Gal C and MBP, were highly immunoreactive to monospecific polyclonal antibodies to the cytochrome P-450scc, enzyme involved in the synthesis of pregnenolone from cholesterol. After incubation of glial cultures with [3H]mevalonolactone in the presence of mevinoline and trilostane, biosynthesis of [3H]cholesterol, [3H]pregnenolone (P) and [3H]pregn-5-ene-3 beta, 20 alpha-diol (20-OHP) was demonstrated. Steroid biosynthesis was related to oligodendroglial differentiation, as the initial and rapid rate of increase in CNPase activity was found to occur at the same time as the onset of steroid synthesis. Both reached a maximum at 3 weeks of culture and remained stable for several weeks. Steroid synthesis was increased by dibutyryl cAMP (0.2 mM), as well as by dexamethasone (10 nM). When aminoglutethimide, a potent inhibitor of cytochrome P-450scc, was added during the incubation of cells with [3H]mevalonolactone, [3H]cholesterol accumulated in the cells. After the release of aminoglutethimide blockade, [3H]20-OHP was the major steroid produced and released in the culture medium. The demonstration of de novo steroid biosynthesis and of the cholesterol side-chain cleavage cytochrome P-450 in normal rat glial cells brings additional support to the concept of "neurosteroids".     

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dst^dt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dst^dt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dst^dt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dst^dt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dst^dt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.     

Genetic galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys (Macaca mulatta)

Globoid cell leukodystrophy, or Krabbe disease, is a severe disorder of the peripheral and central nervous system myelin caused by deficient galactocerebrosidase (GALC) activity. This autosomal recessive disease affects humans and animals including dogs, mice, and rhesus monkeys. Cloning of the human and animal GALC genes opened opportunities for therapeutic trials using animal models. We describe the clinical, pathologic, and biochemical features of the affected rhesus monkey. Affected monkeys had very low GALC activity and a two base pair deletion in both copies of the GALC gene. Clinical signs of tremors, hypertonia, and incoordination led to humane euthanasia by 5 months of age. At necropsy, peripheral nerves were enlarged. Microscopically, the cerebral, cerebellar, and spinal cord white matter was infiltrated with periodic acid-Schiff-positive multinucleated globoid cells, and there was a striking lack of myelin. Peripheral nerve fibers were decreased in number and separated by Alcian blue- and safranin O-positive material. Myelin sheaths were greatly diminished. Lipid analysis of brains of 12-day-old and 158-day-old affected monkeys revealed a great excess of psychosine in white matter. The rhesus monkey model will be especially useful for exploring treatment options, including prenatal bone marrow transplantation and various approaches to gene therapy.     

Remyelination properties of human embryonic nerve cells in the course of long-term cultivation

We studied the mitogenic and differentiation potential and the remyelination properties of human embryonic nerve cells in culture. After 1 month of cultivation without differentiation agents, a 3.6-fold increase in the number of CNP-positive cells (the mitotically active precursors of oligodendrocytes) was observed. At the same time, the number of GalC-positive cells (mature oligodendrocytes) remained low. The remyelination properties of embryonic nerve cells can thus be explained by a high density of oligodendrocyte precursors. After cultivation, the population of cells maintained and enhanced their remyelination potential by increasing the number of CNP-positive cells under conditions of experimental demyelination.