Epigenome-wide base-resolution profiling of DNA methylation in chorionic villi of fetuses with Down syndrome by methyl-capture sequencing.

BACKGROUND: Epigenetic mechanisms provide an interface between environmental factors and the genome and are influential in various diseases. These mechanisms, including DNA methylation, influence the regulation of development, differentiation, and establishment of cellular identity. Here, we performed high-throughput methylome profiling to determine whether differential patterns of DNA methylation correlate with Down syndrome (DS). MATERIALS AND METHODS: We extracted DNA from the chorionic villi cells of five normal and five DS fetuses at the early developmental stage (12-13 weeks of gestation). Methyl-capture sequencing (MC-Seq) was used to investigate the methylation levels of CpG sites distributed across the whole genome to identify differentially methylated CpG sites (DMCs) and regions (DMRs) in DS. New functional annotations of DMR genes using bioinformatics tools were predicted. RESULTS: DNA hypermethylation was observed in DS fetal chorionic villi cells. Significant differences were evident for 4,439 DMCs, including hypermethylation (n = 4,261) and hypomethylation (n = 178). Among them, 140 hypermethylated DMRs and only 1 hypomethylated DMR were located on 121 genes and 1 gene, respectively. One hundred twenty-two genes, including 141 DMRs, were associated with heart morphogenesis and development of the ear, thyroid gland, and nervous systems. The genes were significantly associated with DS and various diseases, including hepatopulmonary syndrome, conductive hearing loss, holoprosencephaly, heart diseases, glaucoma, and musculoskeletal abnormalities. CONCLUSIONS: This is the first study to compare the whole-epigenome DNA methylation pattern of the chorionic villi cells from normal and DS fetuses at the early developmental-stage using MC-seq. Overall, our results indicate that the chorionic villi cells of DS fetuses are hypermethylated in all autosomes and suggested that altered DNA methylation may be a recurrent and functionally relevant downstream response to DS in human cells. This study provides basic information for future research focused on the pathophysiology of the DS and its potential effects, as well as the role DNA methylation plays in the early developmental stage of DS fetuses.

Integrative analyses of genes and microRNA expressions in human trisomy 21 placentas.

BACKGROUND: The most frequent chromosomal aneuploidy is trisomy 21 (T21) that is caused by an extra copy of chromosome 21. The imbalance of whole genome including genes and microRNAs contributes to the various phenotypes of T21. However, the integrative association between genes and microRNAs in the T21 placenta has yet to be determined. METHODS: We analyzed the expressions of genes and microRNAs in the whole genomes of chorionic villi cells from normal and T21 human fetal placentas based on our prior studies. The functional significances and interactions of the genes and microRNAs were predicted using bioinformatics tools. RESULTS: Among 110 genes and 34 microRNAs showing significantly differential expression between the T21 and normal placentas, the expression levels of 17 genes were negatively correlated with those of eight microRNAs in the T21 group. Of these 17 genes, 10 with decreased expression were targeted by five up-regulated microRNAs, whereas seven genes with increased expression were targeted by three down-regulated microRNAs. These genes were significantly associated with hydrogen peroxide-mediated programmed cell death, cell chemotaxis, and protein self-association. They were also associated with T21 and its accompanying abnormalities. The constructed interactive signaling network showed that seven genes (three increased and four decreased expressions) were essential components of a dynamic signaling complex (P = 7.77e-16). CONCLUSIONS: In this study, we have described the interplay of genes and microRNAs in the T21 placentas and their modulation in biological pathways related to T21 pathogenesis. These results may therefore contribute to further research about the interaction of genes and microRNAs in disease pathogenesis.

Lin28B and miR-142-3p regulate neuronal differentiation by modulating Staufen1 expression.

Staufen1 (STAU1) and Lin28B are RNA-binding proteins that are involved in neuronal differentiation as a function of post-transcriptional regulation. STAU1 triggers post-transcriptional regulation, including mRNA export, mRNA relocation, translation and mRNA decay. Lin28B also has multiple functions in miRNA biogenesis and the regulation of translation. Here, we examined the connection between STAU1 and Lin28B and found that Lin28B regulates the abundance of STAU1 mRNA via miRNA maturation. Decreases in the expression of both STAU1 and Lin28B were observed during neuronal differentiation. Depletion of STAU1 or Lin28B inhibited neuronal differentiation, and overexpression of STAU1 or Lin28B enhanced neuronal differentiation. Interestingly, the stability of STAU1 mRNA was modulated by miR-142-3p, whose maturation was regulated by Lin28B. Thus, miR-142-3p expression increased as Lin28B expression decreased during differentiation, leading to the reduction of STAU1 expression. The transcriptome from Staufen-mediated mRNA decay (SMD) targets during differentiation was analyzed, confirming that STAU1 was a key factor in neuronal differentiation. In support of this finding, regulation of STAU1 expression in mouse neural precursor cells had the same effects on neuronal differentiation as it did in human neuroblastoma cells. These results revealed the collaboration of two RNA-binding proteins, STAU1 and Lin28B, as a regulatory mechanism in neuronal differentiation.

Regulation of Fanconi anemia protein FANCD2 monoubiquitination by miR-302.

Fanconi anemia (FA) is a recessively inherited multigene disease characterized by congenital defects, progressive bone marrow failure, and heightened cancer susceptibility. Monoubiquitination of the FA pathway member FANCD2 contributes to the repair of replication stalling DNA lesions. However, cellular regulation of FANCD2 monoubiquitination remains poorly understood. In the present study, we identified the miR-302 cluster as a potential regulator of FANCD2 by bioinformatics analysis. MicroRNAs (miRNAs) are the major posttranscriptional regulators of a wide variety of biological processes, and have been implicated in a number of diseases. Expression of the exogenous miR-302 cluster (without miR-367) reduced FANCD2 monoubiquitination and nuclear foci formation. Furthermore, miR-302 cells showed extensive chromosomal breakage upon MMC treatment when compared to mock control cells. Taken together, our results suggest that overexpression of miR-302 plays a critical role in the regulation of FANCD2 monoubiquitination, resulting in characteristic defects in DNA repair within cells.

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells.

Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using γ-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.

Granulocyte-colony stimulating factor as a treatment for diabetic neuropathy in rat.

Effective treatment of diabetic neuropathy (DN) remains unsolved. We serendipitously observed dramatic relief of pain in several patients with painful DN receiving granulocyte-colony stimulating factor (G-CSF). The aim of this study was to determine if G-CSF could treat DN in an animal model and to ascertain its mechanism of action. In a rodent model of DN, G-CSF dramatically recovered nerve function, retarded histological nerve changes and increased the expression of neurotrophic factors within nerve. A sex-mismatched bone marrow transplantation (BMT) study revealed that G-CSF treatment increased the abundance of bone marrow (BM)-derived cells in nerves damaged by DN. However, we did not observe evidence of transdifferentiation or cell fusion of BM-derived cells. The beneficial effects of G-CSF were dependent on the integrity of BM. In conclusion, G-CSF produced a therapeutic effect in a rodent model of DN, which was attributed, at least in part, to the actions of BM-derived cells.

Changes in the methylation status of DAT, SERT, and MeCP2 gene promoters in the blood cell in families exposed to alcohol during the periconceptional period.

BACKGROUND: Alcohol exposure has been shown to cause devastating effects on neurobehavioral development in numerous animal and human studies. The alteration of DNA methylation levels in gene-specific promoter regions has been investigated in some studies of human alcoholics. This study was aimed to investigate whether social alcohol consumption during periconceptional period is associated with epigenetic alteration and its generational transmission in the blood cells. METHODS: We investigated patterns of alcohol intake in a prospective cohort of 355 pairs of pregnant women and their spouses who reported alcohol intake during the periconceptional period. A subpopulation of 164 families was established for the epigenetic study based on the availability of peripheral blood and cord blood DNA. The relative methylation changes of dopamine transporter (DAT), serotonin transporter (SERT), and methyl CpG binding protein 2 (MeCP2) gene promoters were analyzed using methylation-specific endonuclease digestion followed by quantitative real-time polymerase chain reaction. RESULTS: The relative methylation level of the DAT gene promoter was decreased in the group of mothers reporting above moderate drinking (p = 0.029) and binge drinking (p = 0.037) during pregnancy. The relative methylation level of the DAT promoter was decreased in the group of fathers reporting heavy binge drinking (p = 0.003). The relative methylation levels of the SERT gene promoter were decreased in the group of newborns of light drinking mothers before pregnancy (p = 0.012) and during pregnancy (p = 0.003). The methylation level in the MeCP2 promoter region of babies whose mothers reported above moderate drinking during pregnancy was increased (p = 0.02). In addition, methylation pattern in the DAT promoter region of babies whose fathers reported heavy binge drinking was decreased (p = 0.049). CONCLUSIONS: These findings suggest that periconceptional alcohol intake may cause epigenetic changes in specific locus of parental and newborn genomes as follows: Alcohol consumption decreases the methylation level of the DAT promoter region of the parent themselves, maternal alcohol drinking during the periconceptional period decreases the methylation level of the SERT promoter region of newborns, and maternal alcohol consumption increases the methylation level of the MeCP2 promoter region of newborns.

Metformin inhibits tumor cell migration via down-regulation of MMP9 in tamoxifen-resistant breast cancer cells.

Metformin is the most widely used anti-diabetic drug in the world. Recent evidence indicates that metformin could potentially inhibit tumorigenesis. In the present study, we found that metformin inhibited cell migration and invasion of phorbol 12-myristate 13-acetate-induced MCF-7 and tamoxifen-resistant MCF-7 breast cancer cells. This inhibition was correlated with the modulation of matrix metalloproteinase-9 (MMP9) via the suppression of its expression and proteolytic activity. These results indicate that metformin leads to the suppression of migration and invasion through regulation of MMP9 and it may have potential as an anticancer drug for therapy in human breast cancer, especially of chemoresistant cancer cells.

The RNA in reticulocytes is not just debris: it is necessary for the final stages of erythrocyte formation.

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

Effect of human parathyroid hormone on hematopoietic progenitor cells in NOD/SCID mice co-transplanted with human cord blood mononuclear cells and mesenchymal stem cells.

PURPOSE: We evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules. MATERIALS AND METHODS: Female NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1 × 10(7) human UCB-derived mononuclear cells (MNCs) and 5 × 10(6) human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH. RESULTS: Engraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance. CONCLUSION: We have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.

Human parathyroid hormone increases the mRNA expression of the IGF system and hematopoietic growth factors in osteoblasts, but does not influence expression in mesenchymal stem cells.

Osteoblasts, which are derived from pluripotent mesenchymal stem cells (MSCs), play an important role in hematopoiesis. Human parathyroid hormone (hPTH) induces osteoblasts to produce many factors that are essential to hematopoietic stem cells. However, little is known about the impact of hPTH on MSCs to enhance hematopoiesis. We determined the optimal dose of hPTH that was necessary in vitro for increased osteoblast function. In addition, we compared MSC and osteoblast function to explore the role of hPTH in hematopoiesis. The mRNA expression levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 6, stromal cell-derived factor 1, insulin-like growth factor 1 (IGF-1), IGF-2, insulin-like growth factor-binding protein 1 (IGFBP-1), IGFBP-2, and IGFBP-3 were comparable in osteoblasts and human cord blood-derived MSCs. However, G-CSF, GM-CSF, IGF-2, IGFBP-1, IGFBP-2, and IGFBP-3 expression levels in osteoblasts were markedly increased after treatment with 50 or 100 nM of hPTH. In conclusion, hPTH does not affect the ability of MSCs to differentiate into osteoblasts. In addition, hPTH may enhance hematopoiesis by activating the IGF system (IGF-2, IGFBP-1, IGFBP-2, and IGFBP-3) and hematopoietic growth factors (G-CSF and GM-CSF) in osteoblasts, but not in MSCs.

Delineation of subtelomeric deletion of the long arm of chromosome 6.

Pure subtelomeric deletion of the long arm of chromosome 6 is rare. The frequency of this deletion accounts for approximately 0.05% of subjects with intellectual disability and developmental delay with or without dysmorphic features. Common phenotypes associated with this deletion include intellectual disability, developmental delay, dysmorphic features, seizure, hypotonia, microcephaly and hypoplasia of the corpus callosum. The smallest overlapped region is approximately 0.4 Mb, and contains three known genes. Of these genes, TBP has been considered as a plausible candidate gene for the phenotype in patients with a subtelomeric 6q deletion. Analysis of the breakpoints in 14 cases revealed a potential common breakpoint interval 8.0-9.0 Mb from the chromosome 6q terminus where the FRA6E fragile site exists and the PARK2 gene is located. This suggests that breakage at the FRA6E fragile site may be the mechanism behind chromosome 6q subtelomeric deletion in some of the cases.

Genetic variation of three autosomal STR loci D21S1435, D21S1411, and D21S1412 in Korean population.

The autosomal tetranucleotide short tandem repeat loci D21S1435, D21S1411 and D21S1412 were analyzed in samples of unrelated 200 Korean individuals. The loci showed no significant deviations from Hardy-Weinberg equilibrium. Alleles were assigned according to the International Society for Forensic Haemogenetics (ISFH) recommendations. The power of discrimination of the analyzed markers was found to be high for the populations, thereby facilitating the validation and efficiency of these STR markers in forensic human identification and paternity testing. To our knowledge, this is the first report of the nomenclature and the allele frequency data for these three STR loci in Korean population.

HY251, a novel cell cycle inhibitor isolated from Aralia continentalis, induces G1 phase arrest via p53-dependent pathway in HeLa cells.

This study was aimed to elucidate the novel structure of HY251 isolated from the roots of Aralia continentalis and to evaluate its detailed inhibition mechanisms on cell cycle progression in HeLa cells. The structure of HY251 was elucidated based on the interpretation of the NMR spectra, as 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol. The flow cytometric analysis revealed an appreciable G(1) phase arrest in HeLa cells treated with 100 microM of HY251. This HY251-induced G(1) phase arrest is associated with decreased expression of cyclin D3 and up-regulation of p21(CIP1) and p27(KIP1), via p53 phosphorylation at Ser-15 by transcriptional up-regulation of ATM, which resulted in increased hypophosphorylated pRb in HeLa cells.

HY253, a novel compound isolated from Aralia continentalis, induces apoptosis via cytochrome c-mediated intrinsic pathway in HeLa cells.

This study was aimed to elucidate the novel structure of HY253 isolated from the roots of Aralia continentalis and to evaluate its detailed mechanisms on apoptotic induction in HY253-treated HeLa cells. The structure of HY253 was elucidated based on the interpretation of the NMR spectra, as 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol. The TUNEL assay using flow cytometer revealed an appreciable apoptotic induction in HeLa cells treated with 100 microM of HY253 for 48 h. This apoptotic induction is associated with cytochrome c release from mitochondria, via up-regulation of pro-apoptotic Bcl-2 proteins, such as Bax and Bak, which, in turn, resulted in the activation of caspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP).

CRBL cells: establishment, characterization and susceptibility to prion infection.

The cerebellum is involved in complex physiological functions including motor control, sensory perception, cognition, language, and emotion. Humans and animals with prion diseases are characterized clinically by ataxia, postural abnormalities and cognitive decline. Pathology in the cerebellum affected by prions includes spongiform degeneration, neuronal loss, and gliosis. To develop an in vitro model system for studying prion biology in cerebellar cells, we established and characterized an immortal cell line (CRBL) isolated from the cerebellum of mice lacking expression of a protein involved in cell cycle arrest. The characteristics of the cells include morphological heterogeneity, rapid proliferation, serum responsiveness during growth, and a change in the number of chromosomes. CRBL cells expressed both neuronal and glial cell markers as well as a considerable level of cellular prion protein, PrP(C). Upon in vitro infection, CRBL cells exhibited selective susceptibility to prions isolated from different sources. These cells chronically propagated prions from SMB cells. Strain-specific prion infection in CRBL cells was not due to instability of the cell line, allelic variance, or mutations in the PrP gene. Molecular properties of prions derived from SMB cells were maintained in the infected CRBL cells. Our results suggest that the specific interaction between a prion strain and hosts determined the selective susceptibility of CRBL cells, which reflects the conditions in vivo. In addition to the future studies revealing cellular and molecular mechanism involved in prion pathogenesis, CRBL cells will contribute to the studies dealing with prion strain properties and host susceptibilities.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

OBJECTIVE: To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. DESIGN: Randomized, prospective study. SETTING: Hospital-based assisted reproductive technology laboratory. ANIMAL(S): F(1) (C57BL/6 x 129P3/J) mice. INTERVENTION(S): Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. MAIN OUTCOME MEASURE(S): Embryogenesis and embryonic stem cell establishment. RESULT(S): The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. CONCLUSION(S): We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Discovery of cyclin-dependent kinase inhibitor, CR229, using structurebased drug screening.

To generate new scaffold candidates as highly selective and potent cyclin-dependent kinase (CDK) inhibitors, structure-based drug screening was performed utilizing 3D pharmacophore conformations of known potent inhibitors. As a result, CR229 (6-bromo-2,3,4,9-tetrahydro-carbolin-1-one) was generated as the hit-compound. A computational docking study using the X-ray crystallographic structure of CDK2 in complex with CR229 was evaluated. This predicted binding mode study of CR229 with CDK2 demonstrated that CR229 interacted effectively with the Leu83 and Glu81 residues in the ATP-binding pocket of CDK2 for the possible hydrogen bond formation. Furthermore, biochemical studies on inhibitory effects of CR229 on various kinases in the human cervical cancer HeLa cells demonstrated that CR229 was a potent inhibitor of CDK2 (IC50: 3 microM), CDK1 (IC50: 4.9 microM), and CDK4 (IC50: 3 microM), yet had much less inhibitory effect (IC50: >20 microM) on other kinases, such as casein kinase 2-1 (CK2- alpha1), protein kinase A (PKA), and protein kinase C (PKC). Accordingly, these data demonstrate that CR229 is a potent CDK inhibitor with anticancer efficacy.

Generation of FISH probes using laser microbeam microdissection and application to clinical molecular cytogenetics.

Chromosome microdissection and the reverse FISH technique is one of the most useful methods for the identification of structurally abnormal chromosomes. In particular, the laser microbeam microdissection (LMM) method allows rapid isolation of a target chromosome or a specific region of chromosomes without damage of genetic materials and contamination. Isolated chromosomes were directly amplified by the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), and then the FISH probes labeled with spectrum green- or spectrum red-dUTP were generated by nick-translation. Whole chromosome painting (WCP) probes were successfully generated from only 5 copies of the chromosome. With this method, we produced 24 WCP probes for each human chromosome. We also tried to characterize a marker chromosome, which seemed to be originated from chromosome 11 on conventional banding technique. The marker chromosomes were isolated by the LMM method and analyzed by reverse FISH. We elucidated that the marker chromosome was originated from the short arm of chromosome 5 (5p11-->pter). A fully automated and computer-controlled LMM method is a very simple laboratory procedure, and enables rapid and precise characterization of various chromosome abnormalities.

Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 x 10(4)-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.