Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.
Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; Cells, Cultured ; DNA-Binding Proteins/genetics/*metabolism ; E-Box Elements ; Gene Expression Regulation/physiology ; Helix-Loop-Helix Motifs ; Human ; Islets of Langerhans/cytology/metabolism ; Mice ; Molecular Sequence Data ; Nerve Tissue Proteins/genetics/*metabolism ; Neurons/cytology/metabolism ; Organ Specificity ; Transcription Factors/genetics/*metabolism ; Two-Hybrid System Techniques

We correct a typo in the title.

Reelin, an extracellular glycoprotein has an important role in the proper migration and positioning of neurons during brain development. Lack of reelin causes not only disorganized lamination of the cerebral and cerebellar cortex but also malpositioning of mesencephalic dopaminergic (mDA) neurons. However, the accurate role of reelin in the migration and positioning of mDA neurons is not fully elucidated. In this study, reelin-deficient reeler mice exhibited a significant loss of mDA neurons in the substantia nigra pars compacta (SNc) and a severe alteration of cell distribution in the retrorubal field (RRF). This abnormality was also found in Dab1-deficinet, yotari mice. Stereological analysis revealed that total number of mDA neurons was not changed compared to wild type, suggesting that the loss of mDA neurons in reeler may not be due to the neurogenesis of mDA neurons. We also found that formation of PSA-NCAM-positive tangential nerve fibers rather than radial glial fibers was greatly reduced in the early developmental stage (E14.5) of reeler. These findings provide direct evidence that the alteration in distribution pattern of mDA neurons in the reeler mesencephalon mainly results from the defect of the lateral migration using tangential fibers as a scaffold.
Animals ; Brain ; Cerebellar Cortex ; Dopaminergic Neurons ; Glycoproteins ; Mesencephalon ; Mice ; Mice, Neurologic Mutants ; Nerve Fibers ; Neurogenesis ; Neurons ; Substantia Nigra

Neural stem cells are multipotent stem cells that can differentiate into neurons and glial cells. Neural stem cells are found in not only developing nervous system but some restricted regions in adult brain. Here, we presented an effective method that allows a long-term preservation of neural stem cells without losing multipotency. First, we isolated neural stem cells from the developing forebrain of nestin-EGFP transgenic mice carrying green fluorescence protein (GFP) driven by nestin promoter and enhancer. Primary neurospheres isolated from these mice highly expressed GFP. The expression of GFP in neurospheres was sustained for several passages. In order to investigate the effect of freezing on the stem cell properties, we cryopreserved the primary neurospheres for 2 wks in liquid nitrogen. GFP expression pattern as well as differentiation potential of the secondary neurosphere formed after cryopreservation were not that different from those of the primary neurosphere formed before cryopreservation. When the same cryopreservation method was applied to neural stem cells isolated from human fetal brain (gestation 13 ~15 wks), the expression of nestin, a stem cell marker, and differentiation patterns were not changed after cryopreservation. We also performed isolation of neural stem cells from long-term cryopreserved human fetal brain tissues. The neurospheres were successfully formed and showed similar differention properties with neurospheres isolated from fresh brain tissue. In addition, we demonstrated multipotentiality of neural stem cells was not changed with the duration of cryopreservation of brain tissue, suggesting the self renewality and multipotentiality of neural stem cells were not affected by long-term cryopreservation, The present results provide an useful information for the development of stem cell expansion which is essential factor in clinical application of stem cells.
Adult ; Animals ; Brain ; Cryopreservation* ; Fluorescence ; Freezing ; Humans ; Mice ; Mice, Transgenic ; Multipotent Stem Cells ; Nervous System ; Nestin ; Neural Stem Cells* ; Neuroglia ; Neurons ; Nitrogen ; Prosencephalon ; Stem Cells

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established an efficient method for gene delivery into these cells. Non-viral transfection reagents that were commercially available yielded 5% efficiency. In contrast, a retroviral vector yielded more than 46% transduction, which was further increased to 90% by repetitive infection. Retroviral transduction did not alter the multipotency of hMSCs. Thus, the cells retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineages. The conditions established in this study will contribute to development of trans-differentiation methods of hMSCs into non-mesodermal lineage cells and thereby facilitate their possible use as vehicles for autologous transplantation in both cell and gene therapy for various diseases.
Autografts ; Genetic Therapy ; Humans* ; Indicators and Reagents ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Retroviridae ; Transfection ; Transplantation, Autologous ; Zidovudine*

The profile of membrane currents was investigated in differentiated neuronal cells derived from human neural stem cells (hNSCs) that were obtained from aborted fetal cortex. Whole-cell voltage clamp recording revealed at least 4 different currents: a tetrodotoxin (TTX)-sensitive Na+ current, a hyperpolarization-activated inward current, and A-type and delayed rectifier-type K+ outward currents. Both types of K+ outward currents were blocked by either 5 mM tetraethylammonium (TEA) or 5 mM 4-aminopyridine (4-AP). The hyperpolarization-activated current resembled the classical K+ inward current in that it exhibited a voltage-dependent block in the presence of external Ba2+ (30micrometer) or Cs+ (3micrometer). However, the reversal potentials did not match well with the predicted K+ equilibrium potentials, suggesting that it was not a classical K+ inward rectifier current. The other Na+ inward current resembled the classical Na+ current observed in pharmacological studies. The expression of these channels may contribute to generation and repolarization of action potential and might be regarded as functional markers for hNSCs-derived neurons.
4-Aminopyridine ; Action Potentials ; Humans ; Membranes ; Neural Stem Cells ; Neurons ; Tetraethylammonium ; Tetrodotoxin

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established conditions that allowed a long term expansion of hMSCs. To search for the optimum culture condition, growth rates of hMSCs were measured in the presence of several growth factors. Hepatic growth factor (HGF) and leukemia inhibitory factor (LIF) did not facilitate proliferation of hMSCs. In contrast, basic fibroblast growth factor (bFGF) effectively promoted growth of the cells in vitro by 3 fold. The growth stimulatory effect of bFGF was dependent on the concentration. The adipogenic potential was dramatically decreased in hMSCs isolated from an aged donor whereas osteogenic potential was minimally decreased. Addition of bFGF resumed the adipogenic and osteogenic differentiation potential. Thus, the cells that expanded in the presence of bFGF retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineage cells. MSCs could be expanded for at least 8 passages with bFGF and the resulting cells retained the normal karyotype. The cells were positive for CD9, CD13, CD15, CD90, CD137, and CD140b; but negative for CD14, CD34, and CD45. Importantly, the cells were found to express a neural stem cell marker, nestin, and a neuronal marker, beta-tubulin III. The results suggest that bFGF promote proliferation while maintaining multi-lineage differentiation potency of hMSCs. Finally, we suggest that it is critical to identify novel markers other than nestin or beta-tubulin III to monitor acquisition of neuronal phenotypes by hMSCs.
Fibroblast Growth Factor 2* ; Humans* ; Intercellular Signaling Peptides and Proteins ; Karyotype ; Leukemia Inhibitory Factor ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Nestin ; Neural Stem Cells ; Neurons ; Phenotype ; Tissue Donors ; Tubulin

NeuroD/BETA2, a basic helix-loop-helix transcription factor, has been known to play a role in terminal differentia-tion during neurogenesis. To gain further insight into the function of NeuroD/BETA2 in the nervous system devel-opment, we examined the expression pattern of NeuroD/BETA2 during embryonic and postnatal development by in situ hybridization. Dynamic changes of NeuroD/BETA2 expression were observed in the developing nervous system. Gene expression of the NeuroD/BETA2 in developing cerebellum and hippocampus increased during the embryonic stages and persisted throughout postnatal development and remained at a stable level in the adult brain. NeuroD/ BETA2 expression was detected in postmitotic cells in the subventricular zone of the cerebrum during embryogenesis. This observation confirms that NeuroD/BETA2 may have a role in terminal differentiation during neurogenesis.
Adult ; Animals ; Brain ; Central Nervous System* ; Cerebellum ; Cerebrum ; Embryonic Development ; Female ; Gene Expression* ; Hippocampus ; Humans ; In Situ Hybridization ; Mice* ; Nervous System ; Neurogenesis ; Pregnancy ; Transcription Factors

Human central neurocytoma is a kind of the brain tumors that are usually found in anterior part of the lateral ventricles. In this study, we established conditions that allowed proliferation of neurocytoma cells culture and analyzed characteristics of neurocytoma cells in vitro. For in vitro, a condition that used for culturing neural stem cells and contained basic fibroblast growth factor (bFGF) provided high proliferation. RT-PCR analaysis showed that nestin was found in neurocytoma cells, indicating that the neurocytomas possess neural stem cell properties. Interestingly, treatment of neurocytoma cells with forskolin increased expression of glial fibrillary acidic protein with a concomitant decrease in the nestin expression. Forskolin also induced morphological changes of neurocytoma cells to adopt an astrocyte-like phenotype. The results suggest that neurocyotma cells may have properties of multipotent neural stem cells.
Animals ; Astrocytes/cytology/*physiology ; Cell Differentiation/*drug effects ; Cell Proliferation ; Cell Shape ; Fibroblast Growth Factor 2/pharmacology ; Forskolin/*pharmacology/*therapeutic use ; Humans ; Intermediate Filament Proteins/metabolism ; Nerve Tissue Proteins/metabolism ; Neurocytoma/*drug therapy/pathology ; Research Support, Non-U.S. Gov't ; Tumor Cells, Cultured

Stroke is one of the common causes of death and disability. Despite extensive efforts in stroke research, therapeutic options for improving the functional recovery remain limited in clinical practice. Experimental stroke models using genetically modified mice could aid in unraveling the complex pathophysiology triggered by ischemic brain injury. Here, we optimized the procedure for generating mouse stroke model using an intraluminal suture in the middle cerebral artery and verified the blockage of blood flow using indocyanine green coupled with near infra-red radiation. The first week after the ischemic injury was critical for survivability. The survival rate of 11% in mice without any treatment but increased to 60% on administering prophylactic antibiotics. During this period, mice showed severe functional impairment but recovered spontaneously starting from the second week onward. Among the various behavioral tests, the pole tests and neurological severity score tests remained reliable up to 4 weeks after ischemia, whereas the rotarod and corner tests became less sensitive for assessing the severity of ischemic injury with time. Further, loss of body weight was also observed for up 4 weeks after ischemia induction. In conclusion, we have developed an improved approach which allows us to investigate the role of the cell death-related genes in the disease progression using genetically modified mice and to evaluate the modes of action of candidate drugs.
Animals ; Anti-Bacterial Agents ; Body Weight ; Brain Injuries ; Brain Ischemia ; Cause of Death ; Disease Progression ; Indocyanine Green ; Ischemia ; Mice* ; Middle Cerebral Artery ; Stroke* ; Survival Rate ; Sutures ; Therapeutic Human Experimentation