HDAC8 regulates neural differentiation through embryoid body formation in P19 cells.

Histone acetylation and deacetylation correlate with diverse biological phenomena through gene transcription. Histone deacetylases (HDACs) regulate deacetylation of histones and other proteins. However, as a member of the HDAC family, HDAC8 function during neurodevelopment is currently unknown. Therefore, we investigated HDAC8 function during neurodevelopment by examining embryoid body (EB) formation in P19 cells. HDAC8-selective inhibitor (NCC-149) (HDAC8i)-treated cells showed smaller EBs than non-treated cells, as well as reduced expression levels of the neuronal marker, NeuN. Additionally, HDAC8i treatment led to inhibition of cellular proliferation by G2/M phase accumulation and downregulated cyclin A2 and cyclin B1 gene expression. Furthermore, two independent HDAC8 knockout cell lines were established by CRISPR-Cas9, which resulted in smaller EBs, similar to HDAC8i-treated cells. These results suggest that HDAC8 regulates neural differentiation by exerting control of EB formation.

Generation of rat induced pluripotent stem cells using a plasmid vector and possible application of a keratan sulfate glycan recognizing antibody in discriminating teratoma formation phenotypes.

Induced pluripotent stem cells (iPSCs) offer an invaluable tool for biological research and regenerative medicine. We report establishment of rat iPSCs (riPSCs) using a plasmid vector encoding four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. Although all riPSC clones were generated and cultured under the same conditions, expressed hallmark pluripotency markers and differentiated successfully in vitro, the expression of a keratan sulfate glycan epitope with unique properties defined by R-10G antibody varied in the riPSC clones. In contrast, tumor rejection antigen (TRA)-1-81 epitope expression was comparable. A clone highly reactive to R-10G antibody formed teratomas in vivo consisting of cells from all three germ layers. However, clones expressing a lower level of the epitope defined by R-10G resulted in tumors with rapid growth consisting of undifferentiated cells. Additionally, riPSCs could be successfully differentiated into a neuronal lineage including glutamate neurons that responded to agonist stimulation. These observations demonstrate a glycophenotypic difference that may potentially serve as a useful probe for riPSC evaluation and to study the role of glycans in pluripotency and carcinogenesis in these cells.

Allele-specific real-time polymerase chain reaction as a tool for urate transporter 1 mutation detection.

Allele-specific polymerase chain reaction (ASPCR) method has long been applied for the detection of nucleotide variations and genotyping, which are detected by the presence or absence of DNA amplification PCR products. Recently, Real-Time PCR genotyping has fast developed and offered a rapid method of detecting mutations without the need of gel electrophoresis as with ASPCR. Here, we describe an easy and rapid touchdown real-time PCR method for the detection of nucleotide variations. Using our method we successfully detect two main mutations in human urate transporter 1 (SLC22A12), W258X and R90H, and validate the results. The method can potentially be applied to genotype of various other nucleotide variations.

Simple and rapid detection method for the mutations in SLC22A12 that cause hypouricemia by allele-specific real-time polymerase chain reaction.

BACKGROUND: Hypouricemia is a disorder that serum urate level is less than 2.0 mg/dl, and relatively common in the Japanese population, where the main genetic cause of hypouricemia is W258X and R90H mutations in human urate trasnsporter 1(SLC22A12). Small scale screening has relied on time-consuming traditional ways like polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Therefore, it is beneficial that we have an easy and rapid detection method for these mutations. METHODS: In this report, we established a touchdown allele-specific real-time polymerase chain reaction (ASPCR) assay for detecting W258X and R90H mutations in SLC22A12, respectively. RESULTS: Quantifiable discrimination was successfully achieved by ∆Ct value. Furthermore, we conducted W258X and R90H screening against 120 control genome sets, whereby frequency was 2.92% for W258X, and not detected for R90H, respectively. CONCLUSIONS: The two mutations, W258X and R90H in SLC22A12 were successfully genotyped by an easy and rapid ASPCR assay.

Promotion of neuronal differentiation through activation of N-methyl-D-aspartate receptors transiently expressed by undifferentiated neural progenitor cells in fetal rat neocortex.

Neural progenitor cell is a generic term for undifferentiated cell populations composed of neural stem, neuronal progenitor, and glial progenitor cells with abilities for self-renewal and multipotentiality. In this study, we have attempted to evaluate the possible functional expression of N-methyl-D-aspartate (NMDA) receptors by neural progenitor cells prepared from neocortex of 18-day-old embryonic rats. Cells were cultured in the presence of basic fibroblast growth factor (bFGF) for different periods up to 12 days under floating conditions. Reverse transcription-polymerase chain reaction and fluorescence imaging analyses revealed transient expression of functional NMDA receptors in neurospheres formed by clustered progenitors during the culture with bFGF. A similarly potent increase was seen in the fluorescence intensity after brief exposure to NMDA in cells differentiated after the removal of bFGF under adherent conditions, and an NMDA receptor antagonist invariably prevented these increases by NMDA. Moreover, sustained exposure to NMDA not only inhibited the formation of neurospheres when exposed for 10 days from day 2 to day 12 but also promoted spontaneous and induced differentiation of neurospheres to cells immunoreactive for a neuronal marker protein on immunocytochemistry and Western blotting analyses. These results suggest that functional NMDA receptors may be transiently expressed to play a role in mechanisms underlying the modulation of proliferation along with the determination of subsequent differentiation fate toward a neuronal lineage in neural progenitor cells of developing rat neocortex.

Group III metabotropic glutamate receptor activation suppresses self-replication of undifferentiated neocortical progenitor cells.

We evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, L-2-amino-4-phosphonobutyrate, drastically reduced proliferation activity at 1-100 microM without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by L-2-amino-4-phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self-renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors.