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 μM 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.

No abstract

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.

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 pluri potency and carcinogenesis in these cells. Key words induced pluripotent stem cell; teratoma; R-10G antibody; glycobiologyThe generation of induced pluripotent stem cells (iPSC) by the forced expression of the exogenous transcription factors, Oct3/4, Sox2, c-Myc and Klf4, in mouse embryonic fibroblasts (MEFs) by Yamanaka's group paved way for a new era in stem cell research and regenerative medicine.1,2) Using reprogramming factors, iPSC have been successfully generated from humans and other mammalian species such as the rat [3][4][5] and monkey.1)The rat is utilized vastly as a model in pharmacology, toxicology, immunology research fields and behavioral science and has been useful in modeling human neural and cardiac system diseases.2) Rat iPSC lines have been derived from a variety of somatic cells mainly via viral transduction of reprogramming factors.3,4) The successful generation of transgenic rats using riPSC shows great promise in their use. 5)Variation in iPSC properties are widely reported 6) and definitive evaluation systems to assess riPSC pluripotency, differentiation propensity and potential for tumorigenesis still remain a challenge to the field. Glycans are considered to be ideal targets for identifying and analyzing cellular phenotype and cell surface epitopes such as stage-specific embryonic antigens (SSEA)-3/4 and tumor rejection antigen (TRA)-1-60/81 are conventionally used to evaluate pluripotency. However, these epitopes are also expressed in embryonal carcinoma (EC) cells. 7,8) In previous work, we generated the monoclonal antibody, R-10G, which recognizes a keratan sulfate epitope with unique structure on podocalyxin in human iPSC and human embryonal stem (ES) cells. 9) Notably, however, the antibody did not react with human EC cells that are known to be of a tumorigenic nature.Here we describe generation of...

No abstract