The conserved THO/TREX complex is participated in pre-mRNA processing and mRNA nuclear export. In Metazoa, it has been reported that THO/TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent manner; however, how THO/TREX functions is poorly understood. To understand the role of THO/TREX in eukaryotic gene expression, we investigated the role of THO/TREX in Drosophila germline, and found that lack of THOC5, a component of THO/TREX, showed defects in the biogenesis of piRNA, a distinct class of small non-coding RNAs that control expression of transposable elements (TE) in the Drosophila germline. Genome wide RNA-seq showed that THOC5 and other TREX components are essential for the biogenesis of piRNA. THO/TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and co-localize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depends on Cutoff, a protein associated with chromatin of piRNA clusters. We also show that TREX is required for accumulation of nascent piRNA precursors, suggesting that TREX is required for their efficient transcription. In addition to the biogenesis of piRNA, both THO and piRNA mutants showed over-proliferation of germline stem cell-like cells outside of stem cell niche. Taken together our study reveals a novel splicing-independent mechanism for THO/TREX loading on nascent RNA and its importance in piRNA biogenesis as well as a role of piRNA in the differentiation of germline stem cell.

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCGprimed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Transforming growth factor (TGF) family is well known to induce the chondrogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondrogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondrogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondrogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondrogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

Primary adenocarcinoma is a rare neoplasm of the urinary bladder, constituting approximately 2.5% of all primary bladder malignancies. Signet ring cell variant is even rarer, constituting approximately 0.5% to 2.0% of all primary carcinoma of the urinary bladder. However, glandular differentiation in the urinary bladder is relatively more common than primary adenocarcinoma of the urinary bladder. Therefore, diagnosis of primary adenocarcinoma of the urinary bladder can be very challenging, but of significant clinical importance. We report on a case of adenocarcinoma with mucinous and signet ring cell differentiation in the bladder dome in a 49-year-old female.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Brown algae is variety of biological compounds, including xanthophyll, pigments, fucoidans, phycocolloids, and phlorotannins. Several studies concerning these types of compounds have pointed out the variety of biological benefits associated with the algae, including antioxidant, anticoagulant, antihypertension, antibacterial, and antitumor activities. Diphlorethohydroxy- carmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. Numerous studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied that effect of DPHC isolated from Ishige okamurae modified the accumulation of fat on preadipocyte, 3T3-L1 cells in vitro. First, the viability of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Second, proliferation of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment through measure doubling time. 3T3-L1 cell differentiation into adipocyte was analyzed after induction in the induction medium containing DPHC. The metabolic activity was suppressed by DPHC in concentration dependent manner. Doubling of 3T3-L1 was delayed by the treatment of DPHC in concentration dependent manner. DPHC also inhibit accumulation of triglyceride in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC suppress proliferation of adipose precursor cell and differentiation into adipocytes.

This study was conducted to investigate the effects of Woohwangcheungsimweun (ox bezoar), deer antlers, and wild ginseng on induction of cardiomyocyte differentiation using the established mouse embryonic stem (ES) cells. The expression of atrial natriuretic peptide (ANP) was highest in Woohwangcheungsimweun treatment group. The expression of rabbit anti-GATA-4(GATA-4) and troponin (TnI) were highest in wild ginseng and Woohwangcheungsimweun treatment groups, respectively. Fluorescence activated cell sorting (FACS) analysis showed that the expression of ANP was highest in Dimethyl sulfoxide(DMSO) and Woohwangcheungsimweun treatment groups. The expression of GATA-4 was relatively high in wild ginseng treatment group. The expression of TnI was highest in Woohwangcheungsimweun treatment group. In the gene expression analysis, DMSO greatly inhibited GATA-4 expression to 25% of control. Woohwangcheungsimweun treatment caused to increase cTnI and cardiac ANP expression significantly. Wild ginseng extract upregulated GATA-4 gene expression. In conclusion, DMSO widely used as cardiomyocyte differentiation inducer did not show significant effects on the expression of ANP, GATA-4 and TnI in this study. Woohwangcheungsimweun showed upregulation of ANP and TnI expression. Wild ginseng extract showed greater effects than DMSO on GATA-4 expression. These results might suggest that the combination of Woohwangcheungsimweun and wild ginseng extract treatment can be expected to increase expressions of all three genes.

Neural precursor cells (NPCs) with abilities to self-renew and differentiate into neurons are born in the subventricular zone of the hippocampus and the subgranular zone in the adult mammalian brain. NPCs maintain their population by symmetric cell division and neuronal cell differentiation started by asymmetric cell division. Asymmetric cell division produces two daughter cells with different cellular fates. It has been shown that multiple transcription factors, like homeodomain transcription factors and basic helix loop helix (bHLH) transcription factors, play cruel role in cell fate determination (Bertrand et al., 2002). Multipotent cortical progenitors are maintained in a proliferative state by bHLH factors including Id and Hes families. The transition from proliferation to neurogenesis involves a coordinate increase in the activity of proneural bHLH factors (Mash1, Neurogenin1, and Neurogenin2). As development proceeds, inhibition of proneural bHLH factors in cortical progenitors promotes the formation of astrocytes. Finally, the formation of oligodendrocytes is triggered by an increase in the activity of bHLH factors Olig1 and Olig2 that may be coupled with a decrease in Id activity. Thus, bHLH factors have key roles in corticogenesis, affecting the timing of differentiation and the specification of cell fate. Hes1 is a vertebrate homologue of the Drosophila bHLH protein Hairy, originally known as a transcriptional repressor that negatively regulates neuronal differentiation. Hes1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene Mash1, a bHLH activator homologus to the proneuronal Achaete-Scute genes in Drosophila (Campuzano and Modolell, 1992). Down regulation of Hes1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neural differentiation. Expression of Hes1 inhibited neurite outgrowth, whereas Mash1 expression increased neurite outgrowth. Mash1 can induce bipolar neuron differentiation (Tomita et al., 1996) and NSCs culture obtained from Mash1-/- mice cannot differentiate into GBAergic neurons (Oishi et al., 2009) Hes1 is an essential effector for Notch signaling, which regulates the maintenance of undifferentiated cells (Artavanis-Tsakonas et al., 1999). In contrast, it is previously reported that platelet-derived growth factor induces the expression of Mash1 mRNA by regulating the phosphorylation of Hes1 and TLE1 (Ju et al., 2004). Hes1 is required for neuronal differentiation in PDGF treated NSC cultures. The major cell types in the cerebral cortex and hippocampus are the glutamatergic neurons and the GABAergic neurons. Cholinergic neurons are important in spatial learning and memory formation and depleted in patient’s brain of early Alzheimer’s disease. It has not been clear, however, whether new born adult NPCs could generate different cell types of neurons with distinct cellular and physiological properties. During the development, glutamatergic neurons consisting of radially migrating neurons are originated from the ventricular zone of the dorsal telenchephalon (pallium) and give rise to pyramidal neurons. Glutamate and glutamate receptors are involved in cognitive functions by forming major excitatory network. GABAergic neurons in the neocortex and hippocampus are in part migrated from the ventral telenchephalon or from the dorsal NPCs and function as local interneurons by forming inhibitory networks which regulate large populations of glutamatergic pyramidal neurons. During the development, spatiotemporal gene expression regulated by extracellular signaling factors is believed to determine the formation of neuronal phenotypes. Platelet derived growth factor B is known to induce the differentiation into neurons rather than glial cells in the rat NPCs. We found that platelet derived growth factor B is expressed in dorsal cortex and hippocampus more than in ventral cortex in the period of pyramidal cell differentiation of the embryonic rat brain. It indeed induces cell type specific differentiation into glutamatergic cells that produce the glutamate transpoter, vGluT1 and glutamate at the late stage of differentiation although it promotes neuronal differentiation at the early stage in NPCs primarily cultured from the rat embryonic hippocampus. Brain-derived neurotrophic factor, however, facilitated GABAergic differentiation in the hippocampal NPCs that generate glutamatergic pyramidal cells in a similar manner. We also found many transcriptional factors such as homeobox genes (Dlx1, Nkx2.1, Pax6) and bHLH genes (NeuroD, Ngn1, Hes1) are involved in cell type specific differentiation into glutamatergic, GABAergic, and cholinergic cells. We observed the expression of Pax6, homeodomain transcription factor, and Hes1, bHLH transcription factor, increased during PDGF-induced early differentiation in neural stem cells. These transcription factors, however, are also expressed in differentiated neurons with specific phenotype at late differentiation stage. We found pax6 is expressed in cholinergic neurons in the adult brains and in cultures. Phosphorylation of neurogenic transcription factors by protein kinases has been reported as predominant strategy in gene regulation during neuronal development and these regulated activities of different transcription factors are known to be involved in cell fate determination. Homeodomaininteracting protein kinases2 (HIPK2) which belongs to HIPK family has been identified as a nuclear serine-threonine kinase and is known to interact with several transcription factors to regulate gene transcriptions. Among several transcription factors, HIPK2 is mainly reported to target the homeodomain transcription factors such as Nkx and Pax6. Considering the importance of homeodomain transcription factors in neurogenesis and differentiation, HIPK2 also seem to play critical roles in those transcriptional regulations during embryogenesis. To define the roles of HIPK2 in neuronal differentiation during embryonic development, we investigated the expression patterns of neurogenic transcription factors such as Pax6, Hes1 and Mash1 in HIPK2 overexpressing NSCs. Hes1 showed different expression patterns between the wild type and mutant HIPK2 overexpressed cells and Mash1, which is reported to be repressed by Hes1, also showed altered expression patterns. We detected the mRNA expression of Hes1 is upregulated by HIPK2 during neuronal differentiation. The overexpressed Pax6 induced differentiation of neural stem cells into cholinergic neurons and suppressed differentiation into GABAnergic neuron both in vitro and in vivo transplantation study. To evaluate the effect of Pax6 on the transcriptional activation of Hes1 promoter, we performed luciferase reporter assay in NIH3T3 cells. Reporter expression of Hes1 promoter was enhanced upon stimulation with wild type Pax6 and wild type HIPK2. Furthermore, the HDAC inhibition mediated by TSA(Trichostatin A) has been shown to repress the reporter expression. The treatment of TSA increased neurofilaments and GAD expression in E14.5 cortical neuronal cell. These findings suggest that Pax6 promotes neuronal subtype differentiation via regulation of Hes1 bHLH transcription factor, which is mediated by HDAC. To examine the effect of Pax6 and HIPK2 on the transcriptional activation of Hes1, efficiency of hes1 promoter was measured by a luciferase reporter assay. When DNA constructs encoding Pax6 and HIPK2 were transfected along with Hes1 promoter, the expression of the reporter was highly increased. Furthermore, the HDAC inhibition mediated by TSA(Trichostatin A) repressed the reporter expression. Interaction of Pax6 and HIPK2 was shown by co-immunoprecipitation and binding of Pax6 to hes1 promoter was detected by chromatin immunoprecipitation. I also found overexpression of HIPK2 and Pax6 facilitated neural stem cells to differentiate into cholinergic cell fate in NSCs primarily cultured from the rat hippocampus. This is also supported by analysis of the brains of sey/neu Pax6 mutant mice and HIPK2 knock out mice. These findings suggest that Pax6 activation by HIPK2 promotes neuronal subtype differentiation via up regulation of Hes1 and down regulation of Mash1 and it is mediated by HDAC.

Ursolic acid, triterpenoid compound has been shown to stimulate osteoblast differentiation and enhance bone formation. In the present study, we examined the effects of similar triterpenoid compounds, oleanolic acid (OA) and its derivatives, such as oleanolic acid acetate (OAA) and oleanolic acetate methyl ester (OAM) on the bone formation in MC3T3-E1 osteoblast cells. We determined cellular proliferation, alkaline phosphatase (ALP) activity, mineralization, and expression of osteoblast specific genes and mitogen activated protein kinase phosphorylation. Treatment of 0.1-10μm OA, OAA, and OAM increased cellular proliferation, but not significantly increased as compared with dimethyl sulfoxide (DMSO). OA, OAA, and OAM at 5uM concentration enhanced ALP expression, mineralization, and osteocalcin (OCN) mRNA level. In conclusion, OA and its derivatives stimulated the osteoblast differentiation by increasing ALP, mineralization, and OCN mRNA expression. However, there were no significantly difference on osteoblast differentiation among treatment of OA, OAA, and OAM.

Estrogens are ubiquitous signaling molecules that influence nearly every cell type, and exert profound effects on embryonic development, and differentiation. Wnt pathway, which recruits β-catenin into nuclei, and activates The Wnt-dependent transcription factors, also plays an important role in embryonic development and stem cell maintenance, and differentiation. Accumulating evidences indicate that potential convergence between these two pathways in carcinoma cells. However, physiological roles of estrogens in development and differentiation of human embryonic stem cells (hESCs) are relatively unknown. Here, we demonstrated that estrogenic compounds 17α-ethinylestradiol (EE2) and genistein (GEN) significantly increased β-catenin expression in undifferentiated hESCs cultured in feeder-free media. Interestingly, GEN treatement induced an increased trend of mesendodermal gene expressions, and significantly inhibited ectodermal gene expressions (Nestin and Pax6) in embrioid body (EB). Expectantly, GEN increased epithelial-mesenchymal transition (EMT) related gene expression (Snail2, and Twist), whereas decreased E-cadherin on day 6 of EB development. Taken together, these suggest that estrogens may in part the powerful effects on normal hESC differentiation. Mechanistic studies of estrogen signaling continue to suggest novel drug targets for stem cells and will also improve screening methods of developmental toxicity.

난자 및 정자형성과정은 성에 따라 차이가 일부 있지만 생식줄기세포 (germ-line stem cells)의 증식을 통해 그 수를 유지하고, 그 중 일부 또는 전부가 감수분열에 진입하여 생식세포인 정자와 난자를 생산한다. 따라서 생식세포의 형성과정은 세포의 증식과 성장, 분화 기작을 연구할 수 있는 매우 훌륭한 시스템으로 여겨져 과거로부터 많은 연구가 진행되어왔다. 더욱이 인간에서 보조생식술과 동물 생명공학의 발전을 통해 그 중요성은 더욱 커지고 있으나, 그 체외배양 모델시스템이 없어 최근까지의 연구에는 많은 어려움이 있었다. 과거에 많은 연구자들에 의해 생식세포의 배양, 정소와 난소의 기관 배양 등이 시도된 바 있으나, 대부분 단기간 내 성공일 뿐 장기간 배양에는 어려움이 있었다. 하지만 교토대학의 Shinohara 교수팀에 의해 체외에서 정원줄기세포의 장기간 증식 배양법이 확립되었고, 또 배양된 정원줄기세포가 정소 세정관 내에 이식을 통해 정자형성과정을 재현할 수 있음이 확인되어 이 분야의 연구와 응용연구는 활성화되기 시작했다. 또한, 최근 들어 배아줄기세포, 성체줄기세포와 같은 여러 종류의 전분화능 줄기세포에서 생식줄기세포 또는 생식세포로의 분화가 일부 성공됨에 따라 생식세포의 증식과 분화의 조절기작이 일부 밝혀지고 있다. 따라서 이러한 연구성과 는 현재까지 치료가 불가능한 비폐쇄성 무정자증환자의 불임치료 방법으로의 발전 가능성을 제시할 뿐 아니라 동물생명공학분야로의 응용가능성을 더욱 높여주고 있다. 본 연제에서는 실험동물과 인간의 줄기세포로부터 생식세포의 분화에 관한 최근 연구를 정리하고, 이를 이용한 기초연구, 산업, 임상치료에의 적용가능성을 제시하고자 한다.

최근 골수와 혈액으로 유래된 중간엽 줄기세포와 비슷한 능력을 가지는 것으로 알려진 지방 유래 중간엽줄기세포가 새로운 세포 치료제로 떠오르고 있다. 하지만 줄기세포를 이용하여 치료하려는 질병은 나이가 들어감에 따라 발병하는 퇴행성 질환들이 대부분인데, 노화가 진행됨에 따라 줄기세포의 능력이 차이가 있다고 알려져 있다. 이에 본 연구에서는 노화가 일어남에 따라 발생되는 신경성 질환을 자가 유래 지방 중간엽 줄기세포를 이용하여 치료함에 있어서 노화가 진행됨에

최근 제 1형 당뇨병을 치료하기 위하여 인슐린-분비성 세포를 이식하는 세포대체요법이 새로운 치료법으로서 주목받고 있다. 그럼에도 불구하고 췌장세포 이식술은 이식원의 절대적인 부족으로 인해 광범위한 시행이 이루어지지 못하고 있는 실정이다. 무한증식과 전분화능을 보유하는 배아줄기세포는 이식할 -세포의 부족을 해결할 수 있는 잠재적 세포공급원이 될 수 있을 것으로 기대된다. 본 종설에서는 인간배아줄기세포로부터 췌장 -세포로의 유도분화방법에 관한 최근 동향을