Cells In Undifferentiated State Research Articles

Posttranscriptional Control of the Stem Cell and Neurogenic Programs by the Nonsense-Mediated RNA Decay Pathway

The mechanisms dictating whether a cell proliferates or differentiates have undergone intense scrutiny, but they remain poorly understood. Here, we report that UPF1, a central component in the nonsense-mediated RNA decay (NMD) pathway, plays a key role in this decision by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGF-β inhibitor SMAD7 and stimulating TGF-β signaling. UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Neural differentiation is triggered when NMD is downregulated byneurally expressed microRNAs (miRNAs). This UPF1-miRNA circuitry is highly conserved and harbors negative feedback loops that act as a molecular switch. Our results suggest that the NMD pathway collaborates with the TGF-β signaling pathway to lock in the stem-like state, a cellular state that is stably reversed when neural differentiation signals that induce NMD-repressive miRNAs are received.

Glioblastoma stem-like cells: approaches for isolation and characterization

Glioblastoma (GBM) stem-like cells (GSCs) represent the most undifferentiated state of malignant cells with distinct biological characteristics. The fraction of these cells within a glial brain tumor, ranging from 2-30%, is correlating with the increasingWHOstageandpoorprognosisofpatients'survival.GSCsrepresenttheleastvulnerable,thusmostpreferentialtarget cellpopulationtobeexposedtovarioustherapeuticmodalities,althoughtheunderlyingmechanismsofthisresistancearenotyet fully understood. For the development of GSC-targeting therapies, further in depth studies are needed using enrichedand stable GSCcellpopulations.Here,wediscussthecurrentapproachesofGSCisolationandvalidationbasedonexpressionofstemness and oncogenic markers as well as on functional assays. The enrichment of GSC phenotypes in established cell lines and/or primary tumor cultures, achieved by different strategies, is reviewed, providing a comprehensive comparison of selected studies and contemplating the characterization of the plethora of variants of reported GBM population exhibiting the GSC phenotype.

저산소 상태에서 우르솔산이 배아줄기세포 성장에 미치는 효과

우르솔산(Ursolic acid)은 다양한 약재, 과일 그리고 야채 등으로 부터 분리되는 식물성 생리활성물질로서, 천연 항산화제로 널리 알려져 있지만, 배아줄기세포에서 우르솔산의 기능에 대한 연구는 아직까지 잘 이해되지 않고 있다. 본 연구에서는 저산소 상태에서 우르솔산이 배아줄기세포의 성장에 미치는 효과에 대해 조사하였다. 48시간 동안의 저산소 환경은 배아줄기세포의 미분화상태 및 전능성을 유지에 있어서 영향을 미치지 않았지만, 세포 내 활성산소종의 지속적인 생산과 이로 인한 lactate dehydrogenase 활성을 촉진 시켰다. 저산소에 의해 유도된 배아줄기세포의 산화적 스트레스는 <TEX>$30{\mu}M$</TEX>의 우르솔산의 처리로 인해 유의적으로 감소되었으며, 이러한 우르솔산의 활성산소 소거능력은 항산화제인 N-acetyl-cysteine (NAC)의 효과와 유사하였다. 저산소 환경은 또한 유의적으로 배아줄기세포의 생존과 증식을 감소 시킬 뿐만 아니라, 세포의 자살 및 노화를 유도함이 관찰되었지만, 강한 항산화 능력을 지닌 우르솔산은 세포를 저산소로부터 보호하여 정상수준으로 세포의 생존과 증식을 유지시키고, 세포 자살 관련 단백질(cleaved caspase-3, Bcl-2, 그리고 cIAP) 및 세포 노화 관련 단백질(beta-galactosidase)을 조절하는 탁월한 약리학적 효과를 가지고 있었다. 따라서 본 연구결과를 통해, 우르솔산은 강력한 천연 항산화제이며, 저산소에 의해 유도된 유해 기작을 제어함으로서, 배아줄기세포의 전능성을 유지시킬 수 있는 기능성 물질이라는 것을 알 수 있었다. Ursolic acid (UA) a bio-active ingredient found in a variety of fruits and vegetables, and it has potent antioxidant activity. However, the role of UA in mouse embryonic stem (ES) cells is poorly understood. This study investigated the functional role of UA in regulating the development of mouse ES cells under hypoxia. Hypoxia did not exert a significant effect on the undifferentiated state of mouse ES cells. However, it induced reactive oxygen species (ROS) generation and increased the level of lactate dehydrogenase (LDH) production at 48 h of hypoxic exposure. Conversely, oxidative stress induced by hypoxia was significantly inhibited by UA (<TEX>$30{\mu}M$</TEX>) pretreatment. Hypoxia significantly decreased cell survival and the level of [<TEX>$^3H$</TEX>] thymidine incorporation, both of which recovered following pretreatment of UA. In addition, UA decreased the apoptotic effect of hypoxia by attenuating caspase-3 cleavage or by recovering cellular inhibition of the apoptotic protein (cIAP)-2 and Bcl-2 expression. We further found that UA decreased senescence-associated beta-galactosidase activity. We suggest that UA is a natural antioxidant and one of the functional modulators of hypoxia-induced survival, apoptosis, proliferation, and aging in mouse ES cells.

Cellular network entropy as the energy potential in Waddington's differentiation landscape.

Differentiation is a key cellular process in normal tissue development that is significantly altered in cancer. Although molecular signatures characterising pluripotency and multipotency exist, there is, as yet, no single quantitative mark of a cellular sample's position in the global differentiation hierarchy. Here we adopt a systems view and consider the sample's network entropy, a measure of signaling pathway promiscuity, computable from a sample's genome-wide expression profile. We demonstrate that network entropy provides a quantitative, in-silico, readout of the average undifferentiated state of the profiled cells, recapitulating the known hierarchy of pluripotent, multipotent and differentiated cell types. Network entropy further exhibits dynamic changes in time course differentiation data, and in line with a sample's differentiation stage. In disease, network entropy predicts a higher level of cellular plasticity in cancer stem cell populations compared to ordinary cancer cells. Importantly, network entropy also allows identification of key differentiation pathways. Our results are consistent with the view that pluripotency is a statistical property defined at the cellular population level, correlating with intra-sample heterogeneity, and driven by the degree of signaling promiscuity in cells. In summary, network entropy provides a quantitative measure of a cell's undifferentiated state, defining its elevation in Waddington's landscape.

WNT3 Is a Biomarker Capable of Predicting the Definitive Endoderm Differentiation Potential of hESCs

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs.

Analysis of oxygen-dependent cytokine expression in human mesenchymal stem cells derived from umbilical cord

Efficient cell expansion is a basic requirement for obtaining clinically relevant numbers of mesenchymal stem cells designed for cell-based therapies or tissue-engineering application. Previous studies have demonstrated that mesenchymal stem cells (MSC) cultivated under reduced atmospheric oxygen concentrations (2.5% O2) possess enhanced proliferation potential and can maintain their differentiation properties. We have analyzed the oxygen-dependent cytokine expression of human MSC derived from umbilical cord and attempted to link the results to the proliferation and differentiation capacities of these cells. By quantitative reverse transcription plus the polymerase chain reaction and by protein microarray, we measured the gene expression and intracellular protein concentration of several growth factors and growth factor receptors. Fibroblast growth factor-7, two growth factor receptors (vascular endothelial growth factor receptor 2 and stem cell factor receptor), and two growth-factor-binding proteins (insulin-like growth-factor-binding proteins 3 and 6) were over-expressed under hypoxic conditions, indicating that their signaling pathways participate in cell proliferation. On the other hand, typical differentiation factors such as bone morphogenetic protein-4, endothelial growth factor, and tissue growth factor-β1 were absent in cells cultivated under hypoxic and normoxic conditions. The absolute concentration of some intracellular cytokines was also measured for the first time under hypoxia and normoxia. Our results in combination with previous findings indicate that enhanced proliferation potential and a maintained undifferentiated cell state can be ascribed to the oxygen-dependent expression of a set of cytokines. This knowledge might help in the understanding of MSC physiology and in the achievement of directed cell fate of MSC for clinical application.

Distinct roles for N‐Cadherin linked c‐Src and fyn kinases in lens development

Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. Through inhibition of N-cadherin junction maturation, c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

Downregulated LncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression

Long noncoding RNAs (lncRNAs) are key regulators of diverse biological processes such as transcriptional regulation, cell growth and differentiation. Previous studies have demonstrated that the lncRNA-ANCR (anti-differentiation ncRNA) is required to maintain the undifferentiated cell state within the epidermis. However, little is known about whether ANCR regulates osteoblast differentiation. In this study, we found that the ANCR expression level is significantly decreased during hFOB1.19 cell differentiation. ANCR-siRNA blocks the expression of endogenous ANCR, resulting in osteoblast differentiation, whereas ANCR overexpression is sufficient to inhibit osteoblast differentiation. We further demonstrated that ANCR is associated with enhancer of zeste homolog 2 (EZH2) and that this association results in the inhibition of both Runx2 expression and subsequent osteoblast differentiation. These data suggest that ANCR is an essential mediator of osteoblast differentiation, thus offering a new target for the development of therapeutic agents to treat bone diseases.

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.

Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

BackgroundThe self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.Methodology/Principal FindingsIn this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.Conclusions/SignificanceOur study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

Generation of Tripotent Neural Progenitor Cells from Rat Embryonic Stem Cells

Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem (rES) cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem cells give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) inhibitors (two inhibitors, "2i") with feeder-conditioned medium, we successfully obtained high-quality rat embryoid bodies (rEBs) from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells (rNPCs) were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligodendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for γ-aminobutyric acid (GABA) and tyrosine hydroxylase (TH). In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson's disease and the development of rat nervous system.

Novel Evolutionary-conserved Role for the Activity-dependent Neuroprotective Protein (ADNP) Family That Is Important for Erythropoiesis

ADNP is vital for embryonic development. Is this function conserved for the homologous protein ADNP2? Down-regulation/silencing of ADNP or ADNP2 in zebrafish embryos or mouse erythroleukemia cells inhibited erythroid maturation, with ADNP directly associating with the β-globin locus control region. ADNPs are novel molecular regulators of erythropoiesis. New regulators of globin synthesis are suggested. Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse β-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased β-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of β-globin expression.

Localization of c‐MYB in differentiated cells during postnatal molar and alveolar bone development

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.

STK31 Maintains the Undifferentiated State of Colon Cancer Cells

The expression of serine/threonine kinase (STK) family is frequently altered in human cancers. However, the functions of these kinases in cancer development remain elusive. Here, we report that STK31 is robustly and heterogeneously expressed in colon cancer tissues and plays a critical role in determining the differentiation state of colon cancer cells. Knockdown or overexpression of STK31 induced or inhibited differentiation of colon cancer cells, respectively. Deletion of the STK domain abolished the inhibiting effect of STK31. Associated with differentiation, knockdown of STK31 resulted in significant suppression of tumorigenicity both in vitro and in vivo. Genome microarray analysis showed that knockdown of STK31 altered the expression profile of genes that are known to be involved in germ cell and cancer differentiation. Taken together, these results suggest that STK31 is able to control the differentiation state of colon cancer cells, which critically depends on its STK domain. The present findings may shed light on the new therapeutic approach against cancer by targeting STK31 and cancer differentiation.

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.

Suppression of progenitor differentiation requires the long noncoding RNA ANCR

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

Expression of Oct4A splicing variant in human bladder premalignant lesions predicts its invasiveness

Stem cells are immature, unspecialized cells that are able to proliferate and population self-renewal. In tumor mass, the cells with properties similar to stem cells are present. These cells, so-called cancer stem cells (CSC), are small subpopulation of cells, which can initiate the tumor and cause the recurrence of the tumor. According to published data, CSC have different level of sensitivity to chemo- and radiotherapy, dependent, i. e. on its differentiation state. It seems that complete recovery of the patients is possible only when all CSC will be eliminated. There are many of stem cell markers. In our research we analyzed the expression of OCT4A splicing variant of OCT4 (POU5F1) gene in preinvasive and malignant lesions of the bladder. OCT4A is a marker of embrional stem cells and, as transcription factor, is responsible for sustaining the cells in undifferentiated state, characteristic of the stem cell and potential to self-renewal. Our previous study suggested that its expression is observed in premalignant and malignant lesions and are consistent with results of other authors. We examine archival, buffered formalin-fixed, paraffin-embedded tissue samples of urinary bladder specimens obtained from 113 patients. Expression of OCT4A was detected using immunohistochemistry. The intensity of OCT4A immunostaining was scored as 0 (negative), 1 (faint) or 2 (distinct) and OCT4A-positive cells were analyzed as the numbers of cells per 1000 tumor cells. Based on our results, we found the most intensive and the most extensive (measured by means of percentage of OCT4A-positive cells) expression of this marker in urothelial cells at in situ stage. In papillary tumors (pTa) and in situ cancers the percentage of OCT4A-positive cells was higher when compared to less advanced invasive cancers (pT1 and pT2), whereas the percentage of OCT4A-positive cells in pTa tumors was similar to that observed in invasive tumors at pT4 stage. The observed differences were well-defined and statistically significant. To summarize, the highest number and the most intensive staining of OCT4A-positive cells in non-invasive lesions pTis and pTa could suggest the important role of OCT4A in initiation and promotion of invasive process. It cannot be excluded that, analysis and evaluation of the expression of this transcription factor can have predictive value in the prediction of progression of pre-invasive tumor to invasive form.

B-box and SPRY Domain Containing Protein (BSPRY) is Associated with the Maintenance of Mouse Embryonic Stem Cell Pluripotency and Early Embryonic Development

Mouse embryonic stem (ES) cells consist of heterogeneous populations with differing abilities to proliferate and differentiate. We previously demonstrated that the expression level of platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31 was positively correlated with the undifferentiated state of mouse ES cells. In order to screen for a novel gene(s) involved in ES cell pluripotency, we performed an oligo microarray analysis and identified that B-box and SPRY domain containing protein (BSPRY) was expressed at high levels in PECAM1-positive cells. Two splice isoforms of BSPRY, BSPRY-1 and BSPRY-2, were expressed in undifferentiated ES cells and in blastocysts. Knockdown of BSPRY-1/2 in ES cells significantly reduced the number of undifferentiated colonies and caused increased expression of primitive ectoderm marker gene Fgf5. The overexpression of BSPRY-2 reciprocally increased the number of undifferentiated ES cells in the presence of LIF. Similarly, injection of BSPRY-1/2 siRNAs into 2-cell embryos caused developmental retardation and degeneration of embryos, and a significant decrease in the number of cells, especially in the inner cell mass (ICM), was observed at the blastocyst stage. Furthermore, microinjection of a BSPRY-1 expression vector into pronuclear stage embryos resulted in an increase in the hatching blastocysts rate after 120 h of culture. These results suggest that BSPRY-1 and BSPRY-2 are associated with both ES cell pluripotency and early embryonic development.

Partners in crime: the TGFβ and MAPK pathways in cancer progression.

The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.