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In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.

Κατά τη διάρκεια των αρχικών σταδίων της ανάπτυξης του ποντικού, πλειοδύναμα κύτταρα εντοπίζονται στην προεμφυτευτική, πρώϊμη επιβλάστη, αναμένοντας μετεμφυτευτικά (όψιμη επιβλάστη-πρωτογενές εξώδερμα) την ενεργοποίησή τους από σηματοδοτικά μόρια, για να δώσουν τα τρία βλαστικά δέρματα κατά τη διάρκεια της γαστριδίωσης. H σημασία της in vitro δημιουργίας κυττάρων που προσομοιάζουν με την επιβλάστη από εμβρυονικά βλαστικά κύτταρα ποντικού και της απομόνωσης και καλλιέργειας κυττάρων της μετεμφυτευτικής επιβλάστης έγκειται στη διαπίστωση πως τα κύτταρα αυτά παρουσιάζουν ομοιότητες με τα ανθρώπινα εμβρυονικά βλαστικά κύτταρα. Οι μηχανισμοί δημιουργίας της όψιμης, ενεργοποιημένης επιβλάστης, όμως, δεν είναι γνωστοί. Το γονίδιο Sox2 κωδικοποιεί για ένα μεταγραφικό παράγοντα που περιέχει την περιοχή HMG-box, ο οποίος είναι απαραίτητος για την πλειοδυναμία των εμβρυονικών βλαστικών κυττάρων και για τη δημιουργία της επιβλάστης. Κατά την πρώϊμη ανάπτυξη, το γονίδιο Sox2 εκφράζεται στο μορίδιο, την εσωτερική κυτταρική μάζα της βλαστοκύστης και αργότερα κατά τη διάρκεια της ανάπτυξης εκφράζεται στις αισθητικές πλάκες, το αρχέντερο και το νευρικό σύστημα. Τα έμβρυα, στα οποία έχει απαλειφεί το γονίδιο Sox2, πεθαίνουν στο στάδιο της εμφύτευσης απουσία επιβλάστη, αποδεικνύνοντας πως το Sox2 είναι αναγκαίο για τη δημιουργία της επιβλάστης in vivo. Σε αυτήν την εργασία, χρησιμοποιώντας εμβρυονικά βλαστικά κύτταρα που μας επιτρέπουν να ελέγχουμε τη συνεχή έκφραση του γονιδίου Sox2, δημιουργήσαμε κύτταρα πρωτογενούς εξωδέρματος, τα ΚΠΕ, με χαρακτήρα ενεργοποιημένης - όψιμης επιβλάστης, αποδεικνύοντας πως η συνεχής έκφραση του Sox2 είναι ικανή να δημιουργήσει επιβλάστη απο εμβρυονικά βλαστικά κύτταρα in vitro. Η μετάβαση αυτή συνοδεύεται, σε συμφωνία και με ό,τι έχει παρατηρηθεί μέχρι τώρα σε άλλες εργασίες,απο την ισχυρή παρουσία των μεταγραφικών επιπέδων του γονιδίου Οct4 και την αύξηση των επιπέδων του Nanog, δύο γονιδίων που κωδικοποιούν για παράγοντες πλειοδυναμίας,καθώς και από την ομοιόμορφη και ισχυρή παρουσία των πρωτεϊνικών μορίων που αυτά κωδικοποιούν. Ταυτόχρονα, παρατηρείται άνοδος των μεταγραφικών επιπέδων των γονιδίων Fgf5 και Nodal, φαινόμενο το οποίο συνδέεται στενά με τη δημιουργία της επιβλάστης. Ο κυτταρικός πληθυσμός των ΚΠΕ παρουσιάζει ίχνη μεταγραφικών επιπέδων των γονιδίων του μεταγραφικού παράγοντα Τ και του σηματοδοτικού μορίου Wnt3a, μόρια υπεύθυνα για τη δημιουργία του οπίσθιου άκρου της επιβλάστης, που θα δώσει το μεσόδερμα κατά τη γαστριδίωση. Τα ΚΠΕ δεν εκφράζουν άλλα μεσοδερμικά γονίδια, όπως τα Cer, Gscl, Meox1 και Ttr. Παρόλα αυτά, διατηρούν την ικανότητά τους να διαφοροποιούνται ομοιογενώς σε διαφοροποιημένα κύτταρα μεσοδερμικής προέλευσης, και συγκεκριμένα σε κοινούς προδρόμους των ενδοθηλιακών και αιμοποιητικών κυττάρων, αποκρινόμενα στη δράση του σηματοδοτικού μορίου BMP4. Ακόμα, η απουσία των πρώϊμων ενδοδερμικών παραγόντων AFP, Foxa1 και Foxa2 στα ΚΠΕ δεν εμποδίζει τη δυνατότητά τους να διαφοροποιούνται σε κύτταρα μεσενεδοδερμικής προέλευσης, που εκφράζουν τον ηπατικό παράγοντα FOXA2, κάτω από την επίδραση του σηματοδοτικού μορίου Α-activin. Σε πλήρη συμφωνία με τον εξωδερμικό χαρακτήρα των κυττάρων πρωτογενούς εξωδέρματος, τα ΚΠΕ εκφράζουν εξωδερμικά γονίδια, όπως η Nestin και η NeuroD και διαφοροποιούνται κάτω από την επίδραση του ρετινοϊκού οξέος σε νευρώνες και ολιγοδενδροκύτταρα. Η πλαστικότητα που επιδεικνύουν τα ΚΠΕ δε συνοδεύεται από την ικανότητα γένεσης τερατωμάτων, χαρακτηριστική ιδιότητα των πλειοδύναμων κυττάρων, όπως είναι τα εμβρυονικά βλαστικά κύτταρα και οι κυτταρικές σειρές που απομονώνονται από τη μετεμφυτευτική επιβλάστη. Αυτό αποδίδεται στο ότι έχουν ενεργοποιηθεί από άγνωστα μέχρι στιγμής σηματοδοτικά μόρια, με αποτέλεσμα να έχουν απωλέσει μέρος του αναπτυξιακού δυναμικού της πλειοδύναμης πρώϊμης επιβλάστης. Επιπλέον,τα ΚΠΕ έχουν τη δυνατότητα να μεταπίπτουν σε ΕΒΚ,όταν καλλιεργηθούν υπό συνθήκες ανάλογες, κάτι το οποίο είναι χαρακτηριστικό των κυττάρων πρωτογενούς εξωδέρματος. Συνοψίζοντας, από την παρούσα εργασία δημιουργήθηκαν αρχέγονα κύτταρα που αντιπροσωπεύουν την ενεργοποιημένη πλειοδύναμη κατάσταση της επιβλάστης και μπορούν να χρησιμοποιηθούν για την μελέτη των αρχικών σταδίων της ανάπτυξης και για βιοιατρικές εφαρμογές.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(e-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

This study by Khrimian et al. demonstrates that the bone-derived hormone osteocalcin is necessary and sufficient to correct age-related cognitive decline in the mouse. It also provides genetic, molecular, and neurophysiological evidence that Gpr158 is the receptor mediating osteocalcin’s regulation of cognition., That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein–coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN’s regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes.

Mouse embryonic stem cells (ESCs) are pluripotent mammalian cells derived from the inner cell mass (ICM) of mouse blastocysts, which give rise to all three embryonic germ layers both in vivo and in vitro. Mouse ESCs share a distinct epigenetic landscape and a more decondensed chromatin compared to differentiated cells. Numerous studies have shown that distinct histone modifications in ESCs serve as landmarks of pluripotency. However, so far it is still unknown whether the total histone amount (as opposed to histone modifications) in cells of different developmental stage and differentiation capacity remains the same. In this work we show that the total histone content differs between pluripotent and differentiated cells. In vitro spontaneous differentiation from ESCs to Embryoid Bodies (EBs) and directed differentiation towards neuronal and endodermal cells entails an increase in histone content. Primary MEFs also contain more histones than ESCs. We suggest that the difference in histone content is an additional landmark of pluripotency, in addition to and besides histone modifications.

Sox genes encode for a group of transcription factors that share the presence of an HMG-type (high mobility group) box. This is a 79 amino acid motif that encodes their DNA binding domain. They were identified soon after the discovery of the mammalian testis-determining gene Sry, which also possesses an HMG box, and obtained their name from Sry (“Sox” Sry-related box genes). The members of the Sox family are highly conserved among vertebrates and so far 26 different genes have been identified (Schepers et al., 2002; Wilson and Koopman, 2002). Sox proteins activate or repress target gene transcription, via interactions with a partner factor in a cell-type specific manner (Kondoh and Kamachi, 2010). Based on sequence homology, within and outside the HGM box, they have been classified into seven groups (SoxA to SoxG groups) (Wilson and Koopman, 2002). SoxB genes, divided in SoxB1 (Sox1-3) and SoxB2 (Sox14 and 21) subgroups, code for a group of highly related transcription factors expressed mainly in the developing and adult Central Nervous System (CNS) in mammals (Wood and Episkopou, 1999; Schepers et al., 2002; Avilion et al., 2003; Sarkar and Hochedlinger, 2013). During embryonic development, the nervous system arises from primitive neuroepithelial cells that give rise to neural stem cells (NSCs), cells that have the potential to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. Neuroepithelial progenitors (NEP) represent the cells with a more restricted potential, while precursors describe cells that exist in an earlier developmental state than others (McKay, 1997). In the developing CNS, Sox1-3 and Sox21 are expressed both in embryonic and adult NEPs and in subsets of differentiated neurons of the adult brain and spinal cord, whereas Sox14 is expressed only in selected differentiated neurons of the embryonic and adult CNS. Proteins encoded by Sox14 and Sox21 genes are very similar to each other but distinct from those encoded by Sox1-3, in areas outside the HMG domain and Group B homology that lies immediately C-proximal of the HMG domain (Uchikawa et al., 1999). Sox1-3 proteins are thought to function as transcriptional activators, while Sox14/21 are believed to function as repressors. However, Sox1 and Sox2 have been shown to be involved in repression of transcription as well (Cavallaro et al., 2008; Elkouris et al., 2011). Despite the wide distribution in the expression of SoxB proteins in the developing CNS, the effort to unravel their distinct function has been hampered by redundancy issues, eventually leading to functional compensation, but insights into the function of each factor have been gained from studies in areas where unique expression of a single factor is detected. SoxB function has been studied extensively during neurogenesis, the process of neuron formation, which involves continuous cycles of proliferation and differentiation of NEPs. During CNS development, dividing NEPs, residing in the ventricular zone (VZ), either self-renew or exit the cell cycle and differentiate. NEPs make cell fate decisions controlled by extrinsic and/or intrinsic determinants (Ramasamy et al., 2013). Neurons are generated in two sequential steps. In the specification step, which takes place within the VZ, some NEPs up-regulate the Notch receptors and remain as uncommitted progenitors while others down-regulate Notch receptors, up-regulate Notch ligands and then the proneural bHLH genes, Neurogenins 1-3 (Ngn1-3)/Mash1 and progress to terminal differentiation (Guillemot, 2007; Martynoga et al., 2012; Alberi et al., 2013). Accordingly, loss of Notch signaling invariably leads to over-production of neurons and depletion of NEPs (Pierfelice et al., 2011). In the commitment step, which is a transitionary phase and takes place on the lateral margins of the VZ known as intermediate zone (IZ), specified neuronal progenitors switch on another class of bHLH factors like NeuroD, Prox1, and Nscl1, which consolidate the specification step and allow the cells to progress to terminal differentiation (Christie et al., 2013). Ectopic expression of any of the latter can by-pass the specification phase and drive NEPs out of the cell cycle. The transcription factors Sox1-3 have been proposed to block neuronal commitment and thus prevent differentiation (Bylund et al., 2003; Holmberg et al., 2008; Oosterveen et al., 2013). In contrast Sox21 opposes Sox1-3 function in NEPs, where they are co-expressed, and promotes neurogenesis in vivo (Sandberg et al., 2005). Given that SoxB1 and Sox21 factors are co-expressed in the NEPs and have been shown to bear antagonizing activities, there was an open question how a cell decides to remain uncommitted or exit the cell cycle and how the antagonizing function of these factors is modulated. One model would propose that the relative activities of Sox1-3 vs. Sox21 are thought to determine whether a cell will remain undifferentiated or commit to the neuronal lineage. Accordingly, loss of Sox21 could lead to a predominance of SoxB1 activity leading to a block on neurogenesis in the embryo. However, this is not the case during development, since loss of Sox21 leads to a block in the progression of neuronal specification in the hippocampus only in adult mice (Matsuda et al., 2012). For Sox1, the data had also been conflicting. In vitro studies suggested that Sox1, like Sox21, could promote neurogenesis while Sox2 or Sox3 can block it. Initial reports suggested that over-expression of Sox1, but not Sox2 or Sox3, could promote neurogenesis when over-expressed in primary cultures of neural stem/progenitor cells cultured as neurospheres and the carcinoma cell line P19 (Pevny et al., 1998; Kan et al., 2004, 2007). However, this model was not supported by in vivo loss-of-function analysis in mice. In contrast, the role of Sox1 has been best studied during neuronal differentiation in the developing ventral forebrain and spinal cord, where a unique role has been assigned during neuronal subtype specification (Ekonomou et al., 2005; Panayi et al., 2010). We believe that light into the role of SoxB genes during neuronal specification is likely to be shed by the results from double mutant experiments. For instance, despite the fact that Sox1 and Sox21 single mutant mice are born, double Sox1/Sox21 mutants are embryonic lethal. The cause of lethality is yet to be determined (SM, unpublished data). Neuronal fates are determined around the final cell cycle of NEPs during which neural differentiation factors regulate the cell cycle either directly or indirectly. Only recently, SoxB genes have been implicated in the regulation of cell cycle. Studies in the mouse developing cortex showed that Sox1 acts to maintain the undifferentiated state of NEPs via a mechanism involving suppression of Prox1–mediated cell cycle exit and neurogenesis (Elkouris et al., 2011). These results are consistent with previous data from gain-of-function and loss-of-function analysis in the chick spinal cord, according to which SoxB1 factors block neurogenesis downstream of proneural factors (Bylund et al., 2003; Holmberg et al., 2008). Sox2 has found itself in the position of the most celebrated of the Sox proteins due to its role in epiblast formation (Avilion et al., 2003; Mandalos et al., 2012), pluripotency (Masui et al., 2007) and reprogramming (Polo et al., 2013; Sarkar and Hochedlinger, 2013). Sox2 null embryos die soon after implantation, masking a potential role for Sox2 in the CNS (Avilion et al., 2003; Mandalos et al., 2012). However, the function of Sox2 during neurogenesis has only recently begun to be elucidated using conditional mutagenesis in mice (Episkopou, 2005). Sox2 is expressed initially in NEPs and later in NSCs in the neurogenic niches of the embryonic and adult CNS (Wood and Episkopou, 1999; Avilion et al., 2003; Favaro et al., 2009; Remboutsika et al., 2011; Kang and Hebert, 2012; Mandalos et al., 2012; Raitano et al., 2013; Ramasamy et al., 2013). Sox2 becomes down-regulated during the final cell cycle of NEPs, immediately before differentiation and is required for the maintenance of NEPs properties functioning partly through the Shh and wnt3a pathways during embryonic and early postnatal life (Favaro et al., 2009). NSCs are completely lost from the early postnatal hippocampus upon loss of Sox2 but they gradually re-appear, an observation attributed to compensatory functions of Sox1 and Sox3 proteins, which are both expressed in an overlapping manner with Sox2. At this point, it is worth to mention that Sox2 maintains the embryonic cortical NSCs cycling, while at the same time it preserves and regulates their Pax6+ cortical radial identity and plasticity (Remboutsika et al., 2011; Raitano et al., 2013). Nevertheless, the exact role of Sox2 during cell cycle regulation of NSCs and NEPs is yet elusive. Recent evidence suggests that the function of SoxB1 factors, with even overlapping activities, is not strictly redundant, as they induce different sets of genes and are likely to partner with different proteins to maintain progenitor identity (Archer et al., 2011). It is reasonable then to suggest that during neurogenesis post-translational modifications, context-specific selection of partner proteins or competition for the same target binding sites modulate the activity of SoxB transcription factors. Thus, despite the discovery of SoxB transcription factors for over fifteen years their role during cell cycle, neurogenesis, neuronal commitment and specification has just started to be elucidated.