Advanced Stage Of Differentiation Research Articles

Bone Regeneration in a Large Animal Model Featuring a Modular Off-the-Shelf Soft Callus Mimetic.

Implantation of engineered cartilage with soft callus features triggers remodeling to bone tissue via endochondral bone regeneration (EBR). Thus far, EBR has not progressed to the level of large animals on the axis of clinical translation. Herein, the feasibility of EBR is aimed for a critical-sized defect in a large animal model. Chondrogenesis is first induced in goat-derived multipotent mesenchymal stromal cells (MSCs) by fine-tuning the cellular differentiation process. Through a unique devitalization process, two off-the-shelf constructs aimed to recapitulate the different stages of the transient cartilaginous soft callus template in EBR are generated. To evaluate bone regeneration, the materials are implanted in an adapted bilateral iliac crest defect model in goats, featuring a novel titanium star-shaped spacer. After 3 months, the group at the more advanced differentiation stage shows remarkable regenerative capacity, with comparable amounts of bone regeneration as the autograft group. In contrast, while the biomaterial mimicking the earlier stages of chondrogenesis shows improved regeneration compared to the negative controls, this is subpar compared to the more advanced material. Concluding, EBR is attainable in large animals with a soft callus mimetic material that leads to fast conversion into centimeter-scale bone, which prospects successful implementation in the human clinics.

CD5 Suppresses IL-15-Induced Proliferation of Human Memory CD8+ T Cells by Inhibiting mTOR Pathways.

IL-15 induces the proliferation of memory CD8+ T cells as well as NK cells. The expression of CD5 inversely correlates with the IL-15 responsiveness of human memory CD8+ T cells. However, whether CD5 directly regulates IL-15-induced proliferation of human memory CD8+ T cells is unknown. In the current study, we demonstrate that human memory CD8+ T cells in advanced stages of differentiation respond to IL-15 better than human memory CD8+ T cells in stages of less differentiation. We also found that the expression level of CD5 is the best correlate for IL-15 hyporesponsiveness among human memory CD8+ T cells. Importantly, we found that IL-15-induced proliferation of human memory CD8+ T cells is significantly enhanced by blocking CD5 with Abs or knocking down CD5 expression using small interfering RNA, indicating that CD5 directly suppresses the IL-15-induced proliferation of human memory CD8+ T cells. We also found that CD5 inhibits activation of the mTOR pathway, which is required for IL-15-induced proliferation of human memory CD8+ T cells. Taken together, the results indicate that CD5 is not just a correlative marker for IL-15 hyporesponsiveness, but it also directly suppresses IL-15-induced proliferation of human memory CD8+ T cells by inhibiting mTOR pathways.

The metabolic profile of reconstituting T-cells, NK-cells, and monocytes following autologous stem cell transplantation and its impact on outcome

Previous studies indicated a role of the reconstituting immune system for disease outcome upon high-dose chemotherapy (HDCT) and autologous stem cell transplantation (auto-SCT) in multiple myeloma (MM) and lymphoma patients. Since immune cell metabolism and function are closely interconnected, we used flow-cytometry techniques to analyze key components and functions of the metabolic machinery in reconstituting immune cells upon HDCT/auto-SCT. We observed increased proliferative activity and an upregulation of the glycolytic and fatty acid oxidation (FAO) machinery in immune cells during engraftment. Metabolic activation was more pronounced in T-cells of advanced differentiation stages, in CD56bright NK-cells, and CD14++CD16+ intermediate monocytes. Next, we investigated a potential correlation between the immune cells’ metabolic profile and early progression or relapse in lymphoma patients within the first twelve months following auto-SCT. Here, persistently increased metabolic parameters correlated with a rather poor disease course. Taken together, reconstituting immune cells display an upregulated bioenergetic machinery following auto-SCT. Interestingly, a persistently enhanced metabolic immune cell phenotype correlated with reduced PFS. However, it remains to be elucidated, if the clinical data can be confirmed within a larger set of patients and if residual malignant cells not detected by conventional means possibly caused the metabolic activation.

Sera from Patients with NMOSD Reduce the Differentiation Capacity of Precursor Cells in the Central Nervous System.

Introduction: AQP4 (aquaporin-4)–immunoglobulin G (IgG)-mediated neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that affects the central nervous system, particularly the spinal cord and optic nerve; remyelination capacity in neuromyelitis optica is yet to be determined, as is the role of AQP4–IgG in cell differentiation. Material and Methods: We included three groups—a group of patients with AQP4–IgG-positive neuromyelitis optica, a healthy group, and a sham group. We analyzed differentiation capacity in cultures of neurospheres from the subventricular zone of mice by adding serum at two different times: early and advanced stages of differentiation. We also analyzed differentiation into different cell lines. Results and Conclusions: The effect of sera from patients with NMOSD on precursor cells differs according to the degree of differentiation, and probably affects oligodendrocyte progenitor cells from NG2 cells to a lesser extent than cells from the subventricular zone; however, the resulting oligodendrocytes may be compromised in terms of maturation and possibly limited in their ability to generate myelin. Furthermore, these cells decrease in number with age. It is very unlikely that the use of drugs favoring the migration and differentiation of oligodendrocyte progenitor cells in multiple sclerosis would be effective in the context of neuromyelitis optica, but cell therapy with oligodendrocyte progenitor cells seems to be a potential alternative.

Cd5 Suppresses Il-15-Induced Proliferation of Human Memory Cd8+ T Cells by Inhibiting mTOR Pathways

IL-15 induces the proliferation of memory CD8+ T cells. The expression of CD5 inversely correlates with the IL-15 responsiveness of human memory CD8+ T cells. However, whether CD5 directly regulates IL-15-induced proliferation is unknown. Here, we demonstrate that human memory CD8+ T cells in advanced differentiation stages respond to IL-15 better than those in less differentiated stages. Moreover, the expression level of CD5 was found as the best correlate for IL-15 hypo-responsiveness among human memory CD8+ T cells. Importantly, IL-15-induced proliferation is significantly enhanced by blocking CD5 with antibodies or knocking down CD5 expression using siRNA, indicating that CD5 directly suppresses the IL-15-induced proliferation of human memory CD8+ T cells. We also found that CD5 inhibits activation of the mTOR pathway, which is required for IL-15-induced proliferation. Taken together, the results indicate that CD5 is not just a correlative marker for IL-15 hypo-responsiveness, but also directly suppresses IL-15-induced proliferation of human memory CD8+ T cells by inhibiting mTOR pathways.

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

A Comprehensive Analysis of Key Immune Checkpoint Receptors on Tumor-Infiltrating T Cells From Multiple Types of Cancer.

Background: Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects.Method: This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer. With access to fresh surgical samples, this study simultaneously investigated the ex vivo expression level of eight known immune checkpoint receptors [PD-1, cytotoxic T-lymphocyte antigen-4 [CTLA-4], T-cell immunoglobulin and mucin-domain containing-3 [Tim-3], 2B4, killer cell lectin like receptor G1 [KLRG-1], TIGIT, B- and T-lymphocyte attenuator [BTLA], and CD160] on tumor-infiltrating T cells (TILs) and paired circulating T cells in blood from a 131-patient cohort.Results: We found increased an expression of PD-1 and Tim-3 but a decreased expression of BTLA on TILs when compared with peripheral blood from multiple types of cancer. Moreover, our co-expression analysis of key immune checkpoint receptors delineates “shared” subsets as PD-1+Tim-3+TIGIT+2B4+KLRG-1–CTLA-4– and PD-1+TIGIT+2B4+Tim-3–KLRG-1–CTLA-4– from bulk CD8 TILs. Furthermore, we found that a higher frequency of advanced differentiation stage T cells (CD27–CCR7–CD45RA–) among the “shared” subset (PD-1+Tim-3+TIGIT+2B4+KLRG-1–CTLA-4–) in bulk CD8 TILs was associated with poorly differentiated cancer type in cervical cancer patients.Conclusions: To our knowledge, our study is the first comprehensive analysis of key immune checkpoint receptors on T cells in treatment-naïve, primary cancer patients from the eight most prevalent types of cancer. These findings might provide useful information for future design of mono-blockade/combinatorial blockades and/or genetically modified T-cell immunotherapy.

Bone demineralization promotes superior spread of preosteoblast in culture.

Previous studies have shown substances capable of similar effects of demineralization, accelerating the process of bone remodeling. This study investigated preosteoblasts behavior in cell culture after bone demineralization with citric acid and tetracycline. Seventy-four Wistar rats provided 144 calvarial bone samples, 126 of which were randomly divided in seven groups according to the treatment given to the surface: no demineralization (C), citric acid (CA), tetracycline (TCN) during 15, 30, and 60 s. Each group received preosteoblasts cultured for 24, 48, and 72 hr. Eighteen remaining samples were analyzed for the atomic percentage (A%) by energy dispersive spectroscopy (EDS) before and after demineralization. The average percentage of bone area covered by cells increased with time and it was significantly higher after 24 and 48 hr of culture in groups CA15s, CA30s, CA60s, TCN15s, and TCN30s than in groups TCN60 and C (p < 0.05). The cell morphology in all CA and TCN groups was shown to be compatible with more advanced stages of differentiation than in C group. The A% changed after demineralization. We conclude that demineralization with citric acid or tetracycline for 15-30 s increased the area of bone surface covered by preosteoblasts. The A% changes were not sufficient to impair the cells spreading and morphology. Bone demineralization may promote potential benefits in bone regenerative procedures. HIGHLIGHTS: Low pH effects did not interfere on cell growth. Bone demineralization favored the preosteoblasts growth. A possible alternative to improve graft consolidation.

Effect of cell source and osteoblast differentiation on gene expression profiles of mesenchymal stem cells derived from bone marrow or adipose tissue.

Mesenchymal stem cells (MSCs) have been used in therapies for bone tissue healing. The aim of this study was to investigate the effect of cell source and osteoblast differentiation on gene expression profiles of MSCs from bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) to contribute for selecting a suitable cell population to be used in cell-based strategies for bone regeneration. BM-MSCs and AT-MSCs were cultured in growth medium to keep MSCs characteristics or in osteogenic medium to induce osteoblast differentiation (BM-OBs and AT-OBs). The transcriptomic analysis was performed by microarray covering the entire rat functional genome. It was observed that cells from bone marrow presented higher expression of genes related to osteogenesis, whereas cells from adipose tissue showed a higher expression of genes related to angiogenesis and adipocyte differentiation, irrespective of cell differentiation. By comparing cells from the same source, MSCs from both sources exhibited higher expression of genes involved in angiogenesis, osteoblast differentiation, and bone morphogenesis than osteoblasts. The clustering analysis showed that AT-OBs exhibited a gene expression profile closer to MSCs from both sources than BM-OBs, suggesting that BM-OBs were in a more advanced stage of differentiation. In conclusion, our results suggest that in cell-based therapies for bone regeneration AT-MSCs could be considered for angiogenic purposes, whereas BM-MSCs and osteoblasts differentiated from either source could be better for osteogenic approaches.

An Epigenetic Mitotic Score Tracks the Proliferative History and Capacity of CLL Samples at Diagnosis and Is Associated with Clinical Outcome

Background: DNA methylation has been extensively described to be related with gene regulation. However, recent data indicate that DNA methylation changes in repressed regions accumulate in every cell division, and thus, they can reflect the proliferative history of the cell. The evaluation of this epigenetic feature in cancer cells may provide information regarding the tumor cell biological evolution and may assist to predict the outcome of the patients.Aims: The global aim of this study was to develop a DNA methylation-based proliferative history score and determine its possible prognostic value in chronic lymphocytic leukemia (CLL).Methods: We used 450k methylation arrays from normal B cells subpopulations and CLLs at diagnosis. To analyze DNA methylation changes specifically in leukemic cells, we performed stringent data quality checks and corrections including FACS sorting and in silico purification of DNA methylation. Thus, we retained 67 normal B cells and 477 CLLs, comprising 140 naive-like CLLs (nCLL), 106 intermediate CLLs (iCLL) and 231 memory-like CLLs (mCLL) based on a recent epigenetic classification of cellular origin. We mapped CpGs falling in repressive regions using ChIP-Seq data of 6 histone marks from healthy B cell subpopulations and CLL patients. With the methylation levels of CpGs in repressive chromatin, we built one score per each sample which we termed Epigenetic Mitotic History (epiMH, ranging from 0 to 1). The epiMH was integrated with whole genome sequencing (WGS) and microarray expression data of the very same CLL patients (n=126). Finally, we analyzed whether epiMH was related to clinical behavior.Results: We showed that epiMH is widely variable in normal B cells, with more advanced differentiation stages displaying the highest epiMH, presumably due to their greater accumulated proliferative history. Then, we calculated the epiMH in CLLs with different cellular origins, namely, nCLL, iCLL and mCLL, and we correlated it with different biological features. Using mutational signatures from WGS data of the same samples, we identified significant correlations of epiMH with signatures 1, 5 and 9, that have been reported to various cell division related mutational processes. Next, differential gene expression analysis between CLLs with high epiMH as compared to those with low epiMH revealed an enrichment in gene signatures related to active proliferation. Collectively, these results support the concept that the epiMH may reflect the past and present proliferative history of the cell. Finally, we postulated that the epiMH may be related with future proliferative capacity and clinical behavior. To test this, we analyzed the clinical impact of epiMH in patients from each epigenetic subgroup. An univariate analysis with epiMH as quantitative variable revealed that the higher the epiMH the worse clinical behavior. Additionally, we performed a multivariate Cox regression model for both overall survival (OS) and time to first treatment (TTT) including epiMH together with other well established prognostic factors for CLL, including age, epigenetic subgroups and the number of driver alterations. We showed that epiMH was an independent significant variable for both OS (HR=1.40, pvalue=0.03) and TTT (HR=1.41, pvalue<0.001) (HRs corresponding to 0.1 epiMH increments).Conclusions: We have developed a proliferative history score (named epiMH) for CLL based on DNA methylation dynamics in repressed chromatin. Our data suggest that the epiMH of patients within each CLL epigenetic subgroup at diagnosis may reflect their proliferative history and capacity, and represents an independent prognostic factor. DisclosuresNo relevant conflicts of interest to declare.

Osteogenic differentiation of human mesenchymal stem cells from adipose tissue and Wharton's jelly of the umbilical cord.

Induced osteogenesis of mesenchymal stem cells (MSCs) may provide an important tool for bone injuries treatment. Human umbilical cord and adipose tissue are routinely discarded as clinical waste and may be used as noncontroversial MSCs sources. It still remains to be verified which source of MSCs is the most suitable for bone regeneration. The aim of this research was to investigate the osteogenic potential of human MSCs derived from adipose tissue (AT-MSCs) and Wharton's jelly of the human umbilical cord (WJ-MSCs) differentiated under the same conditions. Osteogenic differentiation of MSCs was detected and quantified by alizarin red S (ARS) staining for calcium deposition and alkaline phosphatase (ALP) activity, osteoprotegerin (OPG), and osteocalcin (OC) secretion measurements. Under osteogenic conditions, after 21 days of differentiation, the measured ALP activity and calcium deposition were significantly higher in the AT-MSCs than in the WJ-MSCs, while the OPG and OC secretion were higher in the WJ-MSCs vs. AT-MSCs. Low concentrations of OPG and high levels of OC in AT-MSCs and WJ-MSCs, prove that these cells reached an advanced stage of the osteogenic differentiation. The levels of OC secreted by AT-MSCs were lower than by WJ-MSCs. Both cell types, AT-MSCs and WJ-MSCs possess a potential to differentiate towards the osteogenic lineage. The observed differences in the levels of osteogenic markers suggest that after 21-days of osteogenic differentiation, the AT-MSCs might have reached a more advanced stage of differentiation than WJ-MSCs.

Searching for naïve human pluripotent stem cells.

Normal mouse pluripotent stem cells were originally derived from the inner cell mass (ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells (ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICM-derived epiblast of post-implantation embryos. These mouse epiblast stem cells (EpiSCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into EpiSC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called naïve (or ground state) human ESCs, and the derivation of naïve human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how (and whether) these relate to different stages of embryonic development, and discuss the potential implications of naïve human ESCs in research and therapy.

Effects of Physical Loading on Adipogenic Differentiation in 3T3-L1 Preadipocytes

Adipose tissue is subjected to various types of physical loading. Previous studies show that adipocytes have mechanosensitive properties and are affected by mechanical stimulation. In this study, we investigated the effects of fluid shear stress on adipogenic differentiation using 3T3-L1 preadipocytes. The study included 3 groups: (1) No Loading, (2) Loading-Immediate harvest, and (3) Loading-Post 2 day harvest. In the loading groups, dynamic fluid shear stress of maximum 1 Pa at 1 Hz was applied to the preadipocytes for 30 mins. GAPDH, ERK, p-ERK, C/EBPβ, PPARγ proteins were assessed by Western blot analysis. Loading resulted in increased p-ERK and reduced C/EBPβ expressions compared to the No Loading group. These changes were more significant when the cells were harvested post 2 day loading. However, loading did not result in significant changes in PPARγ expression. C/EBPβ plays a major role in promoting the differentiation process of preadipocytes and also induces PPARγ which shows enhanced expressions in the terminal differentiation stage of adipocytes. These results indicate that fluid shear stress may suppress adipogenic differentiation through C/EBPβ inhibition. Greater decrease in C/EBPβ when cells were harvested post 2 day loading suggests that changes in C/EBPβ are not immediate with loading and therefore longer duration may be necessary to observe changes in PPARγ expression. Further experiments with increased harvest time as well as experiments using 3T3-L1 preadipocytes of more advanced differentiation stage are underway.

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape.

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome-3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice-cultivated region apparently colonized exclusively by An. coluzzii. Notably, this 'intraspecific' differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome-X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human-made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro-tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.

CONSERVED SEX CHROMOSOMES ACROSS ADAPTIVELY RADIATEDANOLISLIZARDS

Vertebrates possess diverse sex-determining systems, which differ in evolutionary stability among particular groups. It has been suggested that poikilotherms possess more frequent turnovers of sex chromosomes than homoiotherms, whose effective thermoregulation can prevent the emergence of the sex reversals induced by environmental temperature. Squamate reptiles used to be regarded as a group with an extensive variability in sex determination; however, we document how the rather old radiation of lizards from the genus Anolis, known for exceptional ecomorphological variability, was connected with stability in sex chromosomes. We found that 18 tested species, representing most of the phylogenetic diversity of the genus, share the gene content of their X chromosomes. Furthermore, we discovered homologous sex chromosomes in species of two genera (Sceloporus and Petrosaurus) from the family Phrynosomatidae, serving here as an outgroup to Anolis. We can conclude that the origin of sex chromosomes within iguanas largely predates the Anolis radiation and that the sex chromosomes of iguanas remained conserved for a significant part of their evolutionary history. Next to therian mammals and birds, Anolis lizards therefore represent another adaptively radiated amniote clade with conserved sex chromosomes. We argue that the evolutionary stability of sex-determining systems may reflect an advanced stage of differentiation of sex chromosomes rather than thermoregulation strategy.

Nervous systems in 3D: A comparison of Caridean, anomuran, and brachyuran zoea‐I (DECAPODA)

Using serial semi-thin sections and digital 3D-reconstructions we studied the nervous systems of zoea-I larvae in three decapod species, Hippolyte inermis (Leach, 1815), Porcellana platycheles (Pennant, 1777), and Pachygrapsus marmoratus (Fabricius, 1787). These taxa represent three decapod lineages, that is, Caridea, Anomura, and Brachyura, each characterized by specific zoea-I morphology. Special attention was paid to development of ganglia, neuropil composition, and segmental nerves. In all zoeae studied, the overall elements, for example, the segmental ganglia, their neuropils and most of the nerves of the adult decapod nervous system are present. Ongoing differentiation processes are observable as well, most obvious in segments with well-developed limbs the ganglia are in a more advanced stage of differentiation and more voluminous compared to segments with only limb buds or without externally visible limb anlagen. Intra- and interspecific comparisons indicate that neuromere differentiation thus deviates from a simple anterior-posterior gradient as, for example, posterior thoracic neuromeres are less developed than those of the pleon. In addition, the differences in the progress of the development of ganglia between the studied taxa can best be attributed to heterochronic mechanisms. Taxon and stage-specific morphologies indicate that neuronal architecture reflects both, morphogenesis to the adult stage and specific larval adaptions, and provides sets of characters relevant to understanding the corresponding phylogeny.

Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma

Centromere protein F (CENPF) is an essential nuclear protein associated with the centromere-kinetochore complex and plays a critical role in chromosome segregation during mitosis. Up-regulation of CENPF expression has previously been detected in several solid tumors. In this study, we aim to study the expression and functional role of CENPF in hepatocellular carcinoma (HCC). We found CENPF was frequently overexpressed in HCC as compared with non-tumor tissue. Up-regulated CENPF expression in HCC was positively correlated with serum AFP, venous invasion, advanced differentiation stage and a shorter overall survival. Cox regression analysis found that overexpression of CENPF was an independent prognosis factor in HCC. Functional studies found that silencing CENPF could decrease the ability of the cells to proliferate, form colonies and induce tumor formation in nude mice. Silencing CENPF also resulted in the cell cycle arrest at G2/M checkpoint by down-regulating cell cycle proteins cdc2 and cyclin B1. Our data suggest that CENPF is frequently overexpressed in HCC and plays a critical role in driving HCC tumorigenesis.

Clonal Isolation of an Intermediate Pluripotent Stem Cell State

Pluripotent stem cells of different embryonic origin respond to distinct signaling pathways. Embryonic stem cells (ESCs), which are derived from the inner cell mass of preimplantation embryos, are dependent on LIF-Stat3 signaling, while epiblast stem cells (EpiSCs), which are established from postimplantation embryos, require activin-Smad2/3 signaling. Recent studies have revealed heterogeneity of ESCs and the presence of intermediate pluripotent stem cell populations, whose responsiveness to growth factors, gene expression patterns, and associated chromatic signatures are compatible to a state in between ESCs and EpiSCs. However, it remains unknown whether such cell populations represent a stable entity at single-cell level. Here, we describe the identification of clonal stem cells from mouse ESCs with global gene expression profiles representing such a state. These pluripotent stem cells display dual responsiveness to LIF-Stat3 and activin-Smad2/3 at single-cell level and thus named as intermediate epiblast stem cells (IESCs). Furthermore, these cells show accelerated temporal gene expression kinetics during embryoid body differentiation in vitro consistent with a more advanced differentiation stage than that of ESCs. The successful isolation of IESCs supports the notion that traverse from naïve ground state toward lineage commitment occurs gradually in which transition milestones can be captured as clonogenic entity. Our finding provides a new model to better understand the multiple pluripotent states.

PETROGENESIS OF CU-NI SULPHIDE ORES FROM O'OKIEP AND KLIPRAND, NAMAQUALAND, SOUTH AFRICA: CONSTRAINTS FROM CHALCOPHILE METAL CONTENTS

The petrogenesis of sulphide ores in the O’okiep district has remained controversial. Based mainly on the concentration of chalcophile metals (PGE, Cu, Ni), it is proposed that the sulphides segregated from a basaltic magma generated during melting of sub-continental lithospheric mantle. Sulphide saturation of the magma was delayed due to relatively high fO2 until crustal contamination occurred during the advanced stages of differentiation. The immiscible sulphide melt was entrained and fractionated in dynamic magma conduits. Sulphides enriched in monosulphide solid solution (mss) component precipitated at depth in the Kliprand area of southern Namaqualand to form the Hondekloof deposits, whereas the O’okiep ores crystallised at shallower levels from highly fractionated residual sulphide liquids enriched in intermediate solid solution (iss). Sulphides of intermediate composition occur at Ezelsfontein. In the context of this model, the O’okiep intrusions could represent the proximal magmatic members of an IOCG suite of deposits, raising the prospect for additional IOCG deposits elsewhere in southern Africa. The model also predicts an enhanced potential at O’okiep for undiscovered Ni sulphide ores at depth.

198 CILOSTAMIDE SUSTAINS GAP JUNCTION-MEDIATED COMMUNICATION AND CHROMATIN REMODELLING IN PIG OOCYTES

In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P &lt; 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P &lt; 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).