Stages Of Cell Differentiation Research Articles

Characterization of senescence biomarkers in rheumatoid arthritis: relevance to disease progression.

Rheumatoid arthritis (RA) has been associated with early senescent features. However, the effects of disease progression on senescence markers are largely unknown. Here, we evaluated key senescence markers in RA, including telomere length and T cell differentiation stages as well as cytomegalovirus (CMV) serology, previously associated with premature aging. In a cross-sectional study, 44 patients with active (Ac-RA), 26 patients with controlled (Co-RA), and 30 healthy controls were recruited. Peripheral blood was collected and differentiation stages of T cells analyzed by multi-color flow cytometry. Enzyme-linked immunosorbent assays were used to evaluate the CMV serology. The telomere length was measured by multiplex quantitative PCR. Patients with Ac-RA presented lower percentage of intermediate-differentiated T cells (CD4+CD27-CD28+ and CD8+CD27-CD28+; p < 0.001). All patients had a reduced proportion of cytotoxic T cells, and higher CD4/CD8 ratio compared with controls (p < 0.001). A lower proportion of CMV IgG+ subjects was found in the Co-RA group, (P < 0.001), although no differences in the CMV IgG titers were observed between groups. The groups had similar leukocyte telomere length. In addition, age was negatively correlated with CD8+CD27+CD28+ T (early-differentiated) cells (P < 0.05). Positive correlations between CMV IgG titers and age (P < 0.05) and CD4+CD27-CD28- T (late-differentiated) cells (P < 0.01) were observed. Furthermore, disease duration was correlated with CD4+CD27+CD28+ T cells (r = - 0.318, p < 0.05) and CD4+CD27-CD28- T cells (r = 0.308, p < 0.05). Our findings indicate that CMV and age may have a similar impact on T cells in both RA patients and controls. KEY POINTS: • Patients and controls were homogenous regarding CMV IgG titers and TL. • A lower proportion of CMV IgG+ subjects was found in the Co-RA group. • Anti-CMV levels were positively correlated with age and percentage of CD4+CD27-CD28- (late-differentiated) T cells.

PB2019 MOLECULAR ANALYSIS OF A COMPOSITE B‐CELL TUMOR – HAIRY CELL LEUKEMIA AND MANTLE CELL LYMPHOMA COMBINATION

Background:Composite lymphomas are unique models to study the clonal transformation process according stages of lymphoid cell differentiation. Clonal relationship is a key point in the research. At the moment, molecular analysis of V(D)J‐recombination is the most accurate method to evaluate related lymphoid populations.Aims:To study clonal rearrangements of IGH genes in isolated tumor populations in order to determine the mechanism of their occurrence during pathogenesis of composite lymphoma – hairy cell leukemia (HCL) and mantle cell lymphoma (MCL) combinationMethods:Patient was diagnosed and treated at the National Research Center for Hematology. Informed consent was obtained. Isolation of MCL and HCL cells was carried out by magnetic selection of bone marrow leukocyte fraction using anti‐CD103 and anti‐CD5 magnetic particles according to the standard Miltenyi Biotec protocol (www.miltenyibiotec.com). B‐cell clonality testing and IGHV sequencing was performed according to BIOMED‐2 protocol. Fragment analysis and Sanger sequencing was performed on ABI PRISM 3100 genetic analyzer, followed by comparison with the germinal sequence databases www.imgt.org.Results:Patient was performed with leukopenia and lymphocytosis, splenomegaly, lymphadenopathy in May, 2018 and was diagnosed with HCL. The diagnosis was confirmed by the BRAF mutation detection and peripheral blood lymphocytes immunophenotyping (table 1). Patient was consistently treated with interferon α2b and cladribine with good partial response. Within 4 months after therapy was completed spleen repeatedly enlarged. Diagnostic splenectomy was performed and MCL was revealed. The diagnosis was confirmed by the t(11;14) detection. Immunophenotyping of spleen suspension allowed to identify not only MCL cells but minimal residual HCL population (table 1). Analysis of clonally rearranged IGH genes is isolated CD103+ and CD5+ population revealed two clonal products corresponding to components of composite lymphoma (figure 1). The sequence analysis identified the dissimilar repertoire of gene segments involved in V(D)J‐recombination in MCL (IGHV3‐9/IGHD2/IGHJ4) and HCL (IGHV1‐69/IGHD3/IGHJ4). This distinction indicated the independence of two clones’ origin, at least at the stage of B‐lymphocyte precursors as we do not have other molecular markers for progenitor lymphoid cells. Nucleotide sequence was 100% germinal in MCL and only 90% matches with the germinal sequence were detected in HCL reflecting the postgerminal phenotype. Thus not only the existence of separate tumor precursors was strictly supposed but also the different way of parallel lymphoid cell differentiation was proven. Patient was treated with R‐BAC cycles for MCL. Complete remission was achieved and has been maintained for 6 months.Summary/Conclusion:At the moment, there is no known description of the synchronous development of MCL and HCL. The presented clinical case is the first observation of the clinical course of such the composite lymphoma. We demonstrated independent origin of two tumor components and proved the different way of their differentiation. The proposed algorithm for the analysis of isolated cell populations and the obtained results make an additional contribution to the lymphomagenesis understanding, which is necessary for the development of effective methods of diagnosis and therapy.image

Lens differentiation is characterized by stage-specific changes in chromatin accessibility correlating with differentiation state-specific gene expression

Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r ​> ​0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (−7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 ​kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.

Stem Cell Differentiation Stage Factors and their Role in Triggering Symmetry Breaking Processes during Cancer Development: A Quantum Field Theory Model for Reprogramming Cancer Cells to Healthy Phenotypes.

A long history of research has pursued the use of embryonic factors isolated during cell differentiation processes for the express purpose of transforming cancer cells back to healthy phenotypes. Recent results have clarified that the substances present at different stages of cell differentiation-which we call stem cell differentiation stage factors (SCDSFs)-are proteins with low molecular weight and nucleic acids that regulate genomic expression. The present review summarizes how these substances, taken at different stages of cellular maturation, are able to retard proliferation of many human tumor cell lines and thereby reprogram cancer cells to healthy phenotypes. The model presented here is a quantum field theory (QFT) model in which SCDSFs are able to trigger symmetry breaking processes during cancer development. These symmetry breaking processes, which lie at the root of many phenomena in elementary particle physics and condensed matter physics, govern the phase transitions of totipotent cells to higher degrees of diversity and order, resulting in cell differentiation. In cancers, which share many genomic and metabolic similarities with embryonic stem cells, stimulated redifferentiation often signifies the phenotypic reversion back to health and nonproliferation. In addition to acting on key components of the cellular cycle, SCDSFs are able to reprogram cancer cells by delicately influencing the cancer microenvironment, modulating the electrochemistry and thus the collective electrodynamic behaviors between dipole networks in biomacromolecules and the interstitial water field. Coherent effects in biological water, which are derived from a dissipative QFT framework, may offer new diagnostic and therapeutic targets at a systemic level, before tumor instantiation occurs in specific tissues or organs. Thus, by including the environment as an essential component of our model, we may push the prevailing paradigm of mutation-driven oncogenesis toward a closer description of reality.

ROS control in human iPS cells reveals early events in spontaneous carcinogenesis.

Reactive oxygen species (ROS) generated during cellular respiration oxidize various cellular constituents, which cause carcinogenesis. Because most studies on the role of ROS in carcinogenesis have mainly been performed using tumor-derived cell lines, which harbor various types of mutation, it has been difficult to determine the molecular details that lead to cancer formation. To overcome this difficulty, we established human-induced pluripotent stem cell lines in which the intracellular ROS levels are controlled at various differentiation stages by manipulating the ROS-yielding mitochondria. By introducing a specific amino acid substitution (I69E) into the succinate dehydrogenase complex, subunit C protein, a component of mitochondrial respiratory chain complex II, the ROS level increased considerably. When ROS-overproducing cells at the early stage of endoderm differentiation were subcutaneously inoculated into the backs of nude mice, we observed tumor formation. These tumor-initiating cells were subjected to a comprehensive analysis by RNA sequencing. It was revealed that tumor-initiating cells showed 27 upregulated transcripts compared with control cells. The newly identified genes include those coding for PAX8 and FOSB (transcription factors) as well as FGF22, whose expressions are known to increase in developing embryos. These results suggest that these genes may play a pivotal role in cancer formation at the very early stages of cell differentiation.

Regulation of MC3T3-E1 differentiation by actin cytoskeleton through lipid mediators reflecting the cell differentiation stage

Actin cytoskeleton is reported to be related in various functions of osteoblast, bone-forming cell. However the function of actin cytoskeleton in osteoblasts is not fully understood, since bone formation is derived from extracellular interactions of functional proteins produced from osteoblasts, including osteocalcin (Ocn), and it is a result of closely and complex organized sequence of biochemical events.In this study, we showed that actin cytoskeleton of MC3T3-E1 cells functioned in recognition of cell condition and regulation of extracellular matrix mineralization, bone formation. Maturation of MC3T3-E1 cells by 14 days of culture reduced F-actin filaments, while induced expression of Ocn mRNA known as late stage differentiation marker and matrix mineralization, terminal stage of cell differentiation. The disruption of actin cytoskeleton with Cyto D in immature MC3T3-E1 cells significantly increased expression of Ocn mRNA in 24 h. Both PTX-induced inhibition of signal transduction through GPCRs and celecoxib-induced suppression of lipid mediators in immature MC3T3-E1 cells reduced actin filaments and suppressed matrix mineralization. Furthermore, addition of lipid mediators extracted from culture mediums of differentiated MC3T3-E1 cells by Bligh-Dyer method induced actin cytoskeleton reorganization and matrix mineralization change in MC3T3-E1 cells. Taken together, our data suggest that actin cytoskeleton of MC3T3-E1 cells regulates activation of developmental pathway reflecting cell differentiation stages through lipid mediators. The function we identified is important for bone formation tightly regulated by mechanical stress, since actin cytoskeleton is also known as a mechanosensor of osteoblasts.

LncRNAs combine and crosstalk with NSPc1 in ATRA-induced differentiation of U87 glioma cells.

Nervous system polycomb 1 (NSPc1) is a member of the polycomb group (PcG) family of proteins and has been demonstrated to maintain the differentiation and pluripotency of stem cells. Long non-coding RNAs (lncRNAs) have been demonstrated to be involved in the control of pluripotency and differentiation in embryonic and pluripotent cells. In the present study, the expression levels of NSPc1 were associated with the malignant potential of various glioma cell lines. Additionally, lncRNAs were differentially expressed in glioblastoma cell lines. Following induced differentiation of U87 glioblastoma cells with all-trans retinoic acid, the expression levels of NSPc1 decreased initially, reaching its lowest point on day 6, but then subsequently increased until day 10. The expression of lncRNA candidates decreased in the cell differentiation stage. Additionally, the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), sex-determining region of the Y chromosome-box 2 overlapping transcript (SOX2OT) and antisense non-coding RNA in the INK4 locus (ANRIL) was significantly altered relative to the expression levels of NSPc1. RNA immunoprecipitation (RIP) assays demonstrated that MALAT1, SOX2OT and ANRIL bind to NSPc1 in U87 glioblastoma cells and the enrichment of ANRIL in anti-NSPc1 antibody group was associated with the expression levels of NSPc1 during U87 cell differentiation. Small interfering RNA mediated downregulation of NSPc1 expression with MALAT1, SOX2OT and ANRIL, inhibited the proliferation, and promoted apoptosis in U87 cells. The results of the present study demonstrate that MALAT1, SOX2OT and ANRIL combine and crosstalk with NSPc1 in U87 cells to affect proliferation and apoptosis.

Single cell RNA-sequencing identified Dec2 as a suppressive factor for spermatogonial differentiation by inhibiting Sohlh1 expression

Gonocyte-to-spermatogonia transition is a critical fate determination process to initiate sperm production throughout the lifecycle. However, the molecular dynamics of this process has not been fully elucidated mainly due to the asynchronized differentiation stages of neonatal germ cells. In this study, we employed single cell RNA sequencing analyses of P1.5–5.5 germ cells to clarify the temporal dynamics of gene expression during gonocyte-to-spermatogonia transition. The analyses identified transcriptional modules, one of which regulates spermatogonial gene network in neonatal germ cells. Among them, we identified Dec2, a bHLH-type transcription factor, as a transcriptional repressor for a spermatogonial differentiation factor Sohlh1. Deficiency of Dec2 in mice induces significant reduction of undifferentiated spermatogonia, and transplantation assay using Dec2-depleted cells also demonstrated the impaired efficiency of engraftment, suggesting its role in maintaining spermatogonial stem cells (SSCs). Collectively, this study revealed the intrinsic role of a new SSC factor Dec2, which protects germ cells from inadequate differentiation during neonatal testis development.

Silica nanoparticle-exposure during neuronal differentiation modulates dopaminergic and cholinergic phenotypes in SH-SY5Y cells

BackgroundSilica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation.ResultsExposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker β-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers β-catenin and GSK-3β were not altered by NP-exposure.ConclusionsThe decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.

D-105 Cellular memory and rare cell variability in cancer

Targeted therapies for cancer are a promising class of drugs that inhibit the specific molecular alterations that underlie the uncontrolled proliferation seen in cancer. The primary shortcoming of targeted therapy is disease relapse, which is driven by a subpopulation of cells that are resistant to these drugs. This phenomenon is generally thought to be genetic in origin; however, our recent work on melanoma shows that non-genetic cellular plasticity may provide a mechanism of resistance to these therapies. Furthermore, we showed that through the addition of the drug itself, cells transition from this transient plasticity into a new, stably resistant cell state via cellular reprogramming, suggesting that the time an individual cell exists in a state is important for producing the divergent resistance phenotype. However, there are currently no methods available to quantify the timescale of these fluctuations for the whole transcriptome. Thus, broadly generalizing this concept of timescales for cellular plasticity, we developed a novel method for genome-wide quantification of the timescales of gene expression memory based on a modern version of the ingenious Luria-Delbrück fluctuation analysis. In melanoma, this method revealed the gene expression state of rare cells resistant to targeted therapy. In a completely new model, triple negative breast cancer, this method revealed a novel rare subpopulation of cells exhibiting resistance to chemotherapy. More generally, this method has the potential to reveal other new phenotypes associated with rare cell biology including metastasis and the early stages of stem cell differentiation. Taken together, our findings in melanoma and novel methods for studying cellular plasticity outline a framework for using single-cell technologies for applications in basic biology and clinical medicine.

Maintenance and Restoration Effect of the Surface Hydrophilicity of Pure Titanium by Sodium Hydroxide Treatment and its Effect on the Bioactivity of Osteoblasts

In recent years, studies on the surface of titanium implants have shown that hydrophilic properties have a positive effect on bone binding, warranting further investigation into the maintenance and restoration of hydrophilic properties. In this work, a hydrophilic surface was obtained by plasma oxidation on the surface of sandblasted and acid-etched (SLA) titanium discs. We aimed to determine the effect of sodium hydroxide (NaOH) treatment on the maintenance and restoration of the surface hydrophilicity of titanium discs, as well as the relationship between the changes in hydrophilic properties on titanium surfaces and their biological properties. The results show that the treatment of hydrophilic surfaces with SLA, plasma oxidation, and NaOH treatments tend to enhance the early stages of cell adhesion, proliferation, and differentiation. Those results provide important guidance that SLA, plasma oxidation, and NaOH treatments can be used to restore the hydrophilic property of Ti that has been stored under room temperature and atmospheric pressure conditions.

Apoptosis and proliferation ofperipheral blood T-cells as alternative processes in pathogenesis of diabetes mellitus type 1

Apoptosis, along with proliferation, is a form of lymphocyte response to activating stimuli. In the early stages of cell differentiation, the apoptotic response prevails and it results to the formation of tolerance to inductor antigen. Mature lymphocytes proliferate in response to stimulation and it means the initial stage in the development of the immune response. Since in this case apoptosis and proliferation acts as alternative processes, their ratio can serve as a measure of the effectiveness of the cellular response to activating signals. The resistance of autoreactive T-cells to apoptosis is the main key point in the development of type 1 diabetes mellitus (T1DM). Autoreactive T-cells migrates from the bloodstream to the islet tissue of the pancreas and take an active part in b cells destruction. The resistance of autoreactive effector T-cells to apoptosis may suggest their high proliferative potential. Therefore, the comparative evaluation of apoptosis and proliferation of peripheral blood lymphocytes can give a more complete picture of their functional state and thus will help to reveal the causes of ineffective peripheral blood T-ceiis apoptosis in patients with T1DM and will help to understand more deeply the pathogenesis of the disease. in this article, the features of proliferative response of peripheral blood T-cells in patients with T1DM and in individuals with high risk of developing T1DM have been studied. Apoptosis of T-cell subpopulations has been investigated. The correlation between the apoptotic markers and the intensity of spontaneous and activation- induced in vitro T-cells proliferation of was revealed. it was determined, that autoreactive peripheral blood T-cells were resistant to apoptosis and demonstrated the increased proliferative potential in patients with T1DM and in individuals with high risk of developing T1DM.

Impact of deoxynivalenol and kaempferol on expression of tight junction proteins at different stages of Caco-2 cell proliferation and differentiation.

The barrier function of intestinal tract is essential to gut health, and the expression of tight junction (TJ) proteins in human epithelial colorectal adenocarcinoma (Caco-2) cells, mimicking the intestinal stem cell proliferation and differentiation, was investigated after treatment by the mycotoxin of deoxynivalenol (DON) and phenolic compound of kaempferol (KAM). The results showed that DON (5 μM) significantly reduced the expression of claudin-4 while kaempferol (100 μM) increased the expression of claudin-3 during Caco-2 cell proliferation. After being cultured for 11 days, the DON treatment, unexpectedly, augmented the expression of claudin-4 with an increased TEER. For differentiated Caco-2 cells after a 21 day culture, both the TEER and claudin-4 levels were significantly reduced by DON while KAM pretreatment alleviated the damage caused by DON accompanying an increase of TEER, claudin-3, and ZO-1. Thus, kaempferol and DON differentially affected the expression of tight junction proteins at different stages of Caco-2 cell proliferation and differentiation, and an increase of the integrity of TJ after KAM pretreatment indicates KAM has the potential to protect the integrity of the barrier function of the intestinal epithelium for improved health.

Гетерогенность и базальная аутофагия в клетках гепатокарциномы-29

The topic of research is the current direction of modern experimental oncology - the problem of studying the role of autophagy in the survival and death of a cancer cell. The purpose of this work was to study the cell heterogeneity of the hepatocarcinoma-29 population and the development of autophagy as a way of surviving tumor cells. The scientific significance of the work lies in obtaining previously unknown facts about the development of basal autophagy in certain types of hepatocarcinoma cells, which is a way of surviving tumor cells. The novelty of this study is to identify the types of hepatocarcinoma cells in which the process of autophagy develops, contributing to the survival of the tumor cell population and obtaining data on the structural features and activity of the autophagy process.The practical significance of the work lies in the possibility of using the obtained data for the development of targeted therapy of hepatocarcinoma through the combined use of inducers of autophagy and cytostatics, which will simultaneously use different cellular signaling pathways to stimulate apoptosis and autophagic cell death. The work was performed on the culture of hepatocarcinoma -29 cells using light and electron microscopy. The heterogeneity of the population of cells was revealed, which is determined by the size of the cells and the volume fraction of the nucleus and cytoplasm. In terms of cell volume and nuclear-cytoplasmic ratio, five types of cells are distinguished, corresponding to five stages of differentiation. In the IV and V cells of the differentiation stages, autophagic structures were revealed: autophagosomes, autolysosomes, and lysosomes. The findings make a significant contribution to cell biology, cytology and histology, as well as oncology. Key words: hepatocarcinoma-29, heterogeneity, stages of cell differentiation, autophag

Monoallelic, antisense and total RNA transcription in an in vitro neural differentiation system based on F1 hybrid mice.

We developed an in vitro system to differentiate embryonic stem cells (ESCs) derived from reciprocally crossed F1 hybrid mice into neurons, and used it to investigate poly(A)+ and total RNA transcription at different stages of cell differentiation. By comparing expression profiles of transcripts assembled from 20 RNA sequencing datasets [2 alleles×(2 cell lines×4 time-points+2 mouse brains)], the relative influence of strain, cell and parent specificities to overall expression could be assessed. Divergent expression profiles of ESCs converged tightly at neural progenitor stage. Patterns of temporal variation of monoallelically expressed transcripts and antisense transcripts were quantified. Comparison of sense and antisense transcript pairs within the poly(A)+ sample, within the total RNA sample, and across poly(A)+ and total RNA samples revealed distinct rates of pairs showing anti-correlated expression variation. Unique patterns of sharing of poly(A)+ and poly(A)- transcription were identified in distinct RNA species. Regulation and functionality of monoallelic expression, antisense transcripts and poly(A)- transcription remain elusive. We demonstrated the effectiveness of our approach to capture these transcriptional activities, and provided new resources to elucidate the mammalian developmental transcriptome.

Semi-Intact Cell System for Reconstituting and Analyzing Cellular Golgi Dynamics.

Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.

Astroglia-mediated regulation of cell development in the model of neurogenic niche in vitro treated with Aβ1-42

Neurogenesis is a complex process which governs embryonic brain development and is importants for brain plasticity throughout the whole life. Postnatal neurogenesis occurs in neurogenic niches that regulate the processes of proliferation and differentiation of stem and progenitor cells under the action of stimuli that trigger the mechanisms of neuroplasticity. Cells of glial and endothelial origin are the key regulators of neurogenesis. It is known that physiological neurogeneses is crucial for memory formation, whereas reparative neurogenesis provides partial repair of altered brain structure and compensation of neurological deficits caused by brain injury. Dysregulation of neurogenesis is a characteristics of various neurodevelopmental and neurodegenerative diseases, particularly, Alzheimer's disease which is very important medical and social problem. In the in vitro model of the neurogenic niche using hippocampal neurospheres as a source of stem/progenitor cells and astrocytes, we studied effects of astrocyte activation on the expression of markers of different stages of cell proliferation and differentiation. We found that aberrant mechanisms of development of stem and progenitor cells, caused by the beta-amyloid (Aβ1-42), can be partially restored by targeted activation of GFAP-expressing cells in the neurogenic niche.

Progress in single-cell analysis of hematopoiesis

The mechanism underlying production of various types of blood cells from hematopoietic stem and progenitor cells has been a central theme in hematology. Conventionally, hematopoietic cell populations are analyzed by cell surface markers to judge cell types and differentiation stages, and by transplantation assays to assess differentiation potential. Recently, however, next-generation sequencing technology has enabled single-cell transcriptome and epigenome analyses and cell barcoding-based lineage tracing during unperturbed hematopoiesis. These innovative assays revealed that each cell population is extensively heterogenous. Many cells within hematopoietic stem cell populations may not be multipotent, and conversely, hematopoietic progenitor cells often display self-renewal capacity. Moreover, cells tend to make their lineage choice much earlier than previously thought. Altogether, these results challenge the current hierarchical differentiation models and propose new continuous models. Single-cell analyses are expected to greatly contribute to our understanding of normal and abnormal hematopoiesis and to the development of new therapies for blood disorders.

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

SummaryExogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Identification and validation of alternative splicing isoforms as novel biomarker candidates in hepatocellular carcinoma.

Alternative splicing(AS) is a transcriptional regulation mechanism that participates in multiple aspects of cancer. The present study aimed to identify differential AS events from tumor and non‑tumor samples and investigate the potential of AS as a source of candidate cancer diagnostic biomarkers. Deep RNA sequencing of three paired hepatocellular carcinoma(HCC) tumors and adjacent non‑tumors was applied to identify AS events. RT‑qPCR was performed on 45HCC clinical samples to validate the splicing differences. The maximal information coefficient was first used to build an association between clinical features and AS changes. We identified 197 significantly differential skipped exon events, of which only 29% overlapped with the differentially expressed genes. The differentially spliced genes were mainly enriched in HCC‑characterized biological processes and pathways, clearly separating tumors from non‑tumors. We also validated the statistically significant splicing differences of three AS candidates (CEACAM1 exon7, VPS29 exon2 and ISOC2 exon3). Furthermore, a clinicopathological analysis revealed that carcinoembryonic antigen‑related cell adhesion molecule1 (CEACAM1) exon7 was significantly correlated with the survival time, and VPS29 exon2 was associated with cell differentiation stages. In conclusion, the findings of the three AS candidates in the present study could be beneficial in HCC prognosis and new treatment strategies.