Stages Of Cell Maturation Research Articles

Comprehensive Phenotyping of Human PB NK Cells by Flow Cytometry.

The NK cell compartment provides powerful innate defenses against virus-infected and tumor cells. Specific NK cell receptors control this process and maintain the immune system homeostasis and prevent autoimmunity. A wide variety of NK cell subsets with different functional capabilities exist and this reflects not only the different maturation stages of NK cells but also different microenvironments in which they can operate. In this review, we will give an overview on the various NK cell subsets present in peripheral blood of healthy donors in order to clearly and univocally identify them on the basis of their phenotypic traits using flow cytometry. © 2020 International Society for Advancement of Cytometry.

Variations in the timing of different phenological stages of cambial activity in Abies pindrow (Royle) along an elevation gradient in the north-western Himalaya

High mountains around the globe are some of the most vulnerable ecosystems to climate change and of great concern for conservation. The Himalayan Mountains are experiencing a higher warming than average global warming, which can significantly impact their biodiversity, vegetation distribution and ecosystem structure. There is a need to study the process of wood formation in Himalayan conifers to have a better understanding of their growth responses to predicted climate change. Variations in the climatic factors regulating cambial activity would result in changes in the timing of cambial phenology. In this study, the variations in the timing of different stages of cambial phenology (cell enlargement stage, wall-thickening stage and cell maturation stages) in pindrow fir (Abies pindrow) were investigated from anatomical observations of wood microcores collected during 2014-15 along an elevation range of c.2300−3000 m asl in the north-western Himalaya. The onset of all three cambial phenological stages was significantly correlated with elevation, with onset of cambial activity happening more than a week earlier at the lowest elevation than at the highest elevation site. Although the termination of wall-thickening and maturation stage appeared minimally related to elevation, the cell-enlargement stage showed significant correlation with elevation, with tracheid formation ceasing approximately three weeks earlier in trees at the highest elevation. The timing of these phenological stages did not show strong variations between the two study years. Our findings provide new data on the timings of cambial phenophases and help to understand tree growth response to ongoing changing climate in the Himalayan region.

AI-powered transmitted light microscopy for functional analysis of live cells

Transmitted light microscopy can readily visualize the morphology of living cells. Here, we introduce artificial-intelligence-powered transmitted light microscopy (AIM) for subcellular structure identification and labeling-free functional analysis of live cells. AIM provides accurate images of subcellular organelles; allows identification of cellular and functional characteristics (cell type, viability, and maturation stage); and facilitates live cell tracking and multimodality analysis of immune cells in their native form without labeling.

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

RECONSTITUTION OF T LYMPHOPOIESIS FROM PLURIPOTENT STEM CELLS BY DEFINED TRANSCRIPTION FACTORS

Pluripotent stem cells (PSC)-generated induced PTP could only reconstitute functional T lymphopoiesis in vivo when pre-embedded in congenic fetal thymic lobes, indicative of a cell-intrinsic flaw of homing capacity in vivo.In this study, we identified certain transcription factors that were absently expressed in induced hemogenic endothelial cells but abundantly expressed in their natural counterparts and further screened their combinatory roles in specifying T lymphopoiesis by tandem-TF-knock-in strategy. Coordinated inducible expression of TF1 and TF2 during endothelial to hematopoietic transition (EHT) and subsequent blood cell maturation stages in vitro preferentially resulted in full T lymphopoiesis in vivo after transplanting the derived ETP-abundant hematopoietic progenitors into conditioned NOD scid gamma mice via intravenous delivery. Remarkably, even a single induced hemogenic endothelial cells (iHEC) was able to generate T cells (iT cells) in vitro and reconstitute T lymphopoiesis in vivo. Following the same protocol, another TF-cocktails led to induced hematopoietic progenitors preferentially giving rise to B lymphopoiesis including B1 cells in vivo, strongly supporting the concept that certain transcription factors can specify lymphoid-lineage potentials at the early developmental stages before the emergence of hematopoietic stem cells. Physiologically, the mature iT cells distributed into lymphoid and other solid organs and exhibited abundant TCRαβ repertoire. Functionally, these regenerated iT cells successfully rejected allogeneic skin grafts and formed immune memory response. Translationally, the tumor-antigen-specific TCR-iT cells derived from gene-edited PSC successfully repressed tumor growth in vivo. This study provides insight into reconstituting T lymphopoiesis using PSC source.

Protein pathway analysis to study development-dependent effects of acute and repeated trimethyltin (TMT) treatments in 3D rat brain cell cultures

Trimethyltin is an organometallic compound, described to be neurotoxic and to trigger neuroinflammation and oxidative stress. Previous studies associated TMT with the perturbation of mitochondrial function, or neurotransmission. However, the mechanisms of toxicity may differ depending on the duration of exposure and on the stage of maturation of brain cells. This study aim at elucidating whether the toxicity pathways triggered by a known neurotoxicant (TMT) differs depending on cell maturation stage or duration of exposure. To this end omics profiling of immature and differentiated 3D rat brain cell cultures exposed for 24 h or 10 days (10-d) to 0.5 and 1 μM of TMT was performed to better understand the underlying mechanisms of TMT associated toxicity.Proteomics identified 55 and 17 proteins affected by acute TMT treatment in immature and differentiated cultures respectively, while 10-day treatment altered 96 proteins in immature cultures versus 353 in differentiated. The results suggest different sensitivity to TMT depending on treatment duration and cell maturation. In accordance with known TMT mechanisms oxidative stress and neuroinflammation was observed after 10-d treatment at both maturation stages, whereas the neuroinflammatory process was more prominent in differentiated cultures than in the immature, no development-dependent difference could be detected for oxidative stress or synaptic neurodegeneration. Pathway analysis revealed that both vesicular trafficking and the synaptic machinery were strongly affected by 10-d TMT treatment in both maturation stages, as was GABAergic and glutamatergic neurotransmission.This study shows that omics approaches combined with pathway analysis constitutes an improved tool-set in elucidating toxicity mechanisms.

S843 THE PROLIFERATIVE HISTORY SHAPES THE DNA METHYLOME OF B‐CELL TUMORS AND PREDICTS CLINICAL OUTCOME

Background:Beyond the known role of DNA methylation in gene regulation, recent studies indicate that most DNA methylation changes in cancer accumulate in silent regions without affecting gene expression. To obtain new insights into the biological and clinical implications of DNA methylation, we systematically analyzed genetic, epigenetic, transcriptional, and clinical data of five categories of B‐cell tumors including 1553 samples. This analysis unraveled that mitotic activity shapes the overall genetic and epigenetic landscapes of B cell tumors and affects the clinical behavior of patients.Aims:To provide a holistic view on the biological and clinical implications of DNA methylation variability in B‐cell tumors, with particular emphasis in non‐regulatory regions.Methods:We used 450k methylation arrays from normal B cell subpopulations as well as ALL, MCL DLBCL, CLL and MM patients (n = 67, 830, 74, 27, 490 and 65, respectively). We integrated ChIP‐seq data of 6 histone marks from normal B cells, MCL, CLL and MM (n = 15, 5, 7 and 5, respectively). This allowed us to construct the epigenetic Cumulative Mitosis (epiCMIT) score. We validated the epiCMIT using whole‐genome sequencing and gene expression data of the same CLL patients (n = 135). We additionally used 477 CLL patients with whole exome sequencing to find genetic driver alterations associated with high epiCMIT. From the clinical perspective, we used a machine‐learning approach to build an accurate pan B‐cell cancer diagnostic tool and related the epiCMIT to clinical outcome of ALL, MCL and CLL patients.Results:We dissected the DNA methylome of B‐cell tumors at different levels, including entity‐specific, subentity‐specific and patient‐specific variability. We showed that virtually the entire methylome (87%) is modulated in normal and/or neoplastic conditions, and subsequently dissected the sources of such variability. We initially identified the presence of differential methylation patterns among B‐cell tumors and their subentities. This analysis allowed us to construct an accurate pan B‐cell cancer diagnostic tool for 14 subentities of tumors requiring different clinical management. Furthermore, of particular interest were the hypomethylation patterns in ALL, MCL and CLL, which we could relate to differential binding of transcription factors important for the pathogenesis of each disease, such as ETS1 and MEIS1A in ALL, TCF/ZEB in MCL and NFAT in CLL. Despite the importance of DNA methylation changes in these regulatory regions, we identified that the majority of the changes were actually located in inactive non‐regulatory regions. By means of genetic and transcriptomic associations, we demonstrated that those changes reflect the proliferative history of cancer specimens. Interestingly, we found that cell mitosis leaves different imprints onto the DNA methylome depending on the maturation stage of cancer cells. Subsequently, we built a mitotic clock called epiCMIT (representing the proliferative history of the tumor) and applied it to 1,553 samples. Putting the epiCMIT in the context of the clinical behavior of the ALL, MCL and CLL patients, we found that it represents a strong and independent prognostic factor. Finally, we identified driver genetic alterations related to higher proliferative capacity, a finding that could explain their well‐established clinical impact.Summary/Conclusion:Our analytic approach reveals DNA methylation as a holistic tracker of B cell tumor developmental history, with implications in the differential diagnosis and prediction of prognosis of the patients

Chronic stress triggers divergent dendritic alterations in immature neurons of the adult hippocampus, depending on their ultimate terminal fields

Chronic stress, a suggested precipitant of brain pathologies, such as depression and Alzheimer’s disease, is known to impact on brain plasticity by causing neuronal remodeling as well as neurogenesis suppression in the adult hippocampus. Although many studies show that stressful conditions reduce the number of newborn neurons in the adult dentate gyrus (DG), little is known about whether and how stress impacts on dendritic development and structural maturation of these newborn neurons. We, herein, demonstrate that chronic stress impacts differentially on doublecortin (DCX)-positive immature neurons in distinct phases of maturation. Specifically, the density of the DCX-positive immature neurons whose dendritic tree reaches the inner molecular layer (IML) of DG is reduced in stressed animals, whereas their dendritic complexity is increased. On the contrary, no change on the density of DCX-positive neurons whose dendritic tree extends to the medial/outer molecular layer (M/OML) of the DG is found under stress conditions, whereas the dendritic complexity of these cells is diminished. In addition, DCX+ cells displayed a more complex and longer arbor in the dendritic compartments located in the granular cell layer of the DG under stress conditions; on the contrary, their dendritic segments localized into the M/OML were shorter and less complex. These findings suggest that the neuroplastic effects of chronic stress on dendritic maturation and complexity of DCX+ immature neurons vary based on the different maturation stage of DCX-positive cells and the different DG sublayer, highlighting the complex and dynamic stress-driven neuroplasticity of immature neurons in the adult hippocampus.

Hormonal Regulation of Follicle-Stimulating Hormone Glycosylation in Males

The Follicle-Stimulating Hormone plays an important role in the regulation of gametogenesis. It is synthesized and secreted as a family of glycoforms with differing oligosaccharide structure, biological action, and half-life. The presence of these oligosaccharides is absolutely necessary for the full expression of hormone bioactivity at the level of the target cell. The endocrine milieu modulates the glycosylation of this hormone. During male sexual development a progressive increase in FSH sialylation and in the proportion of glycoforms bearing complex oligosaccharides are the main features in this physiological condition. In late puberty, FSH oligosaccharides are largely processed in the medial- and trans-Golgi cisternae of the gonadotrope and remain without changes throughout adult life. In experimental models, the absence of gonads severely affects FSH sialylation; androgen administration is able to restore the characteristics observed under physiological conditions. The expression of ST6 beta-galactoside alpha-2,6-sialyltransferase 1 is hormonally regulated in the male rat; it decreases after short periods of castration but increases markedly at longer periods of androgen deprivation. Although ST3 beta-galactoside alpha-2,3-sialyltransferase 3 is expressed in the male rat pituitary it is not influenced by changes in the endocrine milieu. The oligosaccharide structure of FSH has an impact on the Sertoli cell endocrine activity. In more advanced stages of Sertoli cell maturation, both sialylation and complexity of the oligosaccharides are involved in the regulation of inhibin B production; moreover, FSH glycoforms bearing incomplete oligosaccharides may enhance the stimulatory effect exerted by gonadal growth factors. In this review, we discuss available information on variation of FSH glycosylation and its hormonal regulation under different physiological and experimental conditions, as well as the effect on Sertoli cell endocrine activity.

Generation of a triple-fluorescent mouse strain allows a dynamic and spatial visualization of different liver phagocytes in vivo.

Resident and circulating immune cells have been extensively studied due to their almost ubiquitous role in cell biology. Despite their classification under the "immune cell department", it is becoming increasingly clear that these cells are involved in many different non-immune related phenomena, including fetus development, vascular formation, memory, social behavior and many other phenotypes. There is a huge potential in combining high-throughput assays - including flow cytometry and gene analysis - with in vivo imaging. This can improve our knowledge in both basic and clinical cell biology, and accessing the expression of markers that are relevant in the context of both homeostasis and disease conditions might be instrumental. Here we describe how we generated a novel mouse strain that spontaneously express three different fluorescence markers under control of well-studied receptors (CX3CR1, CCR2 and CD11c) that are involved in a plethora of stages of cell ontogenesis, maturation, migration and behavior. Also, we assess the percentage of the expression and co-expression of each marker under homeostasis conditions, and how these cells behave when a local inflammation is induced in the liver applying a cutting-edge technology to image cells by confocal intravital microscopy.

The ETS1 Transcription Factor Is Implicated in Human and Murine Intermediate NK Cell Development Stages

Introduction: While key regulators of early NK cell development and differentiation have been identified, few studies have looked at transcription factor (TF) dynamics and regulatory interactions during subsequent stages of NK cell maturation. Epigenetic landscapes are highly dynamic during cellular differentiation, with TFs playing an important role in the establishment and activation of specific DNA elements, such as enhancers, and subsequent programming of gene expression. ETS1 is a TF that is expressed in adult immune tissues and is critical for the development of lymphoid cells. A role for ETS1 has been described in early NK cell development by activating core transcriptional regulators such as T-BET and ID2. However, despite its continual expression in subsequent stages of NK maturation, the role of ETS1 in NK maturation is not well characterized.Methods and Results: We used FACS to isolate purified human NK cells at various maturation stages as established previously (Freud et. al. Cell Reports, 2016, 16:379-91), ranging from intermediate precursors (Stage 3) through to fully developed and mature peripheral NK cells (Stage 6). Epigenetic programming of cells during lineage maturation allows us to identify critical TFs that are active at each stage of development. We employed Illumina EPIC/850K methylation arrays and RNA sequencing to interrogate epigenetic changes at regulatory elements and TF dynamics at multiple stages along the NK developmental axis. Analysis of TF recognition motifs within hypomethylated regions revealed strong enrichment of specific motif sequences implicating T-box (T-BET and Eomes), along with RUNX and ETS TF families in specific programming of epigenetic patterns during NK development. In studying the expression of TFs that potentially bind these motifs, ETS1 exhibited the highest and most consistent expression throughout NK development. Interestingly, despite consistently high expression, ETS motifs were continually programmed throughout NK maturation, including a significant degree of modification between tonsillar Stages 4A to 4B, where NK cells acquire the ability to produce IFN-γ and significantly gain cytotoxic capability and functional maturity. Among the genes that are upregulated at this stage is the NK-cell-specific gene, NKp46.The progressive hypomethylation of regulatory regions enriched in ETS motifs led us to believe that ETS1 has a continuous role in full NK cell maturation. To test our hypothesis, we developed a novel genetically engineered mouse line with a NK cell intermediate stage-specific conditional deletion of Ets1 mediated by NKp46-driven Cre expression, NKp46-Cre-Ets1fl/fl (NKp46-Ets1fl/fl). This allowed us to study the role of ETS1 in the transition between immature and mature NK cell stages in vivo. Using a comprehensive NK cell development panel for multi-color flow cytometry, we found a drastic reduction of total NK cells in NKp46-Ets1fl/fl mice (n=7) compared to the Ets1fl/fl (n=7) and the NKp46-Cre (n=7) controls in bone marrow (3.2x104 ± 5.9x103, 2.9x105 ± 5.7x104, 2.6x105 ± 8.0x104 total NK cells respectively; p= 0.0007), spleen (3.1x104 ± 7.2x103, 1.2x106 ± 2.4x105, 1.5x106 ± 7.7x105 total NK cells respectively; p= 0.0091) and blood (21 ± 6, 385 ± 35, 185 ± 35 NK cells/uL whole blood respectively; p= 0.0001). Supporting our hypothesis, we indeed observed that while CD11b-/CD27+/- immature NK cell populations in our model are unaltered, the loss of ETS1 is associated with a decrease in CD11b+/CD27+/- mature NK cell populations.Conclusions: Our findings demonstrate that in addition to its role in early NK establishment, persistent ETS1 expression is important in intermediate differentiation stages in both human and murine NK cell development. This constitutes a significant step forward in understanding the role of ETS1 as a master transcriptional regulator in the entire NK cell developmental axis. Current studies are ongoing to dissect the mechanism by which ETS1 affects NK cell development and function in the NKp46-Ets1fl/fl mice.(*EH and JW are recipients of Pelotonia Graduate and Undergraduate student fellowships respectively and contributed equally to this work. This work was partly supported by OCRA, NIH R01 CA159296, NIH R01 CA208353, P01CA95426, R35 CA197734 and OSUCCC Leukemia Tissue Bank and Genetically Engineered Mouse Modeling Core supported by P30CA016058) DisclosuresNo relevant conflicts of interest to declare.

Abstract P155: B Cells Are Not Necessary for the Developmentof Ang II-induced Hypertension and End-Organ Damage

Chronic inflammation characterized by vascular T cell and macrophage infiltration is associated with angiotensin II (Ang II)-induced hypertension and end-organ damage. However, the role of B cells in the development of hypertension is not well established. Elevated levels of serum immunoglobulins have been observed in experimental and human hypertension. Prior studies demonstrated that B-cell-activating factor receptor-deficient (BAFF-R -/- ) mice, which lack mature B cells, are protected from Ang II-induced hypertension and vascular remodeling. However, BAFF-R can be expressed on non-B cells as well. Thus, to further investigate the role of B cells in hypertension, we studied mice with targeted disruption of the membrane exon of the immunoglobulin μ heavy chain gene (μMT -/- ) that encodes the constant region of the IgM isotype. In these mice, B cells are arrested at the pre-B cell maturation stage. Serum immunoglobulins were undetectable at a 1:30,000 dilution in μMT -/- mice. Interestingly, μMT -/- mice exhibited similar blood pressure increase in response to 4 weeks of Ang II infusion compared to age-matched wild type (WT) mice (p=0.675). Flow cytometry of aortae from Ang II-treated μMT -/- mice demonstrated fewer CD45+ cells (p=0.047), similar levels of CD8+ and CD4+ T cells (p=0.631 and p=0.721 respectively), a trend for more F4/80+ monocytes/macrophages (p=0.2567), and virtually no CD19+ B cells (p=0.002) compared to WT mice. Endothelium-dependent vasodilatation to acetylcholine was comparable between μMT -/- mice and WT mice after Ang II treatment (p=0.223), however μMT -/- mice displayed enhanced endothelium-independent vasodilatation to sodium nitroprusside than WT mice (p=0.024). Urinary albumin/creatinine ratio was similar between Ang II-treated μMT -/- mice and WT mice (p=0.488), indicating similar renal damage. Taken together, μMT -/- mice are not protected from Ang II-induced hypertension, suggesting that B cells and immunoglobulin production are not critical for hypertension and the associated end-organ damage.

Conditional Selection of B Cells in Mice With an Inducible B Cell Development.

Developing B cells undergo defined maturation steps in the bone marrow and in the spleen. The timing and the factors that control these differentiation steps are not fully understood. By targeting the B cell-restricted mb-1 locus to generate an mb-1 allele that expresses a tamoxifen inducible Cre and another allele in which mb-1 expression can be controlled by Cre, we have established a mouse model with an inducible B cell compartment. With these mice, we studied in detail the kinetics of B cell development and the consequence of BCR activation at a defined B cell maturation stage. Contrary to expectations, transitional 1-B cells exposed to anti-IgM reagents in vivo did not die but instead developed into transitional 2 (T2)-B cells with upregulated Bcl-2 expression. We show, however, that these T2-B cells had an increased dependency on the B cell survival factor B cell activating factor when compared to non-stimulated B cells. Overall, our findings indicate that the inducible mb-1 mouse strain represents a useful model, which allows studying the signals that control the selection of B cells in greater detail.

Sequential Application of Discrete Topographical Patterns Enhances Derivation of Functional Mesencephalic Dopaminergic Neurons from Human Induced Pluripotent Stem Cells

Parkinson’s Disease is a progressive neurodegenerative disorder attributed to death of mesencephalic dopaminergic (DA) neurons. Pluripotent stem cells have great potential in the study for this late-onset disease, but acquirement of cells that are robust in quantity and quality is still technically demanding. Biophysical cues have been shown to direct stem cell fate, but the effect of different topographies in the lineage commitment and subsequent maturation stages of cells have been less examined. Using human induced pluripotent stem cells (iPSCs), we applied topographical patterns sequentially during differentiation stages and examined their ability to influence derivation yield and functionality of regionalized subtype-specific DA neurons. Gratings showed higher yield of DA neurons and may be beneficial for initial lineage commitment. Cells derived on pillars in the terminal differentiation stage have increased neuronal complexity, and were more capable of firing repetitive action potentials, showing that pillars yielded better network formation and functionality. Our topography platform can be applied to patient-derived iPSCs as well, and that cells harbouring LRRK2 mutation were more functionally mature when optimal topographies were applied sequentially. This will hopefully accelerate development of robust cell models that will provide novel insights into discovering new therapeutic approaches for Parkinson’s Disease.

Determination of normal expression patterns of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood and bone marrow cells by flow cytometry

In 2010, new monoclonal antibodies were submitted to the 9th International Workshop on Human Leukocyte Differentiation Antigens, and there are few studies demonstrating normal expression patterns of these markers. Thus, the objective of this study was to determine the normal patterns of cell expression of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood (PB) and bone marrow (BM) samples by flow cytometry. In the present study, CD86 was expressed only in monocytes and B lymphocytes in PB and in monocytes and plasma cells in BM. Regarding CD210a expression, in PB samples, monocytes and NK cells showed weak expression, while neutrophils, B and T lymphocytes, and basophils showed weak and partial expression. In BM samples, expression of CD210a was observed in eosinophils, monocytes, and B and T/NK lymphocytes. Weak expression of CD210a was also observed in neutrophilic cells and plasma cells. All B cell maturation stages had weak expression of CD210a except for immature B cells, which did not express this marker. In the present study, no cell type in PB samples showed positivity for CD261 and, in BM samples, there was very weak expression in neutrophilic series, monocytes, and B lymphocytes. Conversely, plasma cells showed positivity for CD261 with a homogeneous expression. For CD262, there was weak expression in monocytes, neutrophils, and B lymphocytes in PB samples and weak expression in monocytes, B lymphocytes, and plasma cells in BM samples. The evaluation of CD264 showed very weak expression in B cells in PB samples and no expression in BM cells. Very weak expression of CD358 was observed in neutrophils, monocytes, and B lymphocytes in PB and BM samples. In addition, in BM samples, plasma cells and T lymphocytes showed weak expression of CD358. In relation to the maturation stages of B cells, there was weak expression in pro-B cel, pre-B cell, and mature B cell. In the present study, it was possible to observe expression of CD361 in all cell types analyzed in PB and BM samples. The analyzed markers presented varied profiles of expression and, in some cases, these profiles were different from those observed in other studies. Further studies are needed to evaluate these molecules, mainly in relation to a possible application in the diagnosis of hematological malignancies or as new therapeutic targets for the treatment of hematological neoplasms or autoimmune diseases.

Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives.

Human stem cells and their progeny are valuable for a variety of research applications and have the potential to revolutionize approaches to regenerative medicine. However, we currently have limited tools to permit live isolation of homogeneous populations of cells apt for mechanistic studies or cellular therapies. While these challenges can be overcome through the use of immunophenotyping based on accessible cell surface markers, the success of this process depends on the availability of reliable antibodies and well-characterized markers, which are lacking for most stem cell lineages. This chapter outlines an iterative process for the development of new cell surface marker barcodes for identifying and selecting stem cell derived progeny of specific cell types, subtypes, and maturation stages, where antibody-independent identification of cell surface proteins is achieved using a modern chemoproteomic approach to specifically identify N-glycoproteins localized to the cell surface. By taking advantage of a large repository of available cell surfaceome data, proteins that are unlikely to confer cell type specificity can be rapidly eliminated from consideration. Subsequently, targeted quantitation by mass spectrometry can be used to refine candidates of interest, and a bioinformatic visualization tool is key to mapping experimental data to candidate protein sequences for the purpose of epitope selection during the antibody development phase. Overall, the process of developing cell surface barcodes for immunophenotyping is iterative and can include multiple rounds of discovery, refinement, and validation depending on the phenotypic resolution required.

Deformation strain is the main physical driver for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation.

Mesenchymal stem cells play a major role during bone remodelling and are thus of high interest for tissue engineering and regenerative medicine applications. Mechanical stimuli, that is, deformation strain and interstitial fluid-flow-induced shear stress, promote osteogenic lineage commitment. However, the predominant physical stimulus that drives early osteogenic cell maturation is not clearly identified. The evaluation of each stimulus is challenging, as deformation and fluid-flow-induced shear stress interdepend. In this study, we developed a bioreactor that was used to culture mesenchymal stem cells harbouring a strain-responsive AP-1 luciferase reporter construct, on porous scaffolds. In addition to the reporter, mineralization and vitality of the cells was investigated by alizarin red staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Quantification of the expression of genes associated to bone regeneration and bone remodelling was used to confirm alizarin red measurements. Controlled perfusion and deformation of the 3-dimensional scaffold facilitated the alteration of the expression of osteogenic markers, luciferase activity, and calcification. To isolate the specific impact of scaffold deformation, a computational model was developed to derive a perfusion flow profile that results in dynamic shear stress conditions present in periodically loaded scaffolds. In comparison to actually deformed scaffolds, a lower expression of all measured readout parameters indicated that deformation strain is the predominant stimulus for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation.

Vascular Smooth Muscle Cell Maturation Stage and Ki-67 Index are Diagnostic Biomarkers for Pathologic Grade of Ovarian Teratoma.

Although immature teratoma of the ovary is a rare disease, its pathologic grading, especially between low-grade (grade 1) and high-grade (grade 2 or 3) immature teratomas, is important for optimal therapy and prognosis. This grading, however, is currently solely dependent on quantitation of neuroepithelial components as judged by subjective assessments. As we have recently successfully studied the maturation of vascular smooth muscle cells (SMCs) in other organs using an h-caldesmon to α-smooth muscle actin (α-SMA) ratio, we decided to use this ratio to investigate a potential link between teratoma grade and SMC maturation, in combination with Ki-67 index. Sixteen immature teratomas along with 5 mature teratomas of the ovary were studied and stained with antibodies to CD31, α-SMA, h-caldesmon, and Ki-67. The number of vascular SMCs calculated using the α-SMA/CD31 ratio did not differ between teratoma grades (except between grade 0 and 3), whereas the number of mature vascular SMCs calculated using the h-caldesmon/CD31 ratio and maturation state calculated using the h-caldesmon/α-SMA ratio reduced significantly as teratoma grade progressed from 0 to 3. Furthermore, these parameters were significantly lower in high-grade than in low-grade immature teratomas (P<0.05). Ki-67 labeling index, regardless of germ cell layer, also significantly increased with teratoma grade (P<0.05). These results suggest that not only the number of neuroepithelial elements but also vascular immaturity and proliferating cell counts are biomarkers for ovarian teratoma grading. Thus, assessment of the maturity of vascular SMCs may serve as a valuable diagnostic tool for assessing teratoma maturity.

Morphological, molecular and FTIR spectroscopic analysis during the differentiation of kidney cells from pluripotent stem cells

BackgroundKidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells.ResultsIn this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way.ConclusionOur results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.

FGF Signaling Prevents the Terminal Differentiation of Odontoblasts

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis, with conflicting results regarding their effects on odontoblast differentiation. Our recent studies showed that the effects of FGF2 on cells in odontoblast lineage were stage-specific and depended on the stage of cell maturity. Continuous exposure of pulp cells to FGF2 inhibited odontoblast differentiation, whereas early and limited exposure of pulp cells to FGF2 resulted in marked increases in odontoblast differentiation. The purpose of this study was to evaluate the cellular and molecular mechanisms regulating the inhibitory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of the addition of FGF2 during the differentiation/mineralization phase of the in vitro growth of pulp cultures derived from a series of green fluorescent protein reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation. Our results showed that this treatment first stimulated the differentiation of remaining progenitors in pulp cultures into functional odontoblasts but prevented their differentiation into mature odontoblasts. In addition, this treatment inhibited expression of markers of osteogenesis. Furthermore, we demonstrated that the inhibitory effects of FGF2 on odontoblast differentiation were mediated through activation of FGFR/MEK/Erk1/2 signaling and downregulation of bone morphogenetic protein signaling, with negative and positive roles in the expression of Dmp1 and Dspp, respectively, during the advanced stage of odontoblast differentiation.