Stages Of Cell Differentiation Research Articles

The role of autonomously secreted PGE2 and its autocrine/paracrine effect on bone matrix mineralization at the different stages of differentiating MC3T3-E1 cells

Bone is consisted of osteoblast-linage cells, bone-forming cells in various differentiation stages. However, it is not fully understood how communicate and interact these cells immigrated from bone marrow. In this study, we showed that prostaglandin E2 (PGE2) had a role in autonomous modification of matrix mineralization in osteoblastic cell line, MC3T3-E1, and interactions across the cells in different differentiation stages.Analysis using LC-MS/MS and inhibitors showed the autonomous secretion of PGE2 among the prostanoids in differentiation stages and that depend on COX-2, a key enzyme for production of PGE2. Treatment with inhibitors of PGE2 receptors and COX-2 indicated that secreted PGE2 regulates matrix mineralization in an autocrine/paracrine manner. In addition, we showed that the expression profile of PGE2 receptors (EP1-EP4) and PGE2 effects on matrix mineralization derived from it changed during cell differentiation.Treatment with inhibitors of PGE2 signaling in the early differentiation stage of MC3T3-E1 cells induced significant changes in matrix mineralization several days after. Stimulation with the extracts from culture medium of the matured cells including PGE2 and co-culture with the matured cells secreting PGE2 significantly promoted matrix mineralization of the early stage cells, in contrast, treatment with inhibitor of COX-2 and PGE2 receptors failed to do so. These results support that PGE2 plays important roles in the interaction system of osteoblast-linage cells in bone tissue to regulate matrix mineralization reflecting condition of bone-forming cells, that is, population and maturation.

Transcriptome analysis of axolotl oropharyngeal explants during taste bud differentiation stages

The Mexican salamander, Ambystoma mexicanum (Axolotl), is an excellent vertebrate model system to understand development and regeneration. Studies in axolotl embryos have provided important insights into taste bud development. Taste bud specification and determination occur in the oropharyngeal endoderm of axolotl embryos during gastrulation and neurulation, respectively, whereas taste bud innervation and taste cell differentiation occur later in development. Axolotl embryos are amenable to microsurgery, and tissue explants develop readily in vitro. We performed RNA-seq analysis to investigate the differential expression of genes in oropharyngeal explants at several stages of taste cell differentiation. Since the axolotl genome has only recently been sequenced, we used a Trinity pipeline to perform de novo assembly of sequencing reads. Linear models for RNA-seq data were used to identify differentially expressed genes. We found 1234 unique genes differentially expressed during taste cell differentiation stages. We validated four of these genes using RTqPCR and performed GO functional analysis. The differential expression of these genes suggests that they may play a role in taste cell differentiation in axolotls.

AFM Assessment of the Mechanical Properties of Stem Cells During Differentiation

ABSTRACTThe dynamic mechanical force transmitted through microenvironments during tissue formation and regeneration continuously impacts the mechanics of cells and thereby regulates gene and protein expression. The mechanical properties are altered during the process of stem cells differentiating into different lineages. At different stages of differentiation, stem cells display different mechanical properties in response to surrounding microenvironments, which depend on the subcellular structures, especially the cytoskeleton and nucleus. The mechanical properties of the cell nucleus affect protein folding and transport as well as the condensation of chromatin, through which the cell fate is regulated. These findings raise the question as to how cell mechanics change during differentiation. In this study, the mechanical properties of human bone marrow mesenchymal stem cells (hBMSCs) were determined during adipogenic and osteogenic differentiation by atomic force microscopy (AFM). The cytoskeletal structure and the modification of histone were investigated using laser confocal microscope and flow cytometry. The mechanical properties of cell nuclei at different stages of cell differentiation were compared. The stiffness of nuclei increased with time as osteogenesis was induced in hBMSCs. The H3K27me3 level increased during osteogenesis and adipogenesis according to flow cytometry analysis. Our results show conclusively that AFM is a facile and effective method to monitor stem cell differentiation. The measurement of cell mechanical properties by AFM improves our understanding on the connection between mechanics and stem cell fate.

SQLE Promotes Differentiation and Apoptosis of Bovine Skeletal Muscle-Derived Mesenchymal Stem Cells.

In this study, Squalene epoxidase (SQLE) overexpression vector was transfected into bovine skeletal muscle-derived mesenchymal stem/stromal cells (MSCs) to study the molecular mechanism of SQLE regulating meat quality through myogenesis. We initially profiled the expression of SQLE in cattle embryos and adults, in the muscle tissue of four different cattle varieties, and in 11 different tissues/organs of Guangxi cattle variety. Subsequently, we isolated and cultured bovine skeletal muscle-derived MSCs and detected the expression of SQLE during cell proliferation and differentiation. Then, we constructed a bovine SQLE overexpression vector and transfected it into bovine skeletal muscle-derived MSCs by liposome transfection. Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), flow cytometry, immunofluorescence, and quantitative polymerase chain reaction assays were used to characterize cell proliferation and differentiation in detail. The results showed that the relative expression level of bovine SQLE gene in brain tissue was the highest, and in adult muscle tissue was significantly higher than that in embryonic stage. Especially, the expression of SQLE was significantly upregulated in cell differentiation stage. Furthermore, the proliferation, cell cycle, apoptosis, and myoblast differentiation assays indicated that SQLE significantly promoted the differentiation and apoptosis of bovine skeletal muscle-derived MSCs, but inhibited their proliferation. In conclusion, our study reveals the role of SQLE in myoblast differentiation. These results will provide new clues for the regulation network of bovine muscle development.

A microfiber scaffold-based 3D in vitro human neuronal culture model of Alzheimer's disease.

Increasing evidence indicates superiority of three-dimensional (3D) in vitro cell culture systems over conventional two-dimensional (2D) monolayer cultures in mimicking native in vivo microenvironments. Tissue-engineered 3D culture models combined with stem cell technologies have advanced Alzheimer's disease (AD) pathogenesis studies. However, existing 3D neuronal models of AD overexpress mutant genes or have heterogeneities in composition, biological properties and cell differentiation stages. Here, we encapsulate patient induced pluripotent stem cell (iPSC) derived neural progenitor cells (NPC) in poly(lactic-co-glycolic acid) (PLGA) microtopographic scaffolds fabricated via wet electrospinning to develop a novel 3D culture model of AD. First, we enhanced cellular infiltration and distribution inside the scaffold by optimizing various process parameters such as fiber diameter, pore size, porosity and hydrophilicity. Next, we compared key neural stem cell features including viability, proliferation and differentiation in 3D culture with 2D monolayer controls. The 3D microfibrous substrate reduces cell proliferation and significantly accelerates neuronal differentiation within seven days of culture. Furthermore, 3D culture spontaneously enhanced pathogenic amyloid-beta 42 (Aβ42) and phospho-tau levels in differentiated neurons carrying familial AD (FAD) mutations, compared with age-matched healthy controls. Overall, our tunable scaffold-based 3D neuronal culture platform serves as a suitable in vitro model that robustly recapitulates and accelerates the pathogenic characteristics of FAD-iPSC derived neurons.

Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis

ABSTRACTThe discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the co-localization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatids-specific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosome.

Phenotypical T Cell Differentiation Analysis: A Diagnostic and Predictive Tool in the Study of Primary Immunodeficiencies.

Multiparametric flow cytometry (MFC) represents a rapid, highly reproducible, and sensitive diagnostic technology for primary immunodeficiencies (PIDs), which are characterized by a wide range of T cell perturbations and a broad clinical and genetic heterogeneity. MFC data from CD4+ and CD8+ T cell subsets were examined in 100 patients referred for Primary Immunodeficiencies to our center. Naïve, central memory, effector memory, and terminal effector memory cell differentiation stages were defined by the combined expression CD45RA/CD27 for CD4 and CD45RA/CCR7 for CD8. Principal component analysis (PCA), a non-hypothesis driven statistical analysis, was applied to analyze MFC data in order to distinguish the diverse PIDs. Among severe lymphopenic patients, those affected by severe combined and combined immunodeficiency (SCID and CID) segregated in a specific area, reflecting a homogenous, and a more severe T cell impairment, compared to other lymphopenic PID, such as thymectomized and partial DiGeorge syndrome patients. PID patients with predominantly antibody defects were distributed in a heterogeneous pattern, but unexpectedly PCA was able to cluster some patients' resembling CID, hence warning for additional and more extensive diagnostic tests and a diverse clinical management. In conclusion, PCA applied to T cell MFC data might help the physician to estimate the severity of specific PID and to diversify the clinical and diagnostic approach of the patients.

Low-Dose Epo after Tgfβ Blockade Triggers Robust Erythropoiesis and Increased RBC Production

Background: Therapy for chronic anemias is limited to RBC transfusions and Erythropoiesis Stimulating Agents (ESA) which often work on only transiently or not at all. New approaches to treat chronic anemia are needed but development has been limited by our incomplete understanding of erythropoiesis, most of which relates to the terminal maturation of erythroid precursors. Erythropoietin (Epo) acts during a very narrow window of erythropoiesis, well after progenitor commitment to an exclusively erythroid fate. It is not known if the final steps of RBC maturation are coupled to the earlier stages of hematopoietic stem and progenitor cell (HSPC) differentiation; a process that begins almost three weeks earlier when an HSC starts its march towards committed RBC precursors via a series of branching cell fate decisions.We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Results: We deleted TGFβ1 in megakaryocytes (TGFβ1ΔMk/ΔMk) and found that peripheral blood counts were normal in TGFβ1ΔMk/ΔMkmice compared to TGFβ1FL/FLcontrols despite the pool of primitive hematopoietic cells being expanded (Fig. 1a). Similarly total bone marrow cellularity was normal in TGFβ1ΔMk/ΔMkmice (Fig. 1b). Excess HSCs in TGFβ1ΔMk/ΔMkmice appeared capable of robust differentiation because the number of immature lineage-negative (Linneg) hematopoietic progenitor cells was increased in the marrows of TGFβ1ΔMk/ΔMkmice (Fig. 1c). Thus, it remained unexplained why the expanded number of HSPCs (Fig. 1d) do not increase blood counts and marrow cellularity. We hypothesized that the excess progenitors observed in the TGFβ1ΔMk/ΔMkmice failed to increase blood counts because their progeny were unneeded, and inadequately supported by homeostatic levels of late-acting cytokines. Indeed, bone marrow apoptosis was increased in the TGFβ1ΔMk/ΔMkmice compared to controls, as reported by AnnexinV (AV) binding (Fig. 1e-f). Apoptosis of lineage-marker negative (Linneg), Kit+Sca1neg(LKSneg) HPCs and LKS+HSPCs was rare in both TGFβ1ΔMk/ΔMkmice and littermate controls (Fig. 1g). These results suggest that excess, hematopoietic precursors present in the TGFβ1ΔMk/ΔMkmice are pruned by apoptosis during hematopoietic differentiation. We found 10-fold apoptosis in TGFβ1ΔMk/ΔMkprecursors populations BM (Fig. 1h). Epo levels were normal in the serum of these mice, we reasoned that the excess, unneeded cells were not supported physiologic Epo levels. To test this, we treated mice with exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (300U/kg)(Fig. 1i-j). Whereas TGFβ1Flox/Floxmice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMkmice responded with reticulocytosis and erythrocytosis within 6 days (Fig. 1k-l). In contrast, treatment of mice with TGFβ1 worsened the erythroid apoptosis observed in TGFβ1ΔMk/ΔMkmice and caused mild anemia. These results suggest that erythropoiesis is subject to modular regulation with megakaryocytic TGFβ1 constraining the pool of erythroid committed progenitors that are then licensed to mature via Epo signaling. We thought that blockade of TGFβ signaling could phenocopy these effects by inducing overproduction of erythroid committed precursors. To test this, we pre-treated B6 mice with a TGFβ1 neutralizing antibody (1D11) or non-targeting, isotype control antibody (13C4) and then either PBS or low-dose Epo (Fig. 1m). TGFβ neutralization by 1D11 reduced pSmad2/3 MFI in HSPCs in wild-type mice whereas the 13C4 control had no effect, demonstrating on-target activity (Fig. 1n). Low-dose Epo triggered a brisk erythropoietic response in mice treated with 1D11 but not those treated with the 13C4 control (Fig. 1o). Exogenous Epo rescued the erythroid precursor dropout observed in B6 mice treated with 1D11 but did not affect the low apoptosis observed in mice treated with the 13C4 control (Fig. 1p-r). Therefore, the boundary of megakaryocytic TGFβ1 activity is compartmentalized within the marrow with predominant effects on immature HSPCs while excluding their progeny (Fig. 4s). Conclusion: This work also promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Chromatin accessibility and transcription dynamics during in vitro astrocyte differentiation of Huntington\u2019s Disease Monkey pluripotent stem cells

BackgroundHuntington’s Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD.ResultsWe found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed.ConclusionsThe results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.

Computational Analysis of Multiparametric Flow Cytometric Data to Dissect B Cell Subsets in Vaccine Studies.

The generation of the B cell response upon vaccination is characterized by the induction of different functional and phenotypic subpopulations and is strongly dependent on the vaccine formulation, including the adjuvant used. Here, we have profiled the different B cell subsets elicited upon vaccination, using machine learning methods for interpreting high‐dimensional flow cytometry data sets. The B cell response elicited by an adjuvanted vaccine formulation, compared to the antigen alone, was characterized using two automated methods based on clustering (FlowSOM) and dimensional reduction (t‐SNE) approaches. The clustering method identified, based on multiple marker expression, different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their subsets, while this profiling was more difficult with t‐SNE analysis. When undefined phenotypes were detected, their characterization could be improved by integrating the t‐SNE spatial visualization of cells with the FlowSOM clusters. The frequency of some cellular subsets, in particular plasma cells, was significantly higher in lymph nodes of mice primed with the adjuvanted formulation compared to antigen alone. Thanks to this automatic data analysis it was possible to identify, in an unbiased way, different B cell populations and also intermediate stages of cell differentiation elicited by immunization, thus providing a signature of B cell recall response that can be hardly obtained with the classical bidimensional gating analysis. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation.

Interplay between CXCR7 and other CXC receptors, namely CXCR4 or CXCR3, binding such ligands as SDF-1 or ITAC, was shown to regulate multiple cellular processes. The developmental role of signaling pathways mediated by these receptors was proven by the phenotypes of mice lacking either functional CXCR4, or CXCR7, or SDF-1, showing that formation of certain lineages relies on these factors. In this study, using in vitro differentiating mouse embryonic stem cells that lacked the function of CXCR7, we asked the question about the role of CXCR mediated signaling during early steps of differentiation. Our analysis showed that interaction of SDF-1 or ITAC with CXC receptors is necessary for the regulation of crucial developmental regulators expression and that CXCR7 is involved in the control of ESC pluripotency and differentiation into mesodermal lineages.

Thyroid Gland Involvement and the Efficiency of Thyroidectomy in Patients Having Larynx and Hypopharyngeal Cancers Treated with Surgery

Background: Thyroid gland involvement and invasion during larynx and hypopharynx cancers is rare, but due to the anatomical closeness of the thyroid gland and the larynx, it could occur through local invasion. Thyroidectomy is usually performed in patients with thyroid gland involvement having larynx and hypopharyngeal cancers. A lot of controversies exist regarding the thyroid gland involvement and efficiency of a different kind of thyroidectomy intraoperatively in these types of cancers. Objectives: Thus, elucidating the exact role of thyroidectomy, its efficiency, and parameters associated with thyroid gland involvement is critical in treating the patients with the larynx and hypopharyngeal cancers. Methods: In this retrospective study, the medical records of 117 patients admitted to Loghman Hakim Hospital from 2001 to 2015 with laryngeal and hypopharyngeal carcinoma, who underwent laryngectomy with or without thyroidectomy as a primary treatment of cancer, were evaluated. Several parameters, including gender, prevalence, location of the thyroid gland and lymph node involvement, the exact type of malignancy, differentiation stage of the tumor cells, the exact method of thyroidectomy, thyroid function, and its post-surgical cancer recurrence were assessed. Results: The prevalence of laryngeal cancers was significantly higher in males than females and most of the malignancies were squamous cell carcinoma (SCC). The location of the malignancy was mostly at supraglottis, glottis, transglottis, and subglottis, respectively. The rate of thyroid gland involvement was approximately 11.96%. Lobectomy was performed in 57.14 %, total thyroidectomy was performed in 35.71 %, and in 7.14% of the patients, and thyroidectomy was not performed. After the initial surgery, out of 11.96% of all patients with thyroid gland involvement, 28.5% had a recurrence of the thyroid gland. Only 21% of the patients with thyroid gland involvement had hypothyroidism after the surgery. Conclusions: The rate of thyroid gland involvement in the pathology report after surgery was approximately 3.41% of the thyroid gland involvement, which is low. We concluded that thyroidectomy is a very efficient way, but it should be limited to cases of subglottic or transglobic involvement or in cases of bilateral tumors, high staging, post-cricoid involvement, and clear radiological evidence or in the event of muscle involvement.

Transcriptional, Epigenetic and Pharmacological Control of JAK/STAT Pathway in NK Cells.

Differentiation of Natural Killer (NK) cells is a stepwise process having its origin in the bone marrow and proceeding in the periphery, where these cells follow organ specific trajectories. Several soluble factors and cytokines regulate the distinct stages of NK cell differentiation, and ultimately, their functional properties. Cytokines activating the Janus kinases (JAKs) and members of the signal transducer and activator of transcription (STAT) pathway control distinct aspects of NK cell biology, ranging from development, terminal differentiation, activation, and generation of cells with adaptive properties. Here, we discuss how the recent advances of next generation sequencing (NGS) technology have led to unravel novel molecular aspects of gene regulation, with the aim to provide genomic views of how STATs regulate transcriptional and epigenetic features of NK cells during the different functional stages.

Flow Cytometric Analysis of Hematopoietic Populations in Rat Bone Marrow. Impact of Trauma and Hemorrhagic Shock.

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP‐1) lineages. Subsequently, cell populations were fluorescence‐activated cell sorted for morphological assessment. Stage‐specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP‐1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP‐1(−)) and mature (RP‐1+) granulocytes. Multiple B‐cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(−) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

IN EXPERIMENTAL RAT DIABETES TRANSCRIPTION FACTOR MAFA IS SYNTHESIZED IN DIFFERENTIATED PANCREATIC ISLET CELLS AND DOES NOT SERVE AS A MARKER OF PROGENITOR CELLS

Different transcription factors, which are synthesized at different stages of cell differentiation, are often considered markers of p-cell precursors. One of these transcription factors is MafA, the role of which is not fully understood. According to one hypothesis, it activates insulin gene expression in the differentiating p-cells. According to another, this factor is only necessary for the regulation of insulin secretion by already differentiated p-cells. In favor of the latter hypothesis, we showed that MafA is not expressed in the immature p-cells during the prenatal development of the human pancreas. In order to finally determine whether MafA is a marker of differentiating cells or it is synthesized in already mature p-cells the aim of our investigation was the analysis of dynamical changes of MafA-positive cell population and C-kit-positive endocrinocyte precursors in Langerhans islets during experimental diabetes in rats. The study was performed on male Wistar rats (250-300g body weight) which were intraperitoneally injected with alloxan. Animals were sacrificed 1,2, 7, 14, 21 days of the experiment for the morphological analysis of pancreas. Paraffin sections of pancreas were stained immunohistochemically with antibodies against MafA, C-kit, insulin and glucagon. The maximum number of MafA-positive cells in the islets was found during normal prenatal development of pancreas. At all stages of the experimental diabetes the number of MafA-positive cells in the islets decreased, wherein the number of insulin-positive cells in the islets increased by the end of the first and third weeks of the experiment. It was also established that in experimental diabetes, changes in populations of MafA- and C-kit-positive cells occur in different ways. Thus, the results of our research showed that MafA cannot be considered as a marker of progenitor cells and is expressed only in the mature cells of the Langerhans islets, that confirms our previous data obtained during the study of prenatal development of human pancreas.

Cysteine Cathepsins in Tumor-Associated Immune Cells

Cysteine cathepsins are key regulators of the innate and adaptive arms of the immune system. Their expression, activity, and subcellular localization are associated with the distinct development and differentiation stages of immune cells. They promote the activation of innate myeloid immune cells since they contribute to toll-like receptor signaling and to cytokine secretion. Furthermore, they control lysosomal biogenesis and autophagic flux, thus affecting innate immune cell survival and polarization. They also regulate bidirectional communication between the cell exterior and the cytoskeleton, thus influencing cell interactions, morphology, and motility. Importantly, cysteine cathepsins contribute to the priming of adaptive immune cells by controlling antigen presentation and are involved in cytotoxic granule mediated killing in cytotoxic T lymphocytes and natural killer cells. Cathepins'aberrant activity can be prevented by their endogenous inhibitors, cystatins. However, dysregulated proteolysis contributes significantly to tumor progression also by modulation of the antitumor immune response. Especially tumor-associated myeloid cells, such as tumor-associated macrophages and myeloid-derived suppressor cells, which are known for their tumor promoting and immunosuppressive functions, constitute the major source of excessive cysteine cathepsin activity in cancer. Since they are enriched in the tumor microenvironment, cysteine cathepsins represent exciting targets for development of new diagnostic and therapeutic moieties.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.

Abstract 2156: Quantifying cell subsets and heterogeneity in living cultures using real time live-cell analysis

Abstract Considerable heterogeneity exists in the properties and activity of individual cells, even in the simplest cell system. This arises from fundamental differences in the basic cell types present, genetic or epigenetic variations, the stage of cell cycle or differentiation, and the impact of each cell’s unique and dynamic local microenvironment. Such heterogeneity is mirrored by the diversity of pharmacological response at the cellular level, where even seemingly identical cells may respond differently and at different times to drug treatments and perturbagens. Accordingly, analysis at the cell-by-cell level promises valuable and additional biological insight beyond which whole population measures may deliver. Here, we describe new, enabling and industrial-scale, live-cell analysis solutions for quantifying the phenotypic biology of cell subsets in heterogeneous cultures. Time-lapse images of cultured cancer and immune cells in 96-well microplates were automatically collected using an IncuCyte S3 live analyzer (Essen Bioscience). Using new segmentation algorithms, the boundaries of individual cells (typically 300-1000 per image) were identified in each image of the sequence. Parameters and features were extracted from single cells, such as cell area, eccentricity and fluorescence (e.g. with cell labels, cytotoxicity and apoptosis probes). Populations of cells could be identified and classified over time using custom ‘flow cytometry’-like visualisation and classification tools. Using this new approach, we demonstrate cell-by-cell analysis for a range of different primary and immortalised, adherent and non-adherent, living cells for up to 7 days in culture (e.g. Jurkat, A549, human PBMCs). This was coupled with a novel live-cell, fluorescent antibody-based labeling strategy (IncuCyte FabFluor-488) to probe for specific subsets within the cultures. Example data generated in PBMCs during T cell activation (anti-CD3/IL-2, 10 ng/mL) demonstrates the change in cell shape from small, spherical cells (average area 81 ± 0.5 µm2, eccentricity 0.57 ± 0.002) to larger, flatter cells (117 ± 4 µm2, 0.69 ± 0.004) over 120 h. With the addition of FabFluor-488-CD71, it was possible to show an associated, temporal increase in CD71 expression within the activated T cell subset (75 ± 1% of large cells were CD71 positive compared to 12 ± 1% of smaller cells at 48 h, increasing to >90% in the larger cells by 120 h). Other example data sets for subset analysis of proliferation, cell death and cell cycle measurements as well as immuno-phenotyping will be shared to illustrate the value of this approach. Citation Format: Nicola Bevan, Tim Jackson, Clare Szybut, Lauren Kelsey, Hinnah Campwala, Tim Dale, Nicholas Dana, Nevine Holtz, Eric Endsley, Dan Appledorn, Cicely Schramm, Laura Skerlos, Richard Lister, Derek J. Trezise. Quantifying cell subsets and heterogeneity in living cultures using real time live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2156.

Senescence-associated β-galactosidase activity in the developing avian retina.

Senescence-associated β-galactosidase (SA-β-GAL) histochemistry is the most commonly used biomarker of cellular senescence. These SA-β-GAL-positive cells are senescent embryonic cells that are usually removed by apoptosis from the embryo, followed by macrophage-mediated clearance. Some authors have proposed that SA-β-GAL activity in differentiated neurons from young and adult mammals cannot be uniquely attributed to cell senescence, whether in vivo or in vitro. Using the developing visual system of the chicken as a model, the present study found that SA-β-GAL detected in the developing retina corresponded to lysosomal β-galactosidase activity, and that SA-β-GAL activity did not correlate with the chronotopographical distribution of apoptotic cells. However, SA-β-GAL staining in the undifferentiated retina coincided with the appearance of early differentiating neurons. In the laminated retina, SA-β-GAL staining was concentrated in the ganglion, amacrine, and horizontal cell layers. The photoreceptors and pigment epithelial cells also exhibited SA-β-GAL activity throughout retinal development. We have also found that SA-β-GAL staining strongly correlated p21 immunoreactivity. In conclusion, the results clearly show that SA-β-GAL activity cannot be regarded as a specific marker of senescence during retinal development, and that it is mainly expressed in subpopulations of postmitotic neurons, which are nonproliferative cells, even at early stages of cell differentiation.

Expression of serine/threonine protein kinase SGK1F promotes an hepatoblast state in stem cells directed to differentiate into hepatocytes.

The rat pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like (B-13/H) cells in response to glucocorticoid. Since this response is dependent on an induction of serine/threonine protein kinase 1 (SGK1), this may suggest that a general pivotal role for SGK1 in hepatocyte maturation. To test this hypothesis, the effects of expressing adenoviral-encoded flag tagged human SGK1F (AdV-SGK1F) was examined at 3 stages of human induced pluripotent stem cell (iPSC) differentiation to hepatocytes. B-13 cells infected with AdV-SGK1F in the absence of glucocorticoid resulted in expression of flag tagged SGK1F protein; increases in β-catenin phosphorylation; decreases in Tcf/Lef transcriptional activity; expression of hepatocyte marker genes and conversion of B-13 cells to a cell phenotype near-similar to B-13/H cells. Given this demonstration of functionality, iPSCs directed to differentiate towards hepatocyte-like cells using a standard protocol of chemical inhibitors and mixtures of growth factors were additionally infected with AdV-SGK1F, either at an early time point during differentiation to endoderm; during endoderm differentiation to anterior definitive endoderm and hepatoblasts and once converted to hepatocyte-like cells. SGK1F expression had no effect on differentiation to endoderm, likely due to low levels of expression. However, expression of SGK1F in both iPSCs-derived endoderm and hepatocyte-like cells both resulted in promotion of cells to an hepatoblast phenotype. These data demonstrate that SGK1 expression promotes an hepatoblast phenotype rather than maturation of human iPSC towards a mature hepatocyte phenotype and suggest a transient role for Sgk1 in promoting an hepatoblast state in B-13 trans-differentiation to B-13/H cells.