Cell Types Of Origin Research Articles

PD-L1/PD-1 Check Point in Anaplastic Large Cell Lymphoma, ALK+: A Case Report with Immunohistochemical and Molecular Study

Anaplastic large cell lymphoma, ALK+ (ALK+ ALCL) is a T-cell lymphoma consisting of large and pleomorphic lymphoid cells, often with horseshoe-shaped nuclei, with a chromosomal translocation involving the ALK gene and expression of ALK protein and CD30. ALK+ ALCL must be distinguished from primary cutaneous ALCL and from other subtype of T-cell or B-cell lymphoma with anaplastic features and/or CD30 expression. PD-L1/PD-1 check point physiologically plays a key role in induction and maintenance of immune tolerance to self-antigens and limits normal immune response against microorganisms. PD-L1 has been demonstrated in the whole spectrum of normal hematopoietic and non-hematopoietic cells, as well as in a large variety of epithelial cells. Moreover, it is also very commonly expressed by a large number of malignant cell types of epithelial and hematopoietic cell origin, and its activation is one of the major mechanisms exerted by neoplastic cells to evade elimination by the host immune system. Here we report a case of an ALK+ ALCL, with confirmed FISH rearrangement of ALK gene, PD-L1-positive and discuss the molecular mechanism involved in its immunohistochemical profile.

Cell Type of Origin Dictates the Route to Pluripotency

Our current understanding of induced pluripotent stem (iPS) cell generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether or not the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By functionally and molecularly characterizing the reprogramming pathways of fibroblasts, neutrophils and keratinocytes, we unveil that while some aspects of the reprogramming process are conserved, the key events, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion and reactivation of the pluripotency network are to a large degree cell type specific. Thus we reveal universal and cell type specific features of the reprogramming process.

Wnt Signaling in Hematological Malignancies.

Leukemia and lymphoma are a wide encompassing term for a diverse set of blood malignancies that affect people of all ages and result in approximately 23,000 deaths in the United States per year (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Hematopoietic stem cells (HSCs) are tissue-specific stem cells at the apex of the hierarchy that gives rise to all of the terminally differentiated blood cells, through progressively restricted progenitor populations, a process that is known to be Wnt-responsive. In particular, the progenitor populations are subject to uncontrolled expansion during oncogenic processes, namely the common myeloid progenitor and common lymphoid progenitor, as well as the myeloblast and lymphoblast. Unregulated growth of these cell-types leads to mainly three types of blood cancers (i.e., leukemia, lymphoma, and myeloma), which frequently exhibit deregulation of the Wnt signaling pathway. Generally, leukemia is caused by the expansion of myeloid progenitors, leading to an overproduction of white blood cells; as such, patients are unable to make sufficient numbers of red blood cells and platelets. Likewise, an overproduction of lymphocytes leads to clogging of the lymph system and impairment of the immune system in lymphomas. Finally, cancer of the plasma cells in the blood is called myeloma, which also leads to immune system failure. Within each of these three types of blood cancers, there are multiple subtypes, usually characterized by their timeline of onset and their cell type of origin. Of these, 85% of leukemias are encompassed by the four most common diseases, that is, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); AML accounts for the majority of leukemia-related deaths (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Through understanding how HSCs are normally developed and maintained, we can understand how the normal functions of these pathways are disrupted during blood cancer progression; the Wnt pathway is important in regulation of both normal and malignant hematopoiesis. In this chapter, we will discuss the role of Wnt signaling in normal and aberrant hematopoiesis. Our understanding the relationship between Wnt and HSCs will provide novel insights into therapeutic targets.

Epigenetics in Clinical Management of Children and Adolescents with Brain Tumors.

Central nervous system (CNS) tumors represent the second most prevalent group of cancers in children and adolescents, yet account for the majority of childhood cancer-related deaths and considerable morbidity among survivors, due to high-intensity non-selective standard therapies delivered to immature nervous system structures undergoing development. These tumors arise at different ages -not infrequently very early in life-, in different locations and cellular contexts, have varied cell types of origin, and have heterogeneous responses to the "classic" current therapeutic approaches. Demographic, radiologic and morphological characterization have several limitations, putting into the "classic boxes" heterogeneous tumors that are diverse in their genetic and epigenetic background and that will likely behave biologically different. Given that, epigenetic disruption (i.e. DNA methylation, histone modification and chromatin remodeling) is a common feature identified more and more frequently in pediatric cancer, it is logical to speculate that interrogating epigenetic marks may help to further define the molecular profile, and therefore tumor biology, evolution and treatment of these tumors. An integrated approach that incorporates traditional features complemented with genetic and epigenenetic specific markers offers tremendous promise to "risk-group" stratification and better prognostication. Also, it will help unveil the key driver pathways for tumor formation and for the discovery of targeted therapy for neoplasms that appear in the developing brain, facilitating early identification of therapy responders and track accurately disease progression. In this paper, we reviewed the most representative pediatric brain tumors where epigenetic alterations have been identified as initiating or driving events in tumor development, maintenance or progression.

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

ABSTRACTBackground: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity.Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing.Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS.Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures.Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Cell Type of Origin Dictates the Route to Pluripotency

Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

Anticancer potential of Amaryllidaceae alkaloids evaluated by screening with a panel of human cells, real-time cellular analysis and Ehrlich tumor-bearing mice

In this study, twenty-two Amaryllidaceae alkaloids were screened for their anticancer potential. All isolates were evaluated for antiproliferative activities on a panel of 17 human cell types of different tissue origin using WST-1 assay. In addition, we determined the antiproliferative effect with a real-time cell analysis xCELLigence system. Thereafter, to evaluate the barely known in vivo anticancer potential of the most potent molecule haemanthamine, a preliminary study was performed using an Ehrlich tumor-bearing mice model. The results showed that haemanthamine, lycorine and haemanthidine exerted the highest antiproliferative activity. The mean growth percent (GP) value after a single-dose 10 μM treatment was for haemanthamine 21%, for lycorine 21% and for haemanthidine 27% that of untreated control cells (100%). Furthermore, haemanthamine, lycorine and haemanthidine exhibited significant cytotoxicities against all the tested cell lines with individual IC50 values in the micromolar range. Dynamic real-time measures of impedance by xCELLigence indicated that these three compounds suppress cell proliferation after 10 h of treatment at a concentration of 10 μM or higher. Regrettably, in a follow-up in vivo antitumor activity study, haemanthamine showed no statistically significant reduction in the tumor size with no prolongation of survival time of Ehrlich tumor-bearing mice. Taken together, these results provide a new clue and guidance for exploiting Amaryllidaceae alkaloids as anticancer agents.

Abstract 4350: Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal cancer

Abstract Introduction: Colorectal cancer accounts for a significant fraction of cancer-related mortalities, but has proven to be surprisingly refractory to consensus pathological and molecular subclassification. Similarly, little is known about the molecular profiles of colorectal cancer cells-of-origin. DNA methylation is an important epigenomic marker of cellular identity and can therefore be used to infer cancer cells-of-origin. Methods: Infinium 450k data from colorectal cancer samples provided by “The Cancer Genome Atlas” was applied to a consensus clustering algorithm. The identified epigenetic subgroups were tested for features including well established clinical parameters or subgroup specific gene expression. Additionally, the subgroups were compared with Infinium 450k data of non-malignant colorectal adenomas. Finally, clinical significance was addressed by testing for subgroup specific overall survival rates. Results: Analysis of the TCGA dataset defined 5 distinct epigenetic subtypes of human colorectal cancer. These subgroups showed an overlap to the microsatellite instability phenotype and the CpG island methylator phenotype. Gene expression based analysis revealed that the subtypes also form a continuum of epigenetic programs reflecting various intestinal crypt cell differentiation stages. Patient survival correlated with the differentiation stage, with a particular poor prognosis for patients with stem cell-related signatures. Interestingly, its prognostic potential outperformed a recently established gene expression-based classifier. Finally, non-malignant colorectal adenomas could be classified into the same methylation subtypes, reflecting their shared cell-types of origin with carcinomas Conclusion: Our results establish a novel and clinically relevant approach for colorectal cancer classification and illustrate how differences in the cell-type of origin shape the tumor methylome. Citation Format: Felix Bormann, Manuel Rodríguez-Paredes, Yehudit Bergman, Heinz G. Linhart, Frank Lyko. Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2017-4350

Bone Marrow Stromal Stem Cells in Tissue Engineering and Regenerative Medicine.

Bone marrow stromal stem cells (BMSCs) are adult multipotent cells, which have the potential to differentiate into cell types of mesodermal origin, namely osteocytes, adipocytes, and chondrocytes. Due to their accessibility and expansion potential, BMSCs have historically held therapeutic promise in tissue engineering and regenerative medicine applications. More recently, it has been demonstrated that not only can bone marrow stromal stem cells directly participate in tissue regeneration, but they also have the capacity to migrate to distant sites of tissue injury, where they can participate in tissue repair either directly through their differentiation or indirectly through paracrine mechanisms. Additionally, they can elicit various immunomodulatory signals, which can attenuate the inflammatory and immune responses. As such, bone marrow stromal stem cells have been explored clinically for treatment of a wide variety of different conditions including bone defects, graft-vs.-host disease, cardiovascular diseases, autoimmune diseases, diabetes, neurological diseases, and liver and kidney diseases. This review provides an overview of current clinical applications of bone marrow stromal stem cells and discusses their therapeutic properties, while also addressing limitations of their use. PubMed, Ovid, and Google Scholar online databases were searched using several keywords, including "stem cells", "tissue engineering", tissue regeneration" and "clinical trials". Additionally, Clinical trials.gov was used to locate completed clinical trials using bone marrow derived stem cells.

Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines.

Patient-specific induced pluripotent stem cells (iPSCs) facilitate understanding of the etiology of diseases, discovery of new drugs and development of novel therapeutic interventions. A frequently used starting source of cells for generating iPSCs has been dermal fibroblasts (DFs) isolated from skin biopsies. However, there are also numerous repositories containing lymphoblastoid B-cell lines (LCLs) generated from a variety of patients. To date, this rich bioresource of LCLs has been underused for generating iPSCs, and its use would greatly expand the range of targeted diseases that could be studied by using patient-specific iPSCs. However, it remains unclear whether patient’s LCL-derived iPSCs (LiPSCs) can function as a disease model. Therefore, we generated Parkinson’s disease patient-specific LiPSCs and evaluated their utility as tools for modeling neurological diseases. We established iPSCs from two LCL clones, which were derived from a healthy donor and a patient carrying PARK2 mutations, by using existing non-integrating episomal protocols. Whole genome sequencing (WGS) and comparative genomic hybridization (CGH) analyses showed that the appearance of somatic variations in the genomes of the iPSCs did not vary substantially according to the original cell types (LCLs, T-cells and fibroblasts). Furthermore, LiPSCs could be differentiated into functional neurons by using the direct neurosphere conversion method (dNS method), and they showed several Parkinson’s disease phenotypes that were similar to those of DF-iPSCs. These data indicate that the global LCL repositories can be used as a resource for generating iPSCs and disease models. Thus, LCLs are the powerful tools for generating iPSCs and modeling neurological diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0267-6) contains supplementary material, which is available to authorized users.

Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of EVs reflects the type and status of the originating cell and EVs in melanoma patient’s plasma could be indicative for the tumor. Likewise, EVs might influence tumor progression by regulating immune responses. We performed a broad protein characterization of EVs from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer (NK) cells, monocytes, monocyte-derived dendritic cells (moDCs), and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on EVs. Hierarchical clustering of protein intensity patterns grouped EVs according to their originating cell type. The analysis of EVs from stimulated B cells and moDCs revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of EVs from platelets, antigen-presenting cells and NK cells as shown by platelet markers, co-stimulatory proteins, and a NK cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers, indicating a changed vesicle secretion or protein loading of EVs by platelets and a lower CD8 signal that might be associated with a diminished activity of NK cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of EVs in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction.

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators.

Nonadherent Spheres With Multiple Myeloma Surface Markers Contain Cells that Contribute to Sphere Formation and Are Capable of Internalizing Extracellular Double-Stranded DNA.

The most prominent features of cancer stem cells are asymmetric cell division, tumorigenicity, and clonogenicity. Recently one more feature of poorly differentiated cell types of various origin, including cancer stem cells, has been described. Namely, these cells can internalize extracellular DNA natively, without additional transfection procedures. Using our approach to trace internalization of a TAMRA (carboxy tetramethyl-rhodamine [fluorescent dye])-DNA labeled probe by poorly differentiated cell types, we isolated and characterized the cells from free-floating spheres derived from the bone marrow clonogenic aspirate of a multiple myeloma patient. Nonadherent spheres display a B-cell phenotype (CD73/CD20+/CD45+/CD19dim). Further, free-floating spheres contain 1% to 3% cells with a clonogenic potential, and these cells display a marker of poorly differentiated cell types (TAMRA+). Upon association with a group of ∼ 10 free-floating TAMRA- cells, this peculiar cell type forms a sphere-forming cluster that initiates secondary aggregation of cells into a spheric structure. TAMRA+ and TAMRA- cells secrete distinct sets of cytokines indicative of the paracrine regulation. Grafting experiments of intact whole spheres versus cell suspensions prepared from dispersed spheres indicate that successful engraftment only occurs in the former case. Nonadherent 3-D cell colonies (spheres) encompass B cells with CD73/CD20+/CD45+/CD19dim phenotype, as well as double-stranded DNA-internalizing cells. The latter cell type appears to function as a sphere-forming center. Different cells in the spheres communicate with each other by secreting specific sets of cytokines. For successful engraftment and tumor growth in mice, intact spheres containing ∼ 106 cells must be used.

Clinical effectiveness of cell therapies in patients with chronic liver disease and acute-on-chronic liver failure: a systematic review protocol.

BackgroundChronic liver disease (CLD) is a major health burden worldwide. Liver cirrhosis, a form of CLD is the fifth most common cause of death in the UK. Acute-on-chronic liver failure (ACLF) is the result of an acute insult superimposed on patients with liver cirrhosis as a result of precipitating events such as infection or bleeding. ACLF has a high associated mortality as a result of multi-organ failure. The only effective treatment for CLD is liver transplantation, but the treatment is limited by shortage of donor organs. As a result, alternative treatments such as cell therapies have been studied in patients with liver diseases. This study will systematically review the evidence on clinical effectiveness of cell therapies in patients.MethodsAll types of study design that investigate the effectiveness of cell therapies (haematopoietic, mesenchymal and unsorted cell types) of autologous or allogeneic origin and/or the use of granulocyte colony-stimulating factor in patients with CLD including ACLF will be included (except case reports). Both autologous and allogenic cell types will be included. The primary outcomes of interest are survival, model for end-stage liver disease score, quality of life and adverse events. Secondary outcomes include liver function tests, Child-Pugh score and events of liver decompensation. A literature search will be conducted in the following databases: MEDLINE, MEDLINE in Process, EMBASE and Cochrane Library (CENTRAL, CDSR, DARE, HTA databases). Trial registers will be searched for ongoing trials, as will conference proceedings. Reference lists of relevant articles and systematic reviews will be screened. Randomised controlled trial (RCT) evidence is likely to be scant; therefore, controlled trials and concurrently controlled observational studies will be primarily analysed and uncontrolled observational studies will be analysed where primary outcomes are not reported in the control studies or where uncontrolled studies have longer follow-up. Initial screening of studies will be carried by one reviewer with a proportion checked by another reviewer. Full-text selection will be performed by two reviewers independently against the pre-defined selection criteria. The data collection and the risk of bias assessment will be completed by one reviewer and counter checked by another reviewer for all selected studies. Where appropriate, data will be meta-analysed for each study design, therapy and outcome. Data specifically on ACLF will be treated as a subgroup.DiscussionThis systematic review will identify the available evidence on the effectiveness of cell therapies in patients with CLD and in ACLF subgroup. The findings will aid decision-making by clinicians and health service leaders.Systematic review registrationPROSPERO CRD42016016104Electronic supplementary materialThe online version of this article (doi:10.1186/s13643-016-0277-6) contains supplementary material, which is available to authorized users.

Spatially Constrained Growth Enhances Conversional Meltdown

Cells that mutate or commit to a specialized function (differentiate) often undergo conversions that are effectively irreversible. Slowed growth of converted cells can act as a form of selection, balancing unidirectional conversion to maintain both cell types at a steady-state ratio. However, when one-way conversion is insufficiently counterbalanced by selection, the original cell type will ultimately be lost, often with negative impacts on the population’s overall fitness. The critical balance between selection and conversion needed for preservation of unconverted cells and the steady-state ratio between cell types depends on the spatial circumstances under which cells proliferate. We present experimental data on a yeast strain engineered to undergo irreversible conversion: this synthetic system permits cell-type-specific fluorescent labeling and exogenous variation of the relative growth and conversion rates. We find that populations confined to grow on a flat agar surface are more susceptible than their well-mixed counterparts to fitness loss via a conversion-induced “meltdown.” We then present analytical predictions for growth in several biologically relevant geometries—well-mixed liquid media, radially expanding two-dimensional colonies, and linear fronts in two dimensions—by employing analogies to the directed-percolation transition from nonequilibrium statistical physics. These simplified theories are consistent with the experimental results.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Single-cell RNA-seq reveals cell type-specific transcriptional signatures at the maternal-foetal interface during pregnancy.

Growth and survival of the mammalian embryo within the uterine environment depends on the placenta, a highly complex vascularized organ comprised of both maternal and foetal tissues. Recent experiments demonstrate that the zinc finger transcriptional repressor Prdm1/Blimp1 is essential for specification of spiral artery trophoblast giant cells (SpA-TGCs) that invade and remodel maternal blood vessels. To learn more about functional contributions made by Blimp1+ cell lineages here we perform the first single-cell RNA-seq analysis of the placenta. Cell types of both foetal and maternal origin are profiled. Comparisons with microarray datasets from mutant placenta and in vitro differentiated trophoblast stem cells allow us to identify Blimp1-dependent transcripts enriched in SpA-TGCs. Our experiments provide new insights into the functionally distinct cell types present at the maternal–foetal interface and advance our knowledge of dynamic gene expression patterns controlling placental morphogenesis and vascular mimicry.

Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix –related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.