Distribution Of Cell Populations Research Articles

Lipopolysaccharide-induced expansion of histidine decarboxylase-expressing Ly6G+ myeloid cells identified by exploiting histidine decarboxylase BAC-GFP transgenic mice

Histamine is a biogenic amine that is chiefly produced in mast cells and basophils and elicits an allergic response upon stimulation. Histidine decarboxylase (HDC) is a unique enzyme that catalyzes the synthesis of histamine. Therefore, the spatiotemporally specific Hdc gene expression profile could represent the localization of histamine-producing cells under various pathophysiological conditions. Although the bioactivity of histamine is well defined, the regulatory mechanism of Hdc gene expression and the distribution of histamine-producing cell populations in various disease contexts remains unexplored. To address these issues, we generated a histidine decarboxylase BAC (bacterial artificial chromosome) DNA-directed GFP reporter transgenic mouse employing a 293-kb BAC clone containing the entire Hdc gene locus and extended flanking sequences (Hdc-GFP). We found that the GFP expression pattern in the Hdc-GFP mice faithfully recapitulated that of conventional histamine-producing cells and that the GFP expression level mirrored the increased Hdc expression in lipopolysaccharide (LPS)-induced septic lungs. Notably, a CD11b+Ly6G+Ly6Clow myeloid cell population accumulated in the lung during sepsis, and most of these cells expressed high levels of GFP and indeed contain histamine. This study reveals the accumulation of a histamine-producing myeloid cell population during sepsis, which likely participates in the immune process of sepsis.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. P60 may potentiate CIK cell cytotoxicity against tumor cells.

Fabrication of core-shell spheroids as building blocks for engineering 3D complex vascularized tissue

Cell spheroids as building blocks for engineering micro-tissue should be able to mimic the complex structure of natural tissue. However, control of the distribution of multiple cell populations within cell spheroids is difficult to achieve with current spheroid-harvest methods such as hanging-drop and with the use of microwell plates. In this study, we report the fabrication of core-shell spheroids with the ultimate goal to form 3D complex micro-tissue. We used endothelial cells and two types of stem cells (human turbinate mesenchymal stem cells (hTMSCs)/adipose-derived stem cells (ADSCs)). The stem cells and endothelial cells formed layered micro-sized cell sheets (µCSs) on polydopamine micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids by expansion of the hydrogels. The co-cultured spheroids formed a core-shell structure irrespective of stem cell type. In addition, the size of the core-shell spheroids was controlled from 90 ± 1 to 144 ± 3 µm by changing pattern sizes (200, 300, and 400 µm). The shell thickness gradually increased from 12 ± 3 to 30 ± 6 µm by adjusting the endothelial cell seeding density. Finally, we fabricated the micro-tissue by fusion of the co-cultured spheroids, and the spheroids with the core-shell structure rapidly induced in vitro vessel-like network in 3 days. Thus, the position of endothelial cells in co-cultured spheroids may be an important factor for the modulation of the vascularization process, which can be useful for the production of 3D complex micro-tissues using spheroids as building blocks. Statement of significanceThis manuscript describes our work on the fabrication of core-shell spheroids as building blocks to form 3D complex vascularized micro-tissue. Stem cells (human turbinate mesenchymal stem cells (hTMSCs) or adipose-derived stem cells (ADSCs)) and endothelial cells formed layered micro-sized cell sheets (µCSs) on micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids (core – stem cells, shell – endothelial cells), irrespective of stem cell type. In addition, the size and shell thickness of the core-shell spheroids were controlled by modifying pattern size and endothelial cell seeding density. We fabricated the vascularized micro-tissue by fusion of the spheroids and demonstrated that the spheroids with a core-shell structure rapidly induced vessel-like network.

Abstract 52: PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses

Abstract Background The selection of appropriate preclinical models comes always with the major question on how accurately and robustly they can represent the complexity of human disease. Patient-derived xenograft (PDX) models faithfully preserve the biological features and the genetic expression profile of human tumor specimens. However, one limiting aspect of patient-derived models is the replacement of the human host microenvironment by murine stroma within the tumor. Lack of cross-species compatibility compromises the induction of a broad range of signaling pathways that cannot be entirely recapitulated. With our in vitro 3D InSightTMTumor Microtissues derived from PDX lines, we provide a relevant physiological environment and a strategy to assess candidate drugs for novel therapeutic approaches. Aim Development of in vitro 3D InSightTM Tumor Microtissues from PDX lines aimed to retain the cellular heterogeneity found in the original human tumor tissue. Material & Methods and Results PDX cell suspensions of lung, breast and melanoma origin were successfully used to assess 3D aggregation in 96-well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each PDX sample, 3D PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). Furthermore, to provide a more physiological cancer niche, PDX cells were also co-cultured with tumor-matched cancer-associated fibroblasts (CAFs). 3D in vitro tumors were analyzed histologically and cancer phenotypic alterations were evaluated through the analysis of epithelial-to-mesenchymal transition (EMT) markers. The morphology, viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. To assess the distribution of various cell populations within the tumor, 3D PDX samples were screened for standard stromal vs. epithelial-tumor cells markers (e.g. FAP, pan-CK, E-Cadherin), and diagnostic cancer type-specific biomarkers. 3D PDX samples were also employed to investigate the efficacy of specific targeted therapies based on distinct molecular signatures of PDX tumor models. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Conclusion Further efforts will focus on employing this platform to establish more complex co-cultures with integration of additional relevant stromal and immune cells, to enable a reliable preclinical translational research of tumor/immune cell interactions. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment of immunomodulators and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Nicole Buschmann, Chloe' Pichon, Teresa Langova, Armin Maier, Julia Schueler, Patrick Guye. PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses [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 52.

Abstract 1083: Combinatorial strategies for tissue characterization with advanced image analysis and digital spatial profiling

Abstract Characterization of the spatial distribution and molecular profiles of diverse cell populations in the tumor microenvironment is important to understand the conditions required for the development of metastatic disease. The NanoString® GeoMx™ Digitial Spatial Profiling (DSP) platform facilitates these studies by enabling highly multiplexed, spatially resolved characterization of proteins and RNA from FFPE tissue (for Research Use Only). The platform leverages cocktails of fluorophore- and oligonucleotide-conjugated antibodies to visualize and digitally quantify targets from areas of interest (AOI). Here, DSP is combined with advanced tissue profiling using machine learning algorithms to define AOIs based on expression of multiple markers. This strategy enables targeted DSP sampling based on the digital segmentation of heterogeneous cell populations within the tumor microenvironment. We present a series of applications of this combined profiling strategy to enable deep characterization of renal, colon, and bladder cancers to address molecular and cellular interactions between host and tumor that facilitate metastasis and poor patient outcome. Whole slide digital scanning of multiplexed immunofluorescence labelled slides was performed on a Zeiss Axioscan.z1. Digitized scans were imported in Definiens Tissue Studio® software and machine learning-based analysis was performed. The image analysis segmented heterogeneous subpopulations, dependent on biomarker expression and spatial resolution, which acted as a digital spatial map to direct DSP sampling. DSP was performed on a serial section of tissue by selecting AOI within bladder and colorectal cancer sections and a geometric DSP profiling strategy was employed to collect all the protein content from the AOI in a single sample. In another application of the method, a clear cell renal cell carcinoma (ccRCC) tissue microarray comprised of cores from patient matched healthy, primary, venous thrombi and distant metastatic sites was profiled with DSP. We present highly-plexed in situ proteomic data captured from several AOIs across bladder and colorectal cancer tissue sections. This data describes the molecular profile from the densely distributed lymphocytes and macrophages in close proximity to tumor subpopulations such as tumor buds and PDL-1 positive cancer cells. We demonstrate that both tumor and immune cell subpopulation profiles within bladder and CRC alter dependent on their proximity to each other. We further present the altering proteomic landscape of ccRCC as it progresses from primary to metastatic disease. This work demonstrates the feasibility and applicability of combining machine learning and advanced image analysis with multiplexed digital spatial profiling to enable deep characterization of tissues. Analysis approaches such as these will have utility in a variety of pathological research settings. Citation Format: Raffaele De Filippis. Combinatorial strategies for tissue characterization with advanced image analysis and digital spatial profiling [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 1083.

Abstract 5057: Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers

Abstract Tobacco smoke exposure is a risk factor for many human diseases and the global disease burden attributed to smoking remains substantial. In addition to DNA damage, smoking-induced epigenetic changes may contribute to etiology of complex smoking-associated diseases. Smoking alters the epigenome and transcriptome of human blood leukocytes. However, interpretation of bulk genomic approaches is limited because changes could indicate altered distribution of cell (sub)populations or changes in expression within (sub)populations. To characterize smoking-related gene expression changes in primary immune cells, we performed single cell RNA sequencing on human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Transcriptomes of 45,965 cells (78,183 reads per cell) revealed an altered population of Natural Killer (NK)-like T lymphocytes in smokers. Compared to NK cells, the NK-like T cell cluster had elevated expression of CD8A, CD8B, CD3D, CD3E, CD3G, CD6, and CD2, indicative of CD8+ T lymphocytes. Relatively rare in nonsmokers (2.2%), the transcriptionally unique subset of CD8+ T cells comprised 8.7% of PBMCs in smokers. Among CD8+ T cell subtypes, the increase in NK-like CD8+ T cells (Mann-Whitney p = 0.03) corresponded with a decrease in Naïve CD8+ T cells (p = 0.03). We did not observe changes in the frequencies of two additional CD8+ T cell clusters or in the overall frequency of CD8+ T cells. Mass cytometry of a 26-antibody leukocyte panel confirmed no differences in the frequencies of total CD8+ T (CD3+/CD8+/CD56-), total NKT (CD3+/CD56+) or CD8+ NKT (CD3+/CD8+/CD56+) cells between smokers and nonsmokers. This suggests smoking is associated with an increased number of CD8+ T cells that share characteristics with NK cells, but are not NKT cells. Consistent with an NK-like phenotype, altered effector CD8+ T cells had elevated expression of genes reported to be upregulated in T cells reprogrammed to NK-like cells. Compared to other effector CD8+ T cells, altered effector CD8+ T cells had reduced IL7R and increased FCGR3A (CD16), IFNG (interferon gamma), GZMB (granzyme B), and PRF1 (perforin) expression. In mice, granzyme B and perforin expressing CD8+ T cells contribute to the development of atherosclerotic plaques. Our data highlights a potential link between smoking-induced functional changes in human CD8+ T cells and atherosclerosis and /or immune surveillance in cancer. Citation Format: Suzanne N. Martos, Michelle R. Campbell, Marie A. Iannone, Gary S. Pittman, Ma Wan, Douglas A. Bell. Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers [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 5057.

Multiplex immunohistochemistry as a novel tool for the topographic assessment of the bone marrow stem cell niche.

Immunohistochemistry (IHC) using specific antibodies is a well-established method for the visualization of distinct cell populations. With increasing availability of suitable methods for complex tissue analyses, new demands have arisen to provide next to complex quantitative data information on protein expression, spatial distribution and cell-cell interactions in tissue sections. During the last decade, tissue preparation, fluorescent dyes, hardware imaging and software analysis were improved to solve problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining. Automated cell segmentation as well as subcellular multiparameter analysis of fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allows the quantification and localization of multiple proteins in the same tissue section. This technique gives us the opportunity to visualize and record the spatial relationship between different cells and is currently employed for formalin-fixed, paraffin-embedded (FFPE) samples, but has not yet been developed for calcified bone marrow (BM) biopsies. This chapter summarizes a novel protocol developed for decalcified FFPE BM samples. In addition, it discusses the technical aspects and pitfalls using this material thereby extending the use of MSI for analysis of BM malignancies. It provides an overview on the characterization and distribution of cell populations and protein expression patterns regarding their prognostic and predictive value, and their use for guidance of therapeutic decisions.

Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (≈transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies.EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 °C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for <24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes.Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population.

Coupling synthetic biology and programmable materials to construct complex tissue ecosystems.

Synthetic biology combines engineering and biology to produce artificial systems with programmable features. Specifically, engineered microenvironments have advanced immensely over the past few decades, owing in part to the merging of materials with biological mimetic structures. In this review, we adapt a traditional definition of community ecology to describe "cellular ecology", or the study of the distribution of cell populations and interactions within their microenvironment. We discuss two exemplar hydrogel platforms: (1) self-assembling peptide (SAP) hydrogels and (2) Poly(ethylene) glycol (PEG) hydrogels and describe future opportunities for merging smart material design and synthetic biology within the scope of multicellular platforms.

The role of Th17 lymphocytes in drug-resistant epilepsy

Abstract Epilepsy is a chronic neuronal disorder affecting ~65 millions people throughout the world. 1/3 of patients suffer from drug-resistant epilepsy (DRE). Objective to assess the relationship between different Th17 markers and the clinical course of epilepsy to uncover new potential immune biomarkers and therapeutic targets of interest for DRE. Approach Subjects between 19–55 years old with a focal diagnosed epilepsy were recruited at the CHUM epilepsy clinic. The profile of peripheral blood T lymphocytes from DRE patients (n=48) is compared to well-controlled epilepsy (WCE; n=30) and healthy controls (n=35). Peripheral blood mononuclear cells (PBMCs) were isolated by gradient density centrifugation before ex vivo analysis of surface markers (flow cytometry). Total CD4 T lymphocytes were magnetically isolated from PBMCs and stimulated overnight before analysis of cytokine expression or processed for RNA extraction before analysis of transcription factors expression by qRT-PCR. Results Our data suggest that antiepileptic drugs are associated with a relative ‘immunosuppression’, more pronounced in WCE. Epilepsy is associated with an altered distribution of immune cell populations with a higher CD4:CD8 ratio. A higher proportion of CD4 T lymphocytes from DRE subjects express CCR6 and CD161 compared to WCE. The proportion of CD4 T cells expressing pro-inflammatory cytokines from Th17/1 lineage (IL-17A, IL-22, IFN-γ, GM-CSF, TNFα) is higher in DRE than WCE, while anti-inflammatory cytokines (IL-4, IL-10) tend to be lower. Conclusion Our preliminary results suggest an increased frequency of pro-inflammatory Th17/1 lymphocytes and their related cytokines in DRE. Pro-inflammatory CD4 T cells could play a role in DRE.

Immune microenvironment of triple-negative breast cancer in African-American and Caucasian women.

African-American (AA) patients with triple-negative breast cancer (TNBC) are less likely to achieve pathologic complete response from neoadjuvant chemotherapy and have poorer prognosis than Caucasian patients with TNBC, suggesting potential biological differences by race. Immune infiltration is the most consistent predictive marker for chemotherapy response and improved prognosis in TNBC. In this study, we test the hypothesis that the immune microenvironment differs between AA and Caucasian patients. RNA-seq expression data were obtained from The Cancer Genome Atlas (TCGA) database for 162 AA and 697 Caucasian breast cancers. Estrogen receptor (ER)-positive, human epidermal growth factor receptor-2 (HER2)-positive, and TNBC subtypes were included in the analyses. Tumor infiltrating lymphocyte (TIL) counts, immunomodulatory scores, and molecular subtypes were obtained from prior publications for a subset of the TNBC cases. Differences in immune cell distributions and immune functions, measured through gene expression and TIL counts, as well as neoantigen, somatic mutation, amplification, and deletion loads, were compared by race and tumor subtype. Immune metagene analysis demonstrated marginal immune attenuation in AA TNBC relative to Caucasian TNBC that did not reach statistical significance. The distributions of immune cell populations, lymphocyte infiltration, molecular subtypes, and genomic aberrations between AA and Caucasian subtypes were also not significantly different. The MHC1 metagene demonstrated increased expression in AA ER-positive cancers relative to Caucasian ER-positive cancers. This study suggests that the immunological differences between AA and Caucasian breast cancers represented by TCGA data are subtle, if they exist at all. We observed no consistent racial differences in immune gene expression or TIL counts in TNBC by race. However, this study cannot rule out small differences in immune cell subtype distribution and activity status that may not be apparent in bulk RNA analysis.

Novel fluorescence techniques to quantitate renal cell biology.

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Ultrafast Single-Cell Level Enzymatic Tumor Profiling.

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-μFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5-2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of ∼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.

Adaptive metabolic pattern biomarker for disease monitoring and staging of lung cancer with liquid biopsy

In this manuscript, we demonstrate the applicability of a metabolic liquid biopsy for the monitoring and staging of patients with lung cancer. This method provides an unbiased detection strategy to establish a more precise correlation between CTC quantification and the actual burden of disease, therefore improving the accuracy of staging based on current imaging techniques. Also, by applying statistical analysis techniques and probabilistic models to the metabolic status and distribution of peripheral blood mononuclear cell (PBMC) populations “perturbed” by the presence of CTCs, a new category of adaptive metabolic pattern biomarker (AMPB) is described and unambiguously correlated to the different clinical stages of the patients. In fact, this strategy allows for classification of different categories of disease within a single stage (stage IV) before computed tomography (CT) and positron emission tomography (PET) scans and with lower uncertainty.

Abstract 2830: Understanding chemotherapy-induced replicative stress to identify rational combination therapies

Abstract Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multi-scale computational model using the U2OS osteosarcoma cancer cell line that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated based on an extensive data set that comprises cell cycle distribution of the initial cell population, signaling data measured by western blot, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model can predict the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we were able to confirm experimentally. Computational models, like the one presented here, can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death, as well as to rationally identify chemotherapy-potentiating drug combinations. Citation Format: Ozan Alkan, Birgit Schoeberl, Millie Shah, Alexander Koshkaryev, Tim Heinemann, Daryl Drummond, Michael B. Yaffe, Andreas Raue. Understanding chemotherapy-induced replicative stress to identify rational combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2830.

Immunophenotyping of cerebrospinal fluid cells by Chipcytometry

BackgroundThe gold standard in cerebrospinal fluid (CSF) cell immunophenotyping is flow cytometry. Nevertheless, the small amount of CSF cells and the invasive character of lumbar puncture limit the spectrum of possible investigation. Chipcytometry, a modified approach to slide-based cytometry, might be a useful tool for CSF analysis due to the possibility of iterative staining, imaging, and bleaching cycles. The aim of this study was to compare flow cytometric leukocyte subset analysis with Chipcytometry comparing the percentage distribution of distinct cell populations and the T-cell CD4:CD8 ratio. Moreover, this study investigated the interpretability of chips loaded with CSF cells and examined the applicability of Chipcytometry in clinical practice.Methods375 CSF samples from 364 patients were analyzed by Chipcytometry using an automated upright microscope. Cell surface molecules were stained using fluorescence-labeled monoclonal antibodies. For cross-validation experiments, flow cytometry data of six patients were analyzed and matched with Chipcytometry data.ResultsOur experiments showed a better agreement examined by Bland-Altman analysis for samples with CSF pleocytosis than for normocellular CSF samples. Data were more consistent for B cells and CD4:CD8 ratio than for T cells and monocytes. Advantages of Chipcytometry compared to flow cytometry are that cells once fixated can be analyzed for up to 20 months with additional markers at any time. The clinical application of Chipcytometry is demonstrated by two illustrative case reports. However, the low amount of CSF cells limits the analysis of normocellular CSF samples, as in our cohort only 11.7% of respectively loaded chips had sufficient cell density for further investigation compared to 59.8% of all chips loaded with samples with elevated cell counts (≥ 5/μl). Varying centrifuge settings, tube materials and resuspension technique were not able to increase the cell yield.ConclusionIn summary, the results demonstrate the great potential of Chipcytometry of CSF cells for both scientific questions and routine diagnostic. A new chip design optimized to meet the requirements of CSF would greatly enhance the value of this method. Cross-validation results need to be confirmed in a larger cohort.

Backtracked analysis of preleukemic fusion genes and DNA repair foci in umbilical cord blood of children with acute leukemia.

The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.

Multi-Cellular Modelling of Cellular Mechanisms Gives Insights on the Maintenance of Epidermal Tissue Structure

The inter-follicular epidermis (IFE) forms the outer-most layer of the skin. Many individual components fundamental to healthy IFE structure are known: proliferation occurs only in a basal layer; above this layer cells differentiate into keratinocytes forming further distinct layers before they are shed from the surface. However, a definitive understanding of how the balance between proliferation, differentiation, and cell shedding is maintained in IFE tissue during homeostasis does not yet exist. We have developed an agent-based multi-cellular computational model to simulate tissue homeostasis in the skin. Epidermal cells are represented as overlapping spheres, and cell divisions are represented as stochastic time-driven events. Cell movement is determined by adhesive attractive forces and repulsive forces between other cells and the basal membrane. The magnitude of these forces depends upon the types of the interacting bodies, and can vary depending on factors such as cell age and location. Using this model we have analysed the impact of different cell mechanisms and behaviours on the tissue in order to investigate alternate hypotheses around maintenance of tissue structure. Mechanisms investigated include cell division, cell adhesion and stiffness, and cell death. Results of this study provide insights on important considerations in modelling this system. In particular, both choice of biological assumptions and the mathematical representation of cell division impact the spatial distribution of proliferative cell populations. We find that the cell mechanisms and behaviours critical to maintenance of the tissue width are proliferation rate, spatial adhesion variation, and cell loss.

Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

Characterization of myeloid cell populations in human testes collected after sex reassignment surgery

The testis has been described in animal models as a site of immune privilege, which protects spermatids against tissue damage during inflammation. Myeloid cells, including macrophages and dendritic cells (DC), are defined as key players in the testicular immune privilege in animal models. However, their distribution and frequency in human testis remain poorly described. To overcome the challenges related to tissue sampling, we obtained testicular tissue from men under hormonal therapy who elected to have sex reassignment surgery (SRS). We examined the distribution of myeloid cell populations in tissue sections using immunohistofluorescence and evaluated their relative frequencies in fresh testicular cell suspensions compared with matched blood using multi-parametric flow cytometry. We identified 4.9% of CD45+ leucocytes in testicular cell suspensions, of which 0.4% were B cells, demonstrating a low level of blood contamination. Myeloid cells (Lin−HLA-DR+) were located in the testicular interstitium and represented a median of 23.4% of testicular leucocytes, displaying higher HLA-DR expression compared to their counterparts in blood (p=0.001). The frequency of testicular myeloid cells was not linked with the duration of hormonal therapy. Resident macrophages (CD14+CD163+) constituted the most frequent myeloid cell subset and expressed high levels of CD163. Elevated proportion of myeloid DC (CD14−CD11c+) contrasted with the paucity of plasmacytoïd DC (CD14−CD123+) in testis. Myeloid-derived suppressor cells (Lin−HLA-DR−CD33hiCD11bhi) were not detected in the testis while constituting 0.5% of blood leucocytes. For the first time, we characterized myeloid cell subsets in human testes collected after SRS, providing a basis to assess their contribution to immune privilege.