DOCSLIB.ORG

Four-Color Flow Cytometric Analysis of Peripheral Blood Donor Cell Chimerism

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Four-Color Flow Cytometric Analysis of Peripheral Blood Donor Cell Chimerism ELSEVIER Four-Color Flow Cytometric Analysis of Peripheral Blood Donor Cell Chimerism Diana Metes, Alison Logar, William A. Rudert, Adriana Zeevi, J ennifer Woodward, Anthony J. Demetris, Kareem Abu-Elmagd, Bijan Eghtesad, Ron Shapiro, John J. Fung, Massimo Trucco, Thomas E. Starzl, and Noriko Murase ABSTRACT: Passenger leukocytes have been demon­ 0.2%. When the fresh peripheral blood mononuclear cells strated to play significant roles in initiating and also were separated by conventional Ficoll gradienr purifica­ regulating immune reactions after organ transplantation. tion, similar, but slightly lower levels of donor cells were Reliable techniques to detect donor leukocytes in recipi­ detected. Blood samples obtained I ~ 5 months after liver, ents after organ transplantation are essential to analyze the kidney, and intestine transplants revealed that the kind of role, function, and behavior of these leukocytes. In this organ allograft influenced levels and lineage pattern of the report we describe a simple, reliable method to detect circulating donor cells. This procedure provided a simple donor cells with low frequencies using peripheral blood and reliable method in determining early chimerism in samples. Detection of small numbers of major histocom­ transplant recipients. However, the detection of MHC­ patibility complex (MHC) mismatched cells was first mismatched leukocytes of all lineages was much lower studied using four-color flow cytometry in artificially when frozen peripheral blood mononuclear cells were created cell mixtures. By selecting the CD45 + population used. Human Immunology 64, 787~795 (2003), © and simultaneous staining with several leukocyte lineage American Society for Histocompatibility and Immunoge­ markers (CD3, CD4, CDS, CD56, and CD19), MHC­ netics, 20D3. Published by Elsevier Inc. mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood KEYWORDS: flow cytometry; whole blood; chimerism; samples with a threshold sensitivity as low as D.I %~ solid organ transplantation ABBREVIATIONS mAb monoclonal antibody FISH fluorescent in situ hybridization PBMC peripheral blood mononuclear cells SS side scatter WB whole blood FS forward scatter INTRODUCTION Passenger leukocytes in solid organ allografts have been However, the persistence of small numbers of donor conventionally considered to be the main initiators of leukocytes in the recipient for decades after transplanta­ immune reactions leading to allograft rejection (1, 2). tion implies the possible function of these cells as active modulators for graft acceptance (3, 4). The persistence of mobile donor alloantigens, with sustained immune reac­ From the Depc/rtlllents lifSurgery (D.M., AL., W.AR.,]. W,., K.A-E .. tions between donor and recipient leukocytes, and con­ B.E., R,S.. JIF., AI. T., T.E.S., N.M.), Immunology (D.M,), Pathology sequent mutual immunologic regulation, are considered (A,Z .. AI!),), and Department of Pediatrics, Division of ImmunogeneticJ to be the principal factors for allograft acceptance (5, 6}. (W.A R., ;\1. T.), Thomas E. Starzl Tranplantation Institllte, U niz'enity (if Piwb!£rgh Medical Center, Pittsburgh, PA, USA The analysis of the role, function, and behavior of Address reprint reqmstJ to: Dr. N. Murase, University (if Pittsburgh, donor leukocytes after transplantation requires sensitive Thollias E. Starz! TranplantationInstitute, 200 Lothrop Street, Piwimrgh, PA 15261; Tel: (412) 648- 2155; Fax: (412) 624-6666. techniques to distinguish donor and recipient phenotype Received MelY 1, JO()): retised May 14, 2003; accepted ilia) 14, 2003. cells. The detection of small numbers of donor cells long Human Immunology 64, 787-795 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 0198-8859/03/$-see front matter Published by Elsevier Inc. doi:l0.1016/S0198-8859(03)00113-7 788 D. Metes et al. after ttansplantation has only recently become possible. Grand Island, NY, USA), in liquid nitrogen, and thawed Currently, donor cells are analyzed by polymerase chain rapidly for assay in a 37 DC water bath. reaction (PCR), immunohistochemistry, in-situ hybrid­ ization, and flow cytometry by targeting disparities at HLA Typing the human leukocyte antigen (HLA) loci or sex-deter­ HLA typing was performed at the Tissue Typing Labo­ mining region Y [4, 7-9}' Some of these techniques are ratory (Clinical Immunopathology, Central Laboratory either laborious or require particular tissue samples, Services, Inc., University of Pittsburgh Medical Center, thereby precluding their widespread use. Flow cytomet­ Pittsburgh, PA, USA) using genomic DNA extracted ric analysis is technically simple and the procedure can be from PBMC isolated from all patients in the study. completed in a short time. Although this method has been known to have limitations in detecting low fre­ Artificial Mixtures quency events, it could provide valuable information on Whole blood or isolated PBMC from healthy volunteers surface or intracellular expression of various molecules on were mixed in vitro to generate the artificial samples donor cells. Therefore, it was our goal to establish a containing known levels of HLA-mismatched and sex­ standardized flow cytometric technique that can provide mismatched cells (5%,1%,0.3%, and 0.1%). Aliquots reliable results of donor leukocytes after organ transplan­ of PBMC mixtures were cryopreserved and used in par­ tation in a timely manner. allel with WB mixtures or fresh PBMC mixtures for In this report we describe a four-color flow cytometry comparison analysis. method to detect levels and lineages of major histocom­ patibility complex (MHC) mismatched (donor) cells using Antibodies and Reagents variable types of samples, including whole blood, and fresh The panel of FITC-conjugated monoclonal antibodies and frozen peripheral blood mononuclear cells (PBMC) (mAb) specific for HLA-A or -B loci were either pur­ from normal volunteers, artificial mixtures, and samples chased from One Lambda, Inc. (Canoga Park, CA, USA) from organ transplant recipients. The method is quick and or prepared from hybridomas (A TCC, Manassas, VA, reliable, with a detection limit of 0.1 %-0.2% when used USA) and FITC-conjugated at the University of Pitts­ in combination with lineage-specific antibodies. burgh Cancer Institute Hybridoma Facility, directed by Dr. Albert Deleo. Other lineage-specific mAbs included CD3-PE, CD4-PE, CD56-PE, CD19-PC5, CD8-PC5, MATERIALS AND METHODS and CD45-ECD (Beckman Coulter Co., Miami, FL, Blood Collection and Processing USA, and Becton-Dickinson, San Jose, CA, USA). Flu­ Heparinized whole blood (WB) was obtained from orochrome-conjugated isotype-matched non-specific healthy volunteers and transplant recipients in accor­ mAbs were used as negative controls for each assay. dance with the guidelines of the Institutional Review Board at the University of Pittsburgh. Three types of Flow Cytometric Analysis blood samples were used in this study: WB, freshly Whole blood (100,....1) was incubated with relevant mAbs isolated PBMC, and cryopreserved PBMC. PBMC were or isotype controls for 20 minutes in dark at 4 DC, and obtained by Ficoll-Hypaque (Amersham Pharmacia Bio­ then mixed vigorously with FACS lysing solution (Bec­ tech, Piscataway, NJ, USA) standard density gradient ton Dickinson) for 10 minutes at room temperature for centrifugation method [l0}. Aliquots of PBMC were erythrolysis and fixation. Cells were washed twice and cryopreserved in freezing medium containing 10% fixed in 1% paraformaldehyde in PBS. Alternatively, DMSO and 30% FCS in RPMI 1640 (Life Technologies, freshly isolated or frozen PBMC (1 X 105 cells) were TABLE 1 Monoclonal antibodies used in the study Specificity Isotype Dilution Supplier A2/28 mouse IgG2a 1:300 One Lambda, Inc. A3 mouse IgG2a 1:20* ATCC (HB122)/UPCI Hybridoma Facility A9 mouse IgG2b 1:20 One Lambda, Inc. B7 mouse IgG1 1:30* ATCC (HB56)/UPCI Hybridoma Facility B8 mouse IgG2b 1:20 One Lambda, Inc. B12 mouse IgG2b 1:20 One Lambda, Inc. B27 mouse IgG1 1:20 One Lambda, Inc. * Based on the mAb concentrations of 1 mg/ml. Four-Color Flow Cytometry Analysis of Chimerism 789 0.3% 0.10/0 o o 0.01 0.02 0.18 0.16 "., "I '" CD45ECD HLA-A2FITC 0.11 05 6.62 0.04 0. 1 0.80 0.26 0.06 '''''I ,,,' ,;;' «- ..r. HLA-A2FITC FIGURE 1 Detection of major histocompatibility complex scatter (SS) versus forward-scatter (FS) cytograms, 50,000 (MHC) mismatched cells in different artificial mixtures. CD45 + events were collected per sample and analyzed Whole blood from normal volunteers who were mismatched at with EXP032 software (Applied Cytometry System, human leukocyte antigen A (HLA-A) were mixed to generate 1%, 0 . .3%, and 0.1% artificial mixtures. Level and lineage Sheffield, United Kingdom). Daily calibration of the specificity of MHC-mismatched (HLA-A2) cells were analyzed instrument was performed using flow check fluoro­ on gated CD45 (ECD) positive cells with monocloml antibod­ spheres (Beckman Coulter Co.). Fluorescent compensa­ ies for HLA-A2 (FITC), together with CD.3 (PE) and CD19 tion was performed using a normal control and staining (PE). with each fluorochrome separately. Compensation values were set to eliminate spectral overlap. preincubated with 10% goat serum for 20 minutes for the blocking of nonspecific binding, washed three times, Cytospin and Fluorescent In Situ Hybridization and incubated for 20 minutes in dark at 4 °C with for XY relevant mAbs or isotype controls, followed by washing Aliquots of 1 X 105 cells from WB or PBMC were and fixation. A typical four-color flow cytometry analysis cytospun for 5 minutes at 500 rpm using a centrifuge was performed with mAb combinations of CD45-ECD, (Thermo Shandon, Pittsburgh, P A, USA). The slidES anti-HLA-FITC, and two leukocyte lineage markers con­ were air dried and fixed in 95% ethanol for 10 minutes. jugated with PE and PE-Cy5. Data acquisition and anal­ FISH analysis was carried out using directly labeled ysis was performed on a Coulter EPICS XL flow cytom­ probes for X (red) and Y (greC'i1) chromosome alfa-satel­ eter (Beckman Coulter Co.) mounted with four lite regions, according to the manufacturer's recom­ photomultiplier tubes and a single argon laser.
Load more

Recommended publications
  • Stroma-Free, Serum-Free Expansion and Differentiation of Hematopoietic Stem and Progenitor Cells to the T Cell Lineage Nooshin Tabatabaei-Zavareh1, Tim A
    Stroma-free, serum-free expansion and differentiation of hematopoietic stem and progenitor cells to the T cell lineage Nooshin Tabatabaei-Zavareh1, Tim A. Le Fevre1, Alexander J.Y. Man1, Evan A. Karas1, Stephen J. Szilvassy1, Terry E. Thomas1 , Allen C. Eaves1,2, and Albertus W. Wognum1 1 STEMCELL Technologies Inc., Vancouver, BC, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada Introduction The use of T cells for cancer immunotherapy and other therapeutic applications relies on the isolation of T cells from peripheral blood and their subsequent activation and expansion in culture. T cells can also be generated from hematopoietic stem and/or progenitor cells (HSPCs) in cord blood (CB) or bone marrow (BM). This approach not only offers a renewable source of T cells, but also provides a model system to study disease mechanisms or validate new drugs that affect T cell development and/or function. Differentiation of HSPCs to T cells typically requires co-culture with stromal cell lines that have been engineered to express a Notch-ligand. In such cultures, CD34+CD38-/lo HSPCs develop into CD7+CD5+ pro-T cells that further differentiate to T lineage-committed progenitor cells (pre-T cells) characterized by the expression of CD1a. CD7+CD1a+ pre-T cells can then differentiate to express CD4 and become CD4 immature single-positive (CD4ISP) cells. CD4ISP cells give rise to CD4+CD8+ double positive (DP) cells. These finally mature into CD4 and CD8 single-positive (SP) CD3+TCRαβ+ T cells. Here we describe a serum-free culture method that recapitulates these differentiation steps in the absence of stromal cells and generates large numbers of functional T lineage cells from a limited number of purified CD34+ CB cells.
    [Show full text]
  • Multiparameter Phenotype Mapping of Normal and Post-Chemotherapy B
    Leukemia (1997) 11, 1266–1273 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow MN Dworzak, G Fritsch, C Fleischer, D Printz, G Fro¨schl, P Buchinger, G Mann and H Gadner Children’s Cancer Research Institute, St Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria We studied the differentiation profiles of B cell precursors Materials and methods (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the Sample description three major maturational BCP stages in BM. By correlating lin- eage-associated markers, CD45RA, and several adhesion mol- Pediatric bone marrow (BM) samples (n = 44) were obtained ecules, the stage-specific patterns were found to differ in cer- from children undergoing diagnostic BM aspiration for the fol- tain details from previously published concepts. Among the earliest BCP, a subset of CD34+CD10lo precursors was repeat- lowing diagnoses in the absence of BM involvement at the edly observed in addition to the well characterized time of immunologic investigation: suspicion of storage dis- CD34+CD10hiCD19+ majority of cells. Only two-thirds of these ease (n = 1), bacterial lymphadenitis (n = 1), neuroblastoma CD34+CD10lo cells expressed CD19. However, uniformity of (n = 1), severe aplastic anemia in remission (n = 2), single sys- phenotypic features, absence of T lineage markers, and the tem Langerhans cell histiocytosis (n = 1), idiopathic throm- regeneration kinetics after chemotherapy suggest the B lineage = + lo bocytopenic purpura (n 1), early stage (localized) non- affiliation of the CD34 CD10 precursors in general.
    [Show full text]
  • Brain Tumor Cells in Circulation Are Enriched for Mesenchymal Gene Expression
    Author Manuscript Published OnlineFirst on August 19, 2014; DOI: 10.1158/2159-8290.CD-14-0471 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Brain Tumor Cells in Circulation are Enriched for Mesenchymal Gene Expression Authors: James P. Sullivan1,3,*, Brian V. Nahed1,4*, Marissa W. Madden1, Samantha M. Oliveira1, Simeon Springer1, Deepak Bhere5, Andrew S. Chi1,5, Hiroaki Wakimoto1,4, S. Michael Rothenberg1, 3, Lecia V. Sequist1, 3, Ravi Kapur2, Khalid Shah5,6, A. John Iafrate1,7, William T. Curry1,4, Jay S. Loeffler1, Tracy T. Batchelor1,5, David N. Louis1,7, Mehmet Toner2,8, Shyamala Maheswaran1,8†, Daniel A. Haber1,3,9† Author’s Affiliations: 1Massachusetts General Hospital Cancer Center, 2Center for Engineering in Medicine and Departments of 3Medicine, 4Neurosurgery, 5Neurology, 6Radiology, 7Pathology, and 8Surgery, Harvard Medical School, Boston, Massachusetts 02114, USA 9Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA * Denotes equal contribution Corresponding Authors: †Shyamala Maheswaran, Massachusetts General Hospital Cancer Center, Building 149 13th Street, Charlestown, MA 02129. Phone: 617-726-6552; Fax: 617-726-6919; Email: [email protected] †Daniel A. Haber, Massachusetts General Hospital Cancer Center, Building 149 13th Street, Charlestown, MA, 02129. Phone: 617-726-7805; Fax: 617-726-6919; Email: [email protected] Running Title: Mesenchymal Glioblastoma Cells in Circulation. Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2014; DOI: 10.1158/2159-8290.CD-14-0471 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
    [Show full text]
  • LECTURE 07 Immune Cells Surface Receptors and Their Functions
    LECTURE: 07 Title: IMMUNE CELL SURFACE RECEPTORS AND THEIR FUNCTIONS LEARNING OBJECTIVES: The student should be able to: • The chemical nature of the cellular surface receptors. • Define the location of the cellular receptors. • Enumerate the different given terms for the word "receptor". • Define the term "CD" marker. • Describe the location of the immune receptors. • Classify the cellular receptors according to the information they offer. • Classify the cellular receptors according to their families. • Enumerate some common immunoglobulin receptors. • Enumerate some common complement receptors. • Enumerate some adhesion molecules receptors. • Enumerate the main functions of the immune cell receptors. • Realize the importance of the immune receptors. LECTURE REFRENCE: 1. TEXTBOOK: ROITT, BROSTOFF, MALE IMMUNOLOGY. 6th edition. pp. 29, 143, 76, 77, 78, 221, 325. 2. TEXTBOOK: ABUL K. ABBAS. ANDREW H. LICHTMAN. CELLULAR AND MOLECULAR IMMUNOLOGY. 5TH EDITION. Chapter. 6 .pg 105. Chapter 4. pg 65- 80. 1 Lymphocytes can be identified by characteristic markers Lymphocytes and other leukocytes express different surface molecules which can be used for distinguishing cell population. A systemic nomenclature called the CD system has been developed, in which the term CD refers to (cluster of designation). These different cell surface molecules are detected with specific monoclonal antibodies, each of these molecules are given CD number. Molecular markers can be further defined according to the information they offer about the cell. For example; A. Lineage markers are exclusive to a particular cell line, e.g., CD3, which is found only on T cells. B. Maturation markers are transiently expressed during cell differentiation, e.g., CD1, which is only found on cells developing in thymus and is not present on peripheral T cells.
    [Show full text]
  • CD5-Positive Acute Lymphoblastic Leukemia Dawood Ahmed1, Tahir Aziz Ahmed1, Suhaib Ahmed 2, Hamid Nawaz Tipu1 and Muhammad Amin Wiqar1
    CASE REPORT CD5-Positive Acute Lymphoblastic Leukemia Dawood Ahmed1, Tahir Aziz Ahmed1, Suhaib Ahmed 2, Hamid Nawaz Tipu1 and Muhammad Amin Wiqar1 ABSTRACT CD5-positive B-ALL is a rare variant of Acute Lymphoblastic Leukemia (ALL). In literature, only three cases have been reported so far. This fourth case report describes a young lady who was diagnosed as ALL (L-2) on bone marrow examination and was found to be CD5 positive B-cell acute lymphoblastic leukemia on immunophenotyping. Cytogenetic analysis revealed translocation t(9:22). Key words: CD5. Immunophenotyping. Acute lymphoblastic leukemia. INTRODUCTION blood examination. On peripheral blood examination hemoglobin (Hb) was reported 7.6 g/dl, Total Leukocyte CD5 is a 67-kd glycoprotein that is expressed by most Count (TLC) 18,600/µl with 85% lymphocytes, 9% T-cells and a subset of B-cells called B-1 cells. neutrophils, 4% eosinophils and 2% monocytes. CD5-positive B-cells are found in fetal spleen, cord Lymphocytes were described as atypical and abnormal blood and peritoneal cavity of adults. Similar cells are cells and bone marrow aspiration was advised. Blood also present in the marginal zones of lymphoid follicles counts carried out at the time of bone marrow aspiration in spleen. Only 5 to 10% of B-cells belong to this revealed ESR 120 mm after one hour, Hb 5.3 g/dl, TLC category in blood and lymphoid tissues.1 These cells 26,900/µl with 82% lymphocytes and 18% neutrophils in produce IgM antibodies (natural antibodies) against the peripheral blood specimen. Platelets count was polysaccharide and lipid antigens.
    [Show full text]
  • Pre/Pro-B Cells Generate Macrophage Populations During
    Pre/pro-B cells generate macrophage populations PNAS PLUS during homeostasis and inflammation Tatsiana Audzevicha, Rachael Bashford-Rogersb, Neil A. Mabbottc,d, Dan Framptone, Tom C. Freemanc,d, Alexandre Potocnikf, Paul Kellamb, and Derek W. Gilroya,1 aCentre for Clinical Pharmacology and Therapeutics, Division of Medicine, University College London, London WC1E 6JJ, United Kingdom; bDepartment of Medicine, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, United Kingdom; cThe Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom; dRoyal (Dick) School of Veterinary Sciences, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom; eDivision of Infection and Immunity, University College London, London WC1E 6AE, United Kingdom; and fInsitute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom Edited by Harinder Singh, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH and accepted by Editorial Board Member Ruslan Medzhitov March 31, 2017 (received for review October 6, 2016) Most tissue-resident macrophages (Mφs) are believed to be derived publications (16, 18, 19), mature B cells do not differentiate into prenatally and are assumed to maintain themselves throughout Mφs. Instead, we found a population of cells within the early pro- life by self-proliferation. However, in adult mice we identified a B-cell/fraction B-cell compartment with non-rearranged B-cell progenitor within bone marrow, early pro-B cell/fraction B, that receptor (BCR) genes that exit bone marrow under steady state, differentiates into tissue Mφs. These Mφ precursors have non- circulate in peripheral blood, and populate serous cavities where rearranged B-cell receptor genes and coexpress myeloid (GR1, CD11b, they differentiate into Mφs, thereby contributing to the overall and CD16/32) and lymphoid (B220 and CD19) lineage markers.
    [Show full text]
  • Resolving the Spatial and Cellular Architecture of Lung Adenocarcinoma by Multi- Region Single-Cell Sequencing
    Resolving the spatial and cellular architecture of lung adenocarcinoma by multi- region single-cell sequencing SUPPLEMENTARY DATA FILE 2 Supplementary Figures S1-S7 Supplementary Fig. S1 Count matrix Filtering Removal of: Doublets Low quality cells Cells with low complexity libraries/debris Batch effects evaluation and correction Unsupervised clustering, UMAP visualization & major cell lineages annnotation Hierarchical relationship analysis EPCAM- fractions EPCAM+ fractions Subclustering & cell subsets annotation Sublustering & cell subsets annotation Confirmation of subclustering robustness Confirmation of subclustering robustness for specific EPCAM- subsets Stromal inferCNV analysis and clustering Differential gene expression analysis Single-cell trajectory analysis of specific subpopulations Immune Lymphoid Analysis of cell Myeloid states & signatures Cell-Cell communication analysis Supplementary Fig. S1. Schematic view of the bioinformatics analysis workflow. An overview of the quality control steps and analyses done. Supplementary Fig. S2 A 10k 0.15 6k 5k 1k 0.10 4k 3k # of cell 100 0.05 2k 1k # of detected genes fraction of mito-genes 10 0.00 0 4 2 1 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 2 1 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1 Ep+ 4 Ep- 4 Ep+ 3 Ep- 3 Ep+ 2 Ep- 2 Ep+ 1 Ep- 1
    [Show full text]
  • Markers and Methods to Verify Mesenchymal Stem Cell Identity, Potency, and Quality Scott Schachtele, Ph.D., Christine Clouser, Ph.D., and Joy Aho, Ph.D
    WHITE PAPER Markers and Methods to Verify Mesenchymal Stem Cell Identity, Potency, and Quality Scott Schachtele, Ph.D., Christine Clouser, Ph.D., and Joy Aho, Ph.D. ABSTRACT Mesenchymal stem cells (MSCs) are multipotent cells that are functionally defined by their capacity to self-renew and their ability to differentiate into multiple cell types including adipocytes, chondrocytes, and osteocytes. Translation of MSC-based therapies has been confounded by MSC population heterogeneity as well as non-standardized methods for their definition and characterization. This white paper begins by defining and discussing the evolution of MSC nomenclature. It then provides a detailed description of MSC markers and how they may vary by tissue source and species. Ultimately, this review discusses how to standardize MSC characterization by selecting markers for isolation, characterization, and validation. KEY TOPICS • Defining Mesenchymal Stem Cells • MSC Marker Variation by Species • Established and New MSC Markers • Methods for MSC Identification and Characterization • Methods for MSC Isolation • Media Definitions for MSC Expansion INTRODUCTION Mesenchymal stem cells (MSCs) are multipotent cells that adhere to plastic, have a Box 1. Minimal Experimental Criteria for MSC fibroblast-like morphology, express a specific set of surface antigens, and differentiate into as proposed by the International Society for adipocytes, chondrocytes, and osteocytes.1 Clinically, MSCs are of interest for their ability to Cellular Therapy modulate the immune system as well as their potential to regenerate tissues. However, the translation of MSC-based therapies has been hindered by the heterogeneity of the isolated • Adherence to plastic. cells as well as the lack of standardized methods for their definition and characterization.
    [Show full text]
  • Single-Cell Transcriptomics of Human T Cells Reveals Tissue and Activation Signatures in Health and Disease
    ARTICLE https://doi.org/10.1038/s41467-019-12464-3 OPEN Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease Peter A. Szabo 1,6, Hanna Mendes Levitin2,6, Michelle Miron1,3, Mark E. Snyder1, Takashi Senda1,4, Jinzhou Yuan2, Yim Ling Cheng2, Erin C. Bush2, Pranay Dogra1, Puspa Thapa1, Donna L. Farber 1,3,4,7*& Peter A. Sims 2,5,7* 1234567890():,; Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8+ T cells and an interferon-response state for CD4+ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8+ compared to CD4+ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease. 1 Columbia Center for Translational Immunology, Columbia University Irving Medical Center, New York, NY, USA. 2 Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA. 3 Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, USA.
    [Show full text]
  • Hematopoietic Stem Cell & Lineage-Specific Markers
    R&D Systems Tools for Cell Biology Research™ STEM CELL FOCUS: HEMATOPOIETIC STEM CELLS Hematopoietic Stem Cell & Lineage-specific Markers FEATURED DATA: CD14 · CD69 · CXCL12/SDF-1a · CXCR4 · IL-3 Ra · IL-7 Ra/CD127 · LAMP1/CD107a · MPO · MS4A1/CD20 · SCF R/c-kit Hematopoietic stem cells (HSCs) are multipotent, self-renewing progenitor cells from which all differentiated blood cell types arise during the process of hematopoiesis. These cells include lymphocytes, granulocytes, and macrophages of the immune system as well as circulating erythrocytes and platelets. Classically, HSCs are thought to differentiate into two lineage-restricted, lymphoid and myelo-erythroid, oligopotent progenitor cells. An alternative, “myeloid-based” model for blood lineage development from HSCs describes a novel intermediary, a common myelo-lymphoid progenitor cell, which has the capacity to generate progeny from both lineages. The mechanisms controlling HSC homing to the bone marrow, self-renewal, and differentiation are thought to be influenced by a diverse set of cytokines, chemokines, receptors, and intracellular signaling molecules. For a complete listing of tools for HSC identification, expansion, differentiation, and verification, please visit: www.RnDSystems.com/HSC. Hematopoietic Stem Cells CD34 CD38– Flt-3/Flk-2 CD38+/– Flt-3/Flk-2– Sca-1 SCF R/c-kit Common Lymphoid SLAM/CD150 CD48/SLAMF2– 2B4/CD244/SLAMF4– Progenitor Cells IL-7 Rα/CD127 CD10 Flt-3/Flk-2 CD34 T Cells CD2 CD3 CD4 B Cells Lymphocyte Granulocyte-Macrophage Markers Progenitor Cells CD5
    [Show full text]
  • Unravelling Pathobiological Molecular Mechanisms of T-Cell Acute Lymphoblastic Leukemia
    Unravelling Pathobiological Molecular Mechanisms of T-Cell Acute Lymphoblastic Leukemia Rui D. Mendes 1 Unravelling Pathobiological Molecular Mechanisms of T-Cell Acute Lymphoblastic Leukemia Rui D. Mendes 2 The research here described was conducted in the department of Pediatric Oncology/ Hematology at Erasmus Medical Center - Sophia Children’s Hospital, Rotterdam, the Netherlands. The studies described in this thesis were financially supported by Stichting Kinderen Kankervrij (KiKa). Publication of this thesis was financially supported by: Cover: Nathan Sawaya. The original picture displays a two metre sculpture named ‘Building Bricks of Life’ made by the artist Nathan Sawaya. “Just as each brick plays an important part in holding the sculpture together, each gene within the human genome helps us understand more about ourselves as humans and how we are all connected” - Nathan Sawaya Book design: Rui D. Mendes Printed by: ISBN: XXX-XX-XXXX-XXX-X Copyright © 2016 by Rui D. Mendes. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the author. 3 Unravelling Pathobiological Molecular Mechanisms of T-Cell Acute Lymphoblastic Leukemia Het ontrafelen van pathobiologische moleculaire mechanismes in T-cel acute lymphoblastische leukemie Thesis to obtain the degree of doctor from Erasmus University Rotterdam by command of the rector magnificus Prof.dr. H.A.P. Pols and in accordance with the decision of the Doctorate Board. The public defence shall be held on Tuesday December 13, 2016 at 13:30 hours by Rui Daniel Martins Nunes Mendes born in Lisbon, Portugal 4 Doctoral Committee: Promotor: Prof.dr.
    [Show full text]
  • Multicolor Analysis of Cell Surface Marker of Human Leukemia Cell Lines Using Flow Cytometry
    ANTICANCER RESEARCH 34: 4539-4550 (2014) Multicolor Analysis of Cell Surface Marker of Human Leukemia Cell Lines Using Flow Cytometry TOMOKO INOUE1, ANTHONY SWAIN1, YOICHI NAKANISHI2 and DAISUKE SUGIYAMA1 1Department of Research and Development of Next-Generation Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Center for Clinical and Translational Research, Kyushu University Hospital, Fukuoka, Japan Abstract. Background: Leukemia cell lines are utilized as and transcription factor alterations, which lead to anemia, tools for molecular analysis. Their implementation in therapy neutropenia and thrombocytopenia. In almost all cases, will require standards for quality control, including leukemia is classified as one of four types; acute appropriate selection criteria for functional analysis and myelogenous leukemia (AML), acute lymphocytic leukemia efficacy determination. Materials and Methods: (ALL), chronic myelogenous leukemia (CML), and chronic Characteristics of six human leukemia cell lines –Kasumi-1, lymphocytic leukemia (CLL). In acute leukemia, leukocytes NB-4, MOLM-13, MV-4-11, K562, and Jurkat cells– were proliferate rapidly in the bone marrow at an immature state, investigated using multiple color analysis of surface antigen and therefore cannot function normally. On the other hand, in expression and comparative analysis of gene expression. chronic leukemia, blastic cells form more slowly than in Results: Differentiation states of Kasumi-1 and MOLM-13 acute leukemia and consequently cells with abnormal cells are colony-forming units-granulocyte/macrophage function are transported to the hematopoietic tissues. In equivalent cells to myeloblasts with comparatively high AML and CML, myeloid lineage cells such as granulocytes Growth factor independent-1(GFI1) and Transcription factor and monocytes, become malignant and lose the capacity to PU.1 (PU.1) expression, respectively.
    [Show full text]