Multiparameter Fluorescence-activated Cell Sorter Research Articles

Immune involvement of the contralateral hemisphere in a glioblastoma mouse model

BackgroundGlioblastoma (GBM) is the most common and deadliest form of brain cancer in adults. Standard treatment, consisting of surgery and radiochemotherapy, only provides a modest survival benefit and is incapable...

Biological Characteristics of Human Skeletal Muscle-Derived Pericytes/Perivascular Cells and Their Supporting Effects on Hematopoietic Stem Cell in Vitro

Mesenchymal stem/stromal cells (MSCs) as precursor cells of bone marrow stromal cells, in addition to the formation of the bone marrow microenvironment, secrete a variety of blood-related factors, promote hematopoietic recovery and reconstruction. However, mesenchymal stem/stromal cells are extremely rare within human bone marrow, which limit their experimental and clinical applications. Our previous study demonstrated that MSCs were derived from pericytes/perivascular cells (PCs), and PCs were the precursor of MSCs. PCs are widely distributed, especially in skeletal muscle tissue, which are rich in content, easy to be harvested with minimal damage, and rapid cell proliferation. However, very few researches have been done on whether human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) are involved in HSPCs expansion/proliferation, differentiation and possible hematopoietic regulation mechanisms. Based on the above research background and research ideas, in current study, we intend to isolate, purify and characterize the CD146+ hMD-PCs and further investigate their supporting effect on human umbilical cord blood CD34+ cells in vitro. Methods 1. hMD-PCs with phenotype CD146+CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting (MP-FACS), and their biological characteristic were conducted by detecting the surface marker of MSCs and inducing multilineage differentiation. 2. Human UCB CD34+cells were sorted by immunomagnetic beads, and then established the in vitro expansion culture system of UCB CD34+ cells co-culture with CD146+ hMD-PCs (experimental group), human bone marrow MSCs for feeder layer (positive control) and UCB CD34+ cells alone culture system (blank control). After 1 week, 2 weeks and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were analysed. Result 1.CD146+ hMD-PCs were sorted by MP-FACS and the purity was (91.5±1.85)% (n=5); CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell marker CD45, CD34, CD31. After induced culture, CD146+ hMD-PCs can differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts; 2.UCB CD34+ cells were sorted by magnetic beads which number was (9.18±3.50)×105, and then co-cultured with CD146+ hMD-PCs and human BM-MSCs at a density of 5×104/well, respectively. After 1, 2 and 4 weeks of co-culture, there were no statistically significant differences in cell number, colony formation ability and immunophenotype (CD45+cells, CD34+CD33-cells, CD14+cells, CD10+/CD19+cells) between CD146+ hMD-PCs for feeder layer culture system and BM-MSCs for feeder layer culture system (P>0.05, n=6). However, after 1 week, 2 weeks of co-culture, the both cells number of CD146+ hMD-PCs and human BM-MSCs culture system were significantly different from the blank control group, (P<0.01) and (P<0.001) respectively. The colony culture could not be performed due to low cell number of the blank control group. Conclusion CD146+ hMD-PCs, like human BM-MSCs, have hematopoietic support capacity in vitro; therefore CD146+ hMD-PCs can be used as another source of stromal cells for the expansion of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Multidimensional assessment of alveolar T cells in critically ill patients

Pneumonia represents the leading infectious cause of death in the United States. Foxp3+ regulatory T cells promote recovery from severe pneumonia in mice, but T cell responses in patients with pneumonia remain incompletely characterized because of the limited ability to serially sample the distal airspaces and perform multidimensional molecular assessments on the small numbers of recovered cells. As T cell function is governed by their transcriptional and epigenetic landscape, we developed a method to safely perform high-resolution transcriptional and DNA methylation profiling of T cell subsets from the alveoli of critically ill patients. Our method involves nonbronchoscopic bronchoalveolar lavage combined with multiparameter fluorescence-activated cell sorting, unsupervised low-input RNA-sequencing, and a modified reduced-representation bisulfite sequencing protocol. Here, we demonstrate the safety and feasibility of our method and use it to validate functional genomic elements that were predicted by mouse models. Because of its potential for widespread application, our techniques allow unprecedented insights into the biology of human pneumonia.

Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species’ cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34(Low)CD31(Pos)VEGFR-3(Pos)PODOPLANIN(Pos) LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

New Molecular Evidence That Oct-4 Is Truly Expressed In a Rare Population Of Developmental Early Stem Cells In Human Umbilical Cord Blood (UCB) and That Epigenetic Modification Of Imprinting At Igf2-H19 Locus Regulates Their Quiescent State – Potential Implications For Regenerative Medicine

BackgroundOne of the most intriguing questions in stem cell biology is whether human umbilical cord blood (UCB) contains early-development stem cells that express markers of pluripotency and thus could be employed in regenerative medicine. Several groups have reported mRNAs for genes regulating stem cell pluripotency, such as Oct-4A, Nanog, and SSEA-1, in UCB cells. However, detection of the Oct-4A transcript may be hampered by the presence of several pseudogenes and Oct-4B isoform, which is not related to stem cell pluripotency. Another important question is: why are these primitive stem cells that are present in UCB highly quiescent and relatively resistant to ex vivo expansion? We previously identified a population of Oct-4+ CD133+Lin–CD45– cells in human UCB (Leukemia 2007;21:297–303) that may become specified into long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278) and mesenchymal stem cells (Stem Cell & Dev. 2013;22:622). These Oct-4+CD133+Lin–CD45– cells present in UCB correspond to a population of murine Oct-4+Sca-1+Lin+CD45– cells that remain quiescent in bone marrow because of epigenetic modification of parentally imprinted genes, including the Igf-2-H19 tandem gene (Leukemia 2009;23:2042). The quiescence of these cells has been explained by erasure of imprinting in the regulatory differentially methylated region (DMR) at the Igf2-H19 locus. In appropriate animal models, these small cells also give rise to LT-HSCs (Exp. Hematol2011;3:225), mesenchymal stem cells (Stem CellsDev 2010;19:1557), and lung epithelium (Stem Cells2013;doi: 10.1002/stem.1413). Moreover, as we demonstrated, the epigenetic reversal of the maternal type of imprinting to the somatic type in the DMR for the Igf2-H19 locus, which is necessary to maintain balanced expression between insulin-like growth factor 2 (Igf-2) and noncoding H19 RNA (precursor for several inhibitory miRNAs) from paternal and maternal chromosomes, respectively, is required for these cells to enter the cell cycle. The crucial role of Igf2-H19 imprinting in quiescence of the most-primitive stem cells in murine BM has been very recently confirmed by another group (Nature 2013, doi: 10.1038/nature12303). Aim of the studyTo address whether human UCB Oct-4± CD133±Lin–CD45– cells truly express genes regulating pluripotency, we examined the DNA methylation state of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) and of the DMR for Igf2-H19. Materials and MethodsUCB CD133+Lin–CD45– cells were isolated by multiparameter fluorescence-activated cell sorting (FACS) after intra-cellular staining for Oct4 protein in lineage-depleted human UCB mononuclear cells. Bisulfide modification of DNA followed by sequencing was employed to evaluate the methylation state of CpG islands in the promoters for Oct-4, Nanog, and Sall4 as well as in the DMR for the Igf2-H19 locus. Salient ResultsWe observed that Oct4, Nanog, and Sall4 promoters in UCB Oct-4+CD133+Lin–CD45– cells were demethylated to a similar degree as the human teratocarcinoma NTERA2, which is evidence for true expression of these genes. Furthermore, in human UCB we observed Oct-4+CD133+Lin–CD45– cells that, like their murine counterparts, erase the imprinting in the DMR at the Igf2-H19 locus, which demonstrates that genomic imprinting could be a key mechanism for maintaining the quiescence of these cells. This imprinting data was subsequently confirmed by RQ-PCR analysis of gene expression, showing downregulation of autocrine Igf-2 and upregulation of noncoding H19 RNA. ConclusionOur methylation studies of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) provide for the first time strong molecular evidence that UCB contains cells that truly express pluripotent stem cell markers. Moreover, molecular analysis of the methylation state in the DMR for the Igf2-H19 locus also explains for the first time how the quiescent state of these cells is regulated by changes in parental imprinting at the Igf2-H19 locus. Thus, elucidation of this mechanism that controls and modifies genomic imprinting in VSELs will be crucial for developing strategies to expand these cells and employ them more efficiently in regenerative medicine and we are currently working on this. [Display omitted] Disclosures:Ratajczak:Neostem Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

SummaryHematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

BackgroundContinuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.

Higher Frequencies of GARP+CTLA-4+Foxp3+ T Regulatory Cells and Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Are Associated with Impaired T-Cell Functionality

The extent to which T-cell-mediated immune surveillance is impaired in human cancer remains a question of major importance, given its potential impact on the development of generalized treatments of advanced disease where the highest degree of heterogeneity exists. Here, we report the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). Using multi-parameter fluorescence-activated cell sorting analysis, we quantified the cumulative frequency of regulatory T cells (Treg), exhausted CD4(+) helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concurrent views on the overall level of immune dysfunction in these inoperable patients. We documented augmented numbers of Tregs, MDSC, PD-1(+)-exhausted T cells, and increased levels of immunosuppressive cytokines in patients with HCC, compared with normal controls, revealing a network of potential mechanisms of immune dysregulation in patients with HCC. In dampening T-cell-mediated antitumor immunity, we hypothesized that these processes may facilitate HCC progression and thwart the efficacy of immunotherapeutic interventions. In testing this hypothesis, we showed that combined regimens to deplete Tregs, MDSC, and PD-1(+) T cells in patients with advanced HCC restored production of granzyme B by CD8(+) T cells, reaching levels observed in normal controls and also modestly increased the number of IFN-γ producing CD4(+) T cells. These clinical findings encourage efforts to restore T-cell function in patients with advanced stage disease by highlighting combined approaches to deplete endogenous suppressor cell populations that can also expand effector T-cell populations.

Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.

Hepatitis c virus-specific t-cell-derived transforming growth factor beta is associated with slow hepatic fibrogenesis

Hepatitis C virus (HCV)-specific immune effector responses can cause liver damage in chronic infection. Hepatic stellate cells (HSC) are the main effectors of liver fibrosis. TGFβ, produced by HCV-specific CD8(+) T cells, is a key regulatory cytokine modulating HCV-specific effector T cells. Here we studied TGFβ as well as other factors produced by HCV-specific intrahepatic lymphocytes (IHL) and peripheral blood cells in hepatic inflammation and fibrogenesis. This was a cross-sectional study of two well-defined groups of HCV-infected subjects with slow (≤ 0.1 Metavir units/year, n = 13) or rapid (n = 6) liver fibrosis progression. HCV-specific T-cell responses were studied using interferon-gamma (IFNγ)-ELISpot ±monoclonal antibodies (mAbs) blocking regulatory cytokines, along with multiplex, enzyme-linked immunosorbent assay (ELISA) and multiparameter fluorescence-activated cell sorting (FACS). The effects of IHL stimulated with HCV-core peptides on HSC expression of profibrotic and fibrolytic genes were determined. Blocking regulatory cytokines significantly raised detection of HCV-specific effector (IFNγ) responses only in slow fibrosis progressors, both in the periphery (P = 0.003) and liver (P = 0.01). Regulatory cytokine blockade revealed HCV-specific IFNγ responses strongly correlated with HCV-specific TGFβ, measured before blockade (R = 0.84, P = 0.0003), with only a trend to correlation with HCV-specific IL-10. HCV-specific TGFβ was produced by CD8 and CD4 T cells. HCV-specific TGFβ, not interleukin (IL)-10, inversely correlated with liver inflammation (R = -0.63, P = 0.008) and, unexpectedly, fibrosis (R = -0.46, P = 0.05). In addition, supernatants from HCV-stimulated IHL of slow progressors specifically increased fibrolytic gene expression in HSC and treatment with anti-TGFβ mAb abrogated such expression. Although TGFβ is considered a major profibrogenic cytokine, local production of TGFβ by HCV-specific T cells appeared to have a protective role in HCV-infected liver, together with other T-cell-derived factors, ameliorating HCV liver disease progression.

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular‐signal‐regulated kinase and DNA in circulating tumor cells

A simple, selective, and sensitive multiparameter fluorescence activated cell sorting method utilizing density gradient centrifugation and magnetic antibody cell sorting was developed and validated for the determination of phosphorylated extracellular-signal-regulated kinase (pERK) and DNA in circulating tumor cells (CTCs). Cell preparation tubes (CPT) were used for peripheral blood collection and density gradient centrifugation, followed by phosphorylation of ERK with epidermal growth factor (EGF). After fixation with formaldehyde and methanol, magnetic anti epithelial cell adhesion molecule (EpCAM) micro-beads were used for the selective isolation of CTCs from the background, consisting of peripheral blood mononuclear cells and platelets. Subsequently, samples were stained with Hoechst 33342, and fluorescent antibodies against EpCAM, CD45, and pERK. Flow cytometry was used for identification and enumeration of CTCs and determination of their pERK and DNA content. The validation parameters included specificity, recovery, linearity, precision, sensitivity, and stability. The lower limit of quantification was two CTCs per 8 ml peripheral blood. Samples were stable for 4 months in storage at -80°C. The applicability of the method was demonstrated by successful enumeration of CTCs, and the determination of DNA, and pERK before and after stimulation with EGF in 8 ml peripheral blood samples from patients with metastatic cancer.

The CD4+CD28null and the regulatory CD4+CD25High T-cell phenotypes in patients with ulcerative colitis during active and quiescent disease, and following colectomy

The CD4+CD25High T-cell phenotype has an essential immunoregulatory role, while the CD4+CD28null T-cell reflects immune pathology. We investigated the profiles of the CD4+CD25High and the CD4+CD28null T-cell phenotypes in patients with ulcerative colitis (UC) during active and quiescent phases as well as following colectomy. Fifty-nine UC patients, 34 active (UCa) and 25 quiescent (UCq) together with 19 healthy controls (HC) were included. Ten of 34 UCa patients underwent colectomy due to unremitting UC (UCo). Immunohistochemical phenotypic of the peripheral blood lymphocytes bearing CD4, CD25 or CD28 was done for analyzes by a multiparameter fluorescence activated cell sorting technique. The expression of the CD4+CD25High phenotype was higher in UCq (P<0.01) or UCo (P<0.01) group vs UCa group. Further, the expression of the CD4+CD28null phenotype in UCa or UCo group was higher than in the HC group (P<0.05). However, the expression of the CD4+CD28null phenotype up to 12months after colectomy was not significantly different from the levels in the same patients during acute phase. Our impression is that a high CD4+CD25High T-cell reflects alleviation of inflammation, while the expression of the CD4+CD28null T-cell phenotype is an etiologic feature in UC patients, and is maintained after removing the affected colon.

Genetic and Epigenetic Studies Reveal That Murine Oct-4+ Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Present in Adult Tissues Share Several Similarities/Markers with Epiblast-Derived Migratory Primordial Germ Cells (PGCs).

Abstract 2521Poster Board II-498Recently, we identified a population of pluripotent VSELs in murine adult bone marrow (BM; Leukemia 2006:20;857). Compared with other adult stem cells (SCs), VSELs show unique epigenetic features including: i) open chromatin structures in the promoter of Oct-4 and Nanog; and ii) parent-of-origin-specific reprogramming of genomic imprinting. These features explain the pluripotent embryonic-like nature and quiescent status of these cells, respectively (Leukemia 2009:In press), and indicate their relation to an epiblast/germ-line pluripotent (P)SC population. To better understand the developmental origin of VSELs, we examined gene expression profiles and the epigenetic status of epiblast and germ-line related genes in these cells. We employed real time quantitative PCR (RQ-PCR) to evaluate gene expression, a bisulfite sequencing strategy to evaluate DNA methylation, and chromatin immunoprecipitation (ChIP) to elucidate histone codes in genes of interest. VSELs were isolated from murine BM by multiparameter fluorescence-activated cell sorter (FACS) as a population of Sca+lin−CD45− along with Sca+lin−CD45+ hematopoietic (H)SCs and BM mononuclear cells (MNCs). We noticed that VSELs, similarly to epiblast PSCs (EpiSCs), highly express the stemness genes (e.g., Oct-4, Nanog, Sox2, Klf4) and epiblast markers (Gbx2, Fgf5, Nodal). However, the Rex1 gene is expressed at a lower level compared to the murine ESC-D3 line. Moreover, VSELs also highly express the Stella, Blimp1, Dnd1, and Nanos3, which are developmental regulators during specification in the proximal epiblast of PGCs. Accordingly, the Stella promoter in VSELs was partially demethylated and highly enriched for transcriptionally active histones (acetylated H3, trimethylated lysine4 of H3) while being simultaneously less enriched for repressive ones (dimethylated lysine9 and trimethylated lysine27 of H3). In particular, we noticed that VSELs resemble migratory PGCs. To support this notion, VSELs: i) express several markers of migratory PGCs (Dppa2, Dppa4, Mvh); ii) do not express post-migratory PGCs genes (Dazl, Sycp3); and iii) have reprogrammed DNA demethylation in the repetitive sequence (LINE1) and the promoters of Mvh, Dazal, and Sycp3. Finally, VSELs express less the transcripts for cMyc, Stat3, Snai, and Ecat1, which are expressed in early specified PGCs. In conclusion, our previous data showing reprogramming of genomic imprinting in VSELs with the present gene expression profile and epigenetic studies strongly supports VSELs developmentally originating from epiblast-derived germ-line SCs (PGCs), particularly migratory PGCs. We believe VSELs are deposited during embryogenesis in the adult tissues as a backup for tissue-committed SCs and that epigenetic reprogramming tightly controls their proliferative potential. Thus, identification of mechanisms that control and modify the epigenetic marks in VSELs will be crucial for developing more powerful strategies to “unleash the power” of these cells and employ them in regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Accelerated cell line development using two‐color fluorescence activated cell sorting to select highly expressing antibody‐producing clones

The success of engineered monoclonal antibodies as biopharmaceuticals has generated considerable interest in strategies designed to accelerate development of antibody expressing cell lines. Stable mammalian cell lines that express therapeutic antibodies at high levels typically take 6-12 months to develop. Here we describe a novel method to accelerate selection of cells expressing recombinant proteins (e.g., antibodies) using multiparameter fluorescence activated cell sorting (FACS) in association with dual intracellular autofluorescent reporter proteins. The method is co-factor-independent and does not require complex sample preparation. Chinese hamster ovary (CHO) clones expressing high levels of recombinant antibody were selected on the basis of a two-color FACS sorting strategy using heavy and light chain-specific fluorescent reporter proteins. We were able to establish within 12 weeks of transfection cell lines with greater than a 38-fold increase in antibody production when compared to the pool from which they were isolated, following a single round of FACS. The method provides a robust strategy to accelerate selection and characterization of clones and builds a foundation for a predictive model of specific productivity based upon on two-color fluorescence.

Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts

The pathogenesis of bronchiolitis obliterans (BO), a common and devastating obliterative disorder of small airways following lung transplantation, remains poorly understood. Lesions are characterized in their early stages by lymphocyte influx that evolves into dense fibrotic infiltrates. Airway specimens taken from patients with histological BO revealed infiltrating myofibroblasts, which strongly expressed the signaling chain of the high affinity interleukin-13 (IL-13) receptor IL-13Ralpha1. Because IL-13 has proinflammatory and profibrotic actions, a contributory role for IL-13 in BO development was examined using murine models of orthotopic and heterotopic tracheal transplantation. Compared with airway isografts, allografts exhibited a significant increase in relative IL-13 mRNA and protein levels. Allogeneic tracheas transplanted into IL-13-deficient mice were protected from BO in both transplant models. Flow cytometric analysis of orthotopic transplant tissue digests revealed markedly fewer infiltrating mononuclear phagocytes and CD3(+) T lymphocytes in IL-13-deficient recipients. Furthermore, protection from luminal obliteration, collagen deposition, and myofibroblast infiltration was observed in heterotopic airways transplanted into the IL-13(-/-) recipients. Transforming growth factor-beta1 expression was significantly decreased in tracheal allografts into IL-13(-/-) recipients, compared to wild-type counterparts. These human and murine data implicate IL-13 as a critical effector cytokine driving cellular recruitment and subsequent fibrosis in clinical and ex-perimental BO.

Peritoneal macrophages suppress T‐cell activation by amino acid catabolism

T-lymphocyte activation triggered by anti-CD3, endogenous or exogenous superantigen, and mitogens was suppressed in a cell-dose-dependent fashion by peritoneal cavity (PerC) leucocytes. Study of lymphocyte-deficient mice and the use of multiparameter fluorescence-activated cell sorter analyses revealed that macrophages were responsible for this form of immune regulation. Interferon-gamma was essential to trigger suppression, which, by enzyme inhibition studies, was shown to be the result of tryptophan and arginine catabolism. These results illustrate that macrophages, which are classically defined by their innate effector function as antigen-presenting cells, have the potential to temper adaptive immunity.

The road ended up at stem cells.

For me the search for hematopoietic stem cells (HSC) actually started with the discovery by Till, McCulloch, and colleagues (1-3) that bone marrow contained single cells that could give rise to myeloerythroid colonies in the spleen, and sometimes these colonies contained cells that made more spleen colonies as well as radioprotected and reconstituted lethally irradiated mice (3). But in retrospect, it should have started with the remarkable observation of Ray Owen in 1945 that bovine fraternal twins sharing a single placenta and blood circulation retained production of blood cells genetically defined to be from both throughout their life (4). It could be argued that this was the experiment that began both modern experimental hematology as well as modern cellular immunology. The Till, McCulloch, Wu, Becker, and Simonovitch experiments were elegant demonstrations that single, genetically marked cells existed (random DNA breaks and translocations induced by sublethal irradiation of the donor bone marrow) that could both self-renew and differentiate (2, 5). But these experiments did not put the pure cells in the hands of scientists, and so most of their functions for the next 25 years were implied rather than directly analyzed. Just as genetics is the complement to biochemistry (when one considers genes and gene products), cell marking is the complement to cell purification in the fields of developmental and cellular biology. The first attempts at such cellular purification came from the 'school' of Till & McCulloch (6, 7), and independently the school of Van Bekkum in the Netherlands (8). But what was lacking in those experiments and at that time were both a comprehensive approach that would take into account the clonal activity of stem cells in both self-renewal and differentiation to all blood cell outcomes, and the tools with which one could separate what turned out to be an extremely rare population in the bone marrow. And, it wasn't known until much later that most day 8-10 spleen colonies were the progeny of progenitors, not stem cells (9). Two inventions facilitated the technology of purification of HSC: the advent of monoclonal antibody technology by Kohler & Milstein (10), and the development of the multiparameter fluorescence activated cell sorter by the Herzenberg group (11). My laboratory had established assays for the clonal precursors of T cells and B cells, and we had been using the Till-McCulloch spleen colony clonal assays since the mid-1960s. In the late 1970s and early 1980s we began in earnest the search for mouse early hematopoietic progenitors, including HSCs (12-16). The purification of HSCs proved to be much like the purification of an enzyme, or a cell surface receptor, or a gene. Successive enrichments finally led to the isolation of a population, which could no longer be subdivided and which contained precursors that read out in all clonal assays as well as in radioprotection of lethally irradiated hosts (17). Our first experiments transplanting single HSC in 1991 and 1992, led to the definitive demonstration that these were indeed HSCs (18-20). But these experiments and the ideas that led to them were developed in the context of immunology and experimental hematology as they were emerging in the 1950s and 60 s. This volume of Immunological Reviews is a rich testimony to the kinds of ideas and experiments that, at least in retrospect, turned out to be critical. Many roads were taken, but only one ended up at stem cells.