Publications Flow Cytometry

Shared Resource Laboratory Operations: Changes made during Initial Global COVID-19 Lockdown of 2020.
Back JB, Chadick CH, Garcia Vallejo JJ, Orlowski-Oliver E, Patel R, Roe CE, Srivastava J, Walker RV

Undoubtedly, the global pandemic caused by the SARS-CoV-2 virus has had a significant impact on Shared Resource Laboratories (SRL) operations worldwide. Unlike other crises (e.g. natural disasters, acts of war, or terrorism) which often result in a sudden and sustained cessation of scientific research usually affecting one or two cities at a time, this impact is being seen simultaneously in every SRL worldwide albeit to a varying degree. The alterations to SRL operations caused by the COVID-19 pandemic can generally be divided into three categories: i) complete shutdown, ii) partial shutdown, and iii) uninterrupted operations. In many cases SRLs which remained partially or fully operational during the initial wave of global infections saw a concurrent increase in COVID-19-related research coming through their facilities. This forced SRLs to make rapid adjustments to core operations at the same time as infectious disease experts were still developing recommendations for the safety of frontline medical workers. Although many SRLs already had contingency plans in place, this pandemic has highlighted the importance of having such plans for continuity of service, if possible, during a crisis. Immediate changes have occurred in the way SRLs operate due to potential virus transmission and in line with this new "Best Practices" have been established i.e. social distancing, remote working and technology-based meetings and training. Many of these changes are likely to be in place for some time with the threat of further waves of infections toward the end of 2020 and into 2021. Some of these best practices, such as having many training resources recorded and available online, are likely to remain long term. Although many changes have been made in haste, these will alter the future operations of SRLs. In addition we have learnt how to deal with future crises that may be encountered in the workplace.

Cytometry. Part A : the journal of the International Society for Analytical Cytology, 1, 1, , 11 Nov 2020

Cyt-Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops.
Czechowska K, Lannigan J, Aghaeepour N, Back JB, Begum J, Behbehani G, Bispo C, Bitoun D, Fernández AB, Boova ST, Brinkman RR, Ciccolella CO, Cotleur B, Davies D, Dela Cruz GV, Del Rio-Guerra R, Des Lauriers-Cox AM, Douagi I, Dumrese C, Bonilla Escobar DL, Estevam J, Ewald C, Fossum A, Gaudillière B, Green C, Groves C, Hall C, Haque Y, Hedrick MN, Hogg K, Hsieh EWY, Irish J, Lederer J, Leipold M, Lewis-Tuffin LJ, Litwin V, Lopez P, Nasdala I, Nedbal J, Ohlsson-Wilhelm BM, Price KM, Rahman AH, Rayanki R, Rieger AM, Robinson JP, Shapiro H, Sun YS, Tang VA, Tesfa L, Telford WG, Walker R, Welsh JA, Wheeler P, Tárnok A

See publication

Cytometry. Part A : the journal of the International Society for Analytical Cytology, 95, 12, , Dec 2019

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).
Cossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S, Agace WW, Aghaeepour N, Akdis M, Allez M, Almeida LN, Alvisi G, Anderson G, Andrä I, Annunziato F, Anselmo A, Bacher P, Baldari CT, Bari S, Barnaba V, Barros-Martins J, Battistini L, Bauer W, Baumgart S, Baumgarth N, Baumjohann D, Baying B, Bebawy M, Becher B, Beisker W, Benes V, Beyaert R, Blanco A, Boardman DA, Bogdan C, Borger JG, Borsellino G, Boulais PE, Bradford JA, Brenner D, Brinkman RR, Brooks AES, Busch DH, Büscher M, Bushnell TP, Calzetti F, Cameron G, Cammarata I, Cao X, Cardell SL, Casola S, Cassatella MA, Cavani A, Celada A, Chatenoud L, Chattopadhyay PK, Chow S, Christakou E, Čičin-Šain L, Clerici M, Colombo FS, Cooper AM, Corbett AJ, Cosma A, Cosmi L, Coulie PG, Cumano A, Cvetkovic L, Dang VD, Dang-Heine C, Davey MS, Davies D, De Biasi S, Del Zotto G, Dela Cruz GV, Delacher M, Della Bella S, Dellabona P, Deniz G, Dessing M, Di Santo JP, Diefenbach A, Dieli F, Dolf A, Dörner T, Dress RJ, Dudziak D, Dustin M, Dutertre CA, Ebner F, Eckle SBG, Edinger M, Eede P, Ehrhardt GRA, Eich M, Engel P, Engelhardt B, Erdei A, Esser C, Everts B, Evrard M, Falk CS, Fehniger TA, Felipo-Benavent M, Ferry H, Feuerer M, Filby A, Filkor K, Fillatreau S, Follo M, Förster I, Foster J, Foulds GA, Frehse B, Frenette PS, Frischbutter S, Fritzsche W, Galbraith DW, Gangaev A, Garbi N, Gaudilliere B, Gazzinelli RT, Geginat J, Gerner W, Gherardin NA, Ghoreschi K, Gibellini L, Ginhoux F, Goda K, Godfrey DI, Goettlinger C, González-Navajas JM, Goodyear CS, Gori A, Grogan JL, Grummitt D, Grützkau A, Haftmann C, Hahn J, Hammad H, Hämmerling G, Hansmann L, Hansson G, Harpur CM, Hartmann S, Hauser A, Hauser AE, Haviland DL, Hedley D, Hernández DC, Herrera G, Herrmann M, Hess C, Höfer T, Hoffmann P, Hogquist K, Holland T, Höllt T, Holmdahl R, Hombrink P, Houston JP, Hoyer BF, Huang B, Huang FP, Huber JE, Huehn J, Hundemer M, Hunter CA, Hwang WYK, Iannone A, Ingelfinger F, Ivison SM, Jäck HM, Jani PK, Jávega B, Jonjic S, Kaiser T, Kalina T, Kamradt T, Kaufmann SHE, Keller B, Ketelaars SLC, Khalilnezhad A, Khan S, Kisielow J, Klenerman P, Knopf J, Koay HF, Kobow K, Kolls JK, Kong WT, Kopf M, Korn T, Kriegsmann K, Kristyanto H, Kroneis T, Krueger A, Kühne J, Kukat C, Kunkel D, Kunze-Schumacher H, Kurosaki T, Kurts C, Kvistborg P, Kwok I, Landry J, Lantz O, Lanuti P, LaRosa F, Lehuen A, LeibundGut-Landmann S, Leipold MD, Leung LYT, Levings MK, Lino AC, Liotta F, Litwin V, Liu Y, Ljunggren HG, Lohoff M, Lombardi G, Lopez L, López-Botet M, Lovett-Racke AE, Lubberts E, Luche H, Ludewig B, Lugli E, Lunemann S, Maecker HT, Maggi L, Maguire O, Mair F, Mair KH, Mantovani A, Manz RA, Marshall AJ, Martínez-Romero A, Martrus G, Marventano I, Maslinski W, Matarese G, Mattioli AV, Maueröder C, Mazzoni A, McCluskey J, McGrath M, McGuire HM, McInnes IB, Mei HE, Melchers F, Melzer S, Mielenz D, Miller SD, Mills KHG, Minderman H, Mjösberg J, Moore J, Moran B, Moretta L, Mosmann TR, Müller S, Multhoff G, Muñoz LE, Münz C, Nakayama T, Nasi M, Neumann K, Ng LG, Niedobitek A, Nourshargh S, Núñez G, O'Connor JE, Ochel A, Oja A, Ordonez D, Orfao A, Orlowski-Oliver E, Ouyang W, Oxenius A, Palankar R, Panse I, Pattanapanyasat K, Paulsen M, Pavlinic D, Penter L, Peterson P, Peth C, Petriz J, Piancone F, Pickl WF, Piconese S, Pinti M, Pockley AG, Podolska MJ, Poon Z, Pracht K, Prinz I, Pucillo CEM, Quataert SA, Quatrini L, Quinn KM, Radbruch H, Radstake TRDJ, Rahmig S, Rahn HP, Rajwa B, Ravichandran G, Raz Y, Rebhahn JA, Recktenwald D, Reimer D, Reis E Sousa C, Remmerswaal EBM, Richter L, Rico LG, Riddell A, Rieger AM, Robinson JP, Romagnani C, Rubartelli A, Ruland J, Saalmüller A, Saeys Y, Saito T, Sakaguchi S, Sala-de-Oyanguren F, Samstag Y, Sanderson S, Sandrock I, Santoni A, Sanz RB, Saresella M, Sautes-Fridman C, Sawitzki B, Schadt L, Scheffold A, Scherer HU, Schiemann M, Schildberg FA, Schimisky E, Schlitzer A, Schlosser J, Schmid S, Schmitt S, Schober K, Schraivogel D, Schuh W, Schüler T, Schulte R, Schulz AR, Schulz SR, Scottá C, Scott-Algara D, Sester DP, Shankey TV, Silva-Santos B, Simon AK, Sitnik KM, Sozzani S, Speiser DE, Spidlen J, Stahlberg A, Stall AM, Stanley N, Stark R, Stehle C, Steinmetz T, Stockinger H, Takahama Y, Takeda K, Tan L, Tárnok A, Tiegs G, Toldi G, Tornack J, Traggiai E, Trebak M, Tree TIM, Trotter J, Trowsdale J, Tsoumakidou M, Ulrich H, Urbanczyk S, van de Veen W, van den Broek M, van der Pol E, Van Gassen S, Van Isterdael G, van Lier RAW, Veldhoen M, Vento-Asturias S, Vieira P, Voehringer D, Volk HD, von Borstel A, von Volkmann K, Waisman A, Walker RV, Wallace PK, Wang SA, Wang XM, Ward MD, Ward-Hartstonge KA, Warnatz K, Warnes G, Warth S, Waskow C, Watson JV, Watzl C, Wegener L, Weisenburger T, Wiedemann A, Wienands J, Wilharm A, Wilkinson RJ, Willimsky G, Wing JB, Winkelmann R, Winkler TH, Wirz OF, Wong A, Wurst P, Yang JHM, Yang J, Yazdanbakhsh M, Yu L, Yue A, Zhang H, Zhao Y, Ziegler SM, Zielinski C, Zimmermann J, Zychlinsky A

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

European journal of immunology, 49, 1521-4141, , 2019

Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States.
Collier AJ, Panula SP, Schell JP, Chovanec P, Plaza Reyes A, Petropoulos S, Corcoran AE, Walker R, Douagi I, Lanner F, Rugg-Gunn PJ

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.

Cell stem cell, , 1875-9777, , 2017

Aging yeast gain a competitive advantage on non-optimal carbon sources.
Frenk S, Pizza G, Walker RV, Houseley J

Animals, plants and fungi undergo an aging process with remarkable physiological and molecular similarities, suggesting that aging has long been a fact of life for eukaryotes and one to which our unicellular ancestors were subject. Key biochemical pathways that impact longevity evolved prior to multicellularity, and the interactions between these pathways and the aging process therefore emerged in ancient single-celled eukaryotes. Nevertheless, we do not fully understand how aging impacts the fitness of unicellular organisms, and whether such cells gain a benefit from modulating rather than simply suppressing the aging process. We hypothesized that age-related loss of fitness in single-celled eukaryotes may be counterbalanced, partly or wholly, by a transition from a specialist to a generalist life-history strategy that enhances adaptability to other environments. We tested this hypothesis in budding yeast using competition assays and found that while young cells are more successful in glucose, highly aged cells outcompete young cells on other carbon sources such as galactose. This occurs because aged yeast divide faster than young cells in galactose, reversing the normal association between age and fitness. The impact of aging on single-celled organisms is therefore complex and may be regulated in ways that anticipate changing nutrient availability. We propose that pathways connecting nutrient availability with aging arose in unicellular eukaryotes to capitalize on age-linked diversity in growth strategy and that individual cells in higher eukaryotes may similarly diversify during aging to the detriment of the organism as a whole.

Aging cell, , 1474-9726, , 2017

Tracking the embryonic stem cell transition from ground state pluripotency.
Kalkan T, Olova N, Roode M, Mulas C, Lee HJ, Nett I, Marks H, Walker R, Stunnenberg HG, Lilley KS, Nichols J, Reik W, Bertone P, Smith A

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Development (Cambridge, England), , 1477-9129, , 2017

International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.
Barsky LW, Black M, Cochran M, Daniel BJ, Davies D, DeLay M, Gardner R, Gregory M, Kunkel D, Lannigan J, Marvin J, Salomon R, Torres C, Walker R

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

Cytometry. Part A : the journal of the International Society for Analytical Cytology, 89, 1552-4930, , 2016

The spindle assembly checkpoint works like a rheostat rather than a toggle switch.
Collin P,Nashchekina O,Walker R,Pines J

The spindle assembly checkpoint (SAC) is essential in mammalian mitosis to ensure the equal segregation of sister chromatids. The SAC generates a mitotic checkpoint complex (MCC) to prevent the anaphase-promoting complex/cyclosome (APC/C) from targeting key mitotic regulators for destruction until all of the chromosomes have attached to the mitotic apparatus. A single unattached kinetochore can delay anaphase for several hours, but how it is able to block the APC/C throughout the cell is not understood. Present concepts of the SAC posit that either it exhibits an all-or-nothing response or there is a minimum threshold sufficient to block the APC/C (ref. 7). Here, we have used gene targeting to measure SAC activity, and find that it does not have an all-or-nothing response. Instead, the strength of the SAC depends on the amount of MAD2 recruited to kinetochores and on the amount of MCC formed. Furthermore, we show that different drugs activate the SAC to different extents, which may be relevant to their efficacy in chemotherapy.

Nature cell biology, 15, 1476-4679, , 2013

Germline potential of parthenogenetic haploid mouse embryonic stem cells.
Leeb M,Walker R,Mansfield B,Nichols J,Smith A,Wutz A

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.

Development (Cambridge, England), 139, 1477-9129, , 2012

A high-throughput, flow cytometry-based method to quantify DNA-end resection in mammalian cells.
Forment JV,Walker RV,Jackson SP

Replication protein A (RPA) is an essential trimeric protein complex that binds to single-stranded DNA (ssDNA) in eukaryotic cells and is involved in various aspects of cellular DNA metabolism, including replication and repair. Although RPA is ubiquitously expressed throughout the cell cycle, it localizes to DNA replication forks during S phase, and is recruited to sites of DNA damage when regions of ssDNA are exposed. During DNA double-strand break (DSB) repair by homologous recombination (HR), RPA recruitment to DNA damage sites depends on a process termed DNA-end resection. Consequently, RPA recruitment to sub-nuclear regions bearing DSBs has been used as readout for resection and for ongoing HR. Quantification of RPA recruitment by immunofluorescence-based microscopy techniques is time consuming and requires extensive image analysis of relatively small populations of cells. Here, we present a high-throughput flow-cytometry method that allows the use of RPA staining to measure cell proliferation and DNA-damage repair by HR in an unprecedented, unbiased and quantitative manner.

Cytometry. Part A : the journal of the International Society for Analytical Cytology, 81, 1552-4930, , 2012

A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells.
Ng K,Daigle N,Bancaud A,Ohhata T,Humphreys P,Walker R,Ellenberg J,Wutz A

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

Molecular biology of the cell, 22, 1939-4586, , 2011

Oct4 and LIF/Stat3 additively induce Krüppel factors to sustain embryonic stem cell self-renewal.
Hall J,Guo G,Wray J,Eyres I,Nichols J,Grotewold L,Morfopoulou S,Humphreys P,Mansfield W,Walker R,Tomlinson S,Smith A

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Cell stem cell, 5, 1875-9777, , 2009

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.
D Corcos, MJ Osborn, LS Matheson, F Santos, X Zou, JA Smith, G Morgan, A Hutchings, M Hamon, D Oxley, M Brüggemann

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

Blood, 115, 2, , 2010

Transcription of productive and nonproductive VDJ-recombined alleles after IgH allelic exclusion.
J Daly, S Licence, A Nanou, G Morgan, IL Mårtensson

The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.

The EMBO journal, 26, 19, , 2007

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.
C Carter, C Dion, S Schnell, WJ Coadwell, M Graham, L Hepburn, G Morgan, A Hutchings, JC Pascall, H Jacobs, JR Miller, GW Butcher

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a "delayed" apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency ( approximately 30%). This suggests an adaptive significance for this hypomorphic allele.

Journal of immunology (Baltimore, Md. : 1950), 179, 3, , 2007

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.
Richardson SM,Walker RV,Parker S,Rhodes NP,Hunt JA,Freemont AJ,Hoyland JA

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Stem cells (Dayton, Ohio), 24, 1066-5099, , 2006

Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs.
C Dion, C Carter, L Hepburn, WJ Coadwell, G Morgan, M Graham, N Pugh, G Anderson, GW Butcher, JR Miller

Reports suggest that two members of the novel immune-associated nucleotide (Ian) GTPase family, Ian1 and Ian5, play roles in T cell development. We performed real-time PCR analysis of the expression of Ian genes of the rat during T cell maturation, in macrophages and in cell lines. We found that all of the genes were expressed at relatively low levels at the early double-negative thymocyte stage but were expressed more strongly at later cell stages. Our study also revealed the fact that the previously reported Ian9, Ian10 and Ian11 genes are, instead, parts of a single gene for which we retain the name Ian9, potentially encoding a GTPase with a highly unusual triplicated structure. Antisera were developed against both Ian1 and Ian9. We established that Ian9 is produced as an approximately 75-kDa protein in both T cells and thymocytes. We observed that levels of both Ian1 and Ian9 proteins are profoundly reduced in T cells from lymphopenic rats as compared with wild-type rats. It was demonstrated that thymocytes and B cells from lymphopenic rats (Ian5 null) did not show enhanced sensitivity to gamma-irradiation-induced apoptosis.

International immunology, 17, 9, , 2005