Derivation of T Cells <em>In Vitro</em> from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

University of Toronto

The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the â€˜oncometaboliteâ€™ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.

Des mutations dans les g&egrave;nes IDH1 et IDH2 isocitrate d&eacute;shydrog&eacute;nase d&#39;encodage sont fr&eacute;quemment trouv&eacute;es dans les glioblastomes humaines et cytog&eacute;n&eacute;tique normales leuc&eacute;mies my&eacute;lo&iuml;des aigu&euml;s (AML). Ces alt&eacute;rations sont des mutations de gain de fonction qu&#39;ils conduisent &agrave; la synth&egrave;se de la &laquo; oncometabolite &raquo; R-2-hydroxyglutarate (2HG). On ne sait pas comment les mutations IDH1 et IDH2 modifier le d&eacute;veloppement des cellules my&eacute;lo&iuml;des et promouvoir leukaemogenesis. Nous rapportons ici la caract&eacute;risation de souris conditionnelle knock-in (KI) dans lesquelles la mutation la plus courante IDH1, IDH1(R132H), est ins&eacute;r&eacute;e dans le locus de Idh1 murin endog&egrave;ne et s&#39;exprime dans toutes les cellules h&eacute;matopo&iuml;&eacute;tiques (souris Vav-KI), ou plus pr&eacute;cis&eacute;ment dans les cellules de la lign&eacute;e my&eacute;lo&iuml;de (souris LysM-KI). Ces mutants montrent un nombre accru de d&eacute;but prog&eacute;niteurs h&eacute;matopo&iuml;&eacute;tiques et d&eacute;veloppent une spl&eacute;nom&eacute;galie et l&#39;an&eacute;mie avec h&eacute;matopo&iuml;&egrave;se extram&eacute;dullaire, sugg&eacute;rant une niche dysfonctionnelle de la moelle osseuse. En outre, les cellules LysM-KI ont fait histones et changements de m&eacute;thylation de l&#39;ADN semblable &agrave; ceux observ&eacute;s en humain IDH1-IDH2-mutant ou AML. &Agrave; notre connaissance, notre &eacute;tude est le premier &agrave; d&eacute;crire la g&eacute;n&eacute;ration et la caract&eacute;risation de IDH1 conditionnelle (R132H)-KI souris, ainsi que le premier rapport pour d&eacute;montrer l&#39;induction d&#39;une signature de m&eacute;thylation de l&#39;ADN leuc&eacute;miques dans un mod&egrave;le murin. Ainsi notre rapport met en lumi&egrave;re les liens m&eacute;canistes entre mutation IDH1 et AML humaine.

IDH1(R132H) mutation augmente les prog&eacute;niteurs h&eacute;matopo&iuml;&eacute;tiques murins et alt&egrave;re l&#39;&eacute;pig&eacute;n&eacute;tique.

Le mutazioni dei geni IDH1 e IDH2 codifica isocitrate dehydrogenases si trovano frequentemente in glioblastomi umani e citogeneticamente normale leucemie mieloidi acute (LMA). Queste alterazioni sono mutazioni guadagno di funzione che guidano la sintesi di &#39;oncometabolite&#39; R-2-idrossiglutarato (2HG). Rimane poco chiaro come le mutazioni di IDH1 e IDH2 modificare lo sviluppo delle cellule mieloidi e promuovere leukaemogenesis. Qui riportiamo la caratterizzazione dei topi condizionali knock-in (KI) in cui la pi&ugrave; comune mutazione IDH1, IDH1(R132H), viene inserita il locus di Idh1 murino endogeno ed &egrave; espressa in tutte le cellule emopoietiche (topi Vav-KI) o specificamente nelle cellule della linea mieloide (topi LysM-KI). Questi mutanti mostrano aumento del numero di cellule progenitrici ematopoietiche precoce e sviluppano splenomegalia e anemia con emopoiesi extramidollare, suggerendo una nicchia disfunzionale del midollo osseo. Inoltre, le cellule LysM-KI hanno hypermethylated gli istoni e cambiamenti di metilazione del DNA simile a quelle osservate nell&#39;uomo IDH1 - o IDH2-mutante AML. A nostra conoscenza, il nostro studio &egrave; il primo a descrivere la generazione e la caratterizzazione di IDH1 condizionale (R132H)-KI topi e anche il primo rapporto per dimostrare l&#39;induzione di una firma di metilazione del DNA leucemica in un modello del mouse. Il nostro rapporto cos&igrave; getta luce sui link meccanicistica tra mutazione IDH1 e AML umana.

IDH1(R132H) mutazione aumenta progenitori ematopoietici murini e altera di epigenetica.

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

Complete TCR-α gene locus control region activity in T cells derived in vitro from embryonic stem cells.

Hematopoiesis is the highly regulated and complex process by which blood cells are formed. Hematopoiesis can be achieved in vitro by the differentiation of embryonic stem cells (ESCs) into hematopoietic lineage cells. Differentiation of ESCs initially gives rise to mesoderm colonies that go on to form hemangioblast cells, which possess endothelial and hematopoietic lineage potential. While the differentiation of several hematopoietic lineages from ESCs, such as erythrocytes and macrophages, can be easily recapitulated in vitro, T-cell differentiation requires additional Notch-dependent signals for their generation. Keeping with this, ESCs induced to differentiate with OP-9 cells, a bone marrow-derived stromal cell line, give rise to erythro-myeloid cells and B lymphocytes, while the expression of an appropriate Notch ligand, such as Delta-like 1, on OP-9 cells (OP9-DL1) is required to support the generation of T-cells in vitro. Here, we describe an updated and streamlined protocol for the generation of T-lineage cells from mouse ESCs cultured on OP9-DL1 cells. This approach can facilitate studies aimed to assess the effects of environmental and genetic manipulations at various stages of T-cell development.

Directed differentiation of embryonic stem cells to the T-lymphocyte lineage.

Numerous locus control region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and "integration site-independence" would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells.

Roxanne Holmes

Sunnybrook Research Institute,
Department of Immunology,
Sunnybrook Research Institute, Department of Immunology

University of Toronto

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Roxanne Holmes

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University of Toronto

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Sunnybrook Research Institute,
Department of Immunology,
Sunnybrook Research Institute, Department of Immunology