Sten Erik Jacobsen

PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.

Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia.

Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia

Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34⁺ progenitor cells.

The ASXL1 gene encodes a chromatin‐binding protein involved in epigenetic regulation in haematopoietic cells. Loss‐of‐function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral‐based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34+ cells differentiated along the myeloid lineage in vitro. ASXL1‐deficient cells showed a significant decrease in the generation of CD11b+ and CD15+ cells, implicating impaired granulomonocytic differentiation. Furthermore, colony‐forming assays showed a significant increase in the number of multipotent mixed lineage colony‐forming unit (CFU‐GEMM) colonies and a significant decrease in the numbers of granulocyte‐macrophage CFU (CFU‐GM) and granulocyte CFU (CFU‐G) colonies in ASXL1‐deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1‐deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over‐representation of PRC2 targets among the deregulated genes in ASXL1‐deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1.

Summary ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19−IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19−IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.

Modelling the in-utero initation of ETV6-RUNX1 in childhood acute lymphoblastic leukaemia using human pluripotent stem cells

Abstract Background We hypothesise that the clinical differences between adult and childhood acute lymphoblastic leukaemia (cALL) arise partly through their origin in developmentally distinct target cells. Since cALL frequently initiates in utero we aimed to characterise the earliest stages of lymphoid development in human fetal liver. In parallel we used human pluripotent stem cells (hPSC) to recapitulate fetal liver lymphopoiesis and model the impact of the fusion oncogene ETV6-RUNX1 , the commonest genetic aberration in cALL. Methods H1 human embyronic stem cells and MIFF3 human induced hPSCs (provided by University of Sheffield Centre for Stem Cell Biology) were maintained in vitro with mTeSR1/Matrigel. Vectors were produced by recombineering an ETV6 bacterial artificial chromosome with a custom DNA cassette (GeneArt, ThermoFisher, Waltham MA, USA). Vectors were transfected (Nucleofector II, Lonza, Basle, Switzerland) and G418-selected clones were screened by Southern blot. hPSCs were differentiated by sequential OP9/MS5 coculture. Populations sorted by fluorescence-activated cell sorting were analysed by single cell real-time PCR (Biomark 48.48, Fluidigm, San Fransisco, CA, USA) and 200 cell RNA sequencing. Human fetal livers were donated under informed consent with approval of Lund University Ethical Review Board and the Swedish National Board of Health and Welfare. Findings B lymphopoiesis in the fetal liver was distinct from that in adult bone marrow or neonatal cord blood: the earliest fetal liver B cells did not fully express mature lymphoid effectors and the earliest identified lymphoid-capable progenitors coexpressed both lymphoid and myeloid gene expression programmes. Global and single cell gene expression profiling of B lymphopoietic progenitors generated by in vitro differentiation of hPSCs showed that they share the same transcriptional programme. hPSCs that were CRISPR-engineered to express ETV6-RUNX1 were partly arrested in B cell differentiation at the level of the lymphomyeloid progenitor. B cells that passed this block shared the global gene expression signature of the fetal lymphomyeloid progenitor, and single cell real-time PCR showed that they aberrantly coexpressed B and myeloid lineage genes. Interpretation Our results identify a B lymphoid progenitor in human fetal liver characterised by coexpression of lymphoid and myeloid gene expression programmes and suggest that this progenitors B myeloid signature provides a permissive transcriptional context for ETV6-RUNX1 to effect a partial differentiation arrest. The developmental specificity of the gene expression signature of this cell could offer unique therapeutic targets, and the hSPC model might provide a novel drug-screening platform. Funding Wellcome Trust Research Training Fellowship and NIHR Academic Clinical Fellowship (SR); Swedish Childhood Cancer Foundation (CB); Bloodwise, Cancer Research UK, Children with Cancer, and Great Ormond Street Hospital Childrens Charity (TE).

Abstract 2692: ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor

Childhood acute lymphoblastic leukemia (cALL) is distinct from that in adults with higher incidence, better prognosis and a distinct mutational spectrum. One hypothesis for this difference is that cALL arises in transient cells unique to early human development. We explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick testing has shown that this mutation arises in utero and is an initiating event. We characterized B cell development in first trimester human fetal liver (FL) to identify compartments vulnerable to ETV6-RUNX1. Using CD19 as a marker of B lineage commitment we found the first CD19+ B cells emerge in the human FL at Carnegie Stage (CS) 17 and were distinct from adult in that the majority expressed surface IL7 receptor. We used IL7R to identify a CD19-IL7R+ B progenitor compartment that produced B cells in vitro, possessed DJH recombination, but also had monocytic potential. Single cell analysis of CS20 IL7R+ progenitors revealed co-expression of lymphoid and myeloid programmes, whereas at CS17 they were strongly myeloid primed indicating that IL7R+ progenitors acquire lymphoid potential in this developmental window. Some co-expression of lymphoid and myeloid programmes also persisted in CS20 FL B cells. We tested whether FL B cell development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing pro and preB cells as well as an IL7R+ progenitor that switched from myeloid to B-myeloid priming during culture. At the global transcriptional level the hPSC lymphoid hierarchy mapped closely with FL, with both separating from adult suggesting that hPSCs provide a developmentally relevant model of early FL B lymphopoiesis. We next used CRIPSR-directed homologous recombination to engineer the expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B cell differentiation at the level of the IL7R+ progenitor. ETV6-RUNX1 expressing B cells co-expressed an abnormal B-myeloid gene expression signature akin to that seen in the IL7R+ progenitor. Both the transcriptional and differentiation phenotypes were dependent on ETV6-RUNX1 as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model where expression of ETV6-RUNX1 inhibits lymphoid specification in an early FL IL7R+ lymphomyeloid progenitor, arresting B lineage differentiation and resulting in the production of myeloid-primed B cells. This may explain the relatively high levels of myeloid antigen expression lineage promiscuity seen in cALL. ETV6-RUNX1 hPSCs will afford the systematic evaluation of the contribution of additional mutations seen in cALL and may offer a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lymphomyeloid progenitor in the human FL is a candidate target cell for in utero pre-leukemic initiation in cALL. Citation Format: Simon E. Richardson, Charlotta Boiers, Alya Zriwil, Virginia Turati, John Brown, Dapeng Wang, Javier Herrero, Stefan Karlsson, Andrew J. H. Smith, Sten Erik Jacobsen, Tariq Enver. ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2692.