The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 vessels from which RUNX1C blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

IntroductionIn vertebrate species, a prerequisite for the development of the primary germ layers is the commitment of primitive ectoderm (epiblast) cells to gastrulation. [3][4][5][6] In mammalian embryos, this process is accompanied by the formation of the primitive streak, a morphologic structure initiating at the prospective embryonic posterior. [6][7][8] In the mouse epiblast, cells ingressing through the streak emerge as either definitive endoderm or mesoderm, the latter including the progenitors of the hematopoietic system. 9 In the mouse, primitive streak cells are marked by expression of the transcription factor Mixl1 1,2 and mouse embryos deficient in Mixl1 display multiple defects in the formation of mesodermal and endodermal derived structures. 10 Consistent with this, more recent studies have confirmed that Mixl1 expression marks precursors of both mesoderm 11 and endoderm. 12 These latter studies took advantage of embryonic stem cells (ESCs) or mice in which one Mixl1 allele had been replaced by sequences encoding green fluorescent protein (GFP), facilitating the identification and isolation of viable GFP ϩ (Mixl1 ϩ ) primitive streak-like cells. Analysis of Mixl1 GFP/w mouse ESCs showed that a GFP ϩ (Mixl1 ϩ ) population present at differentiation days 3 and 4 contained hematopoietic precursors, 11 supporting previous data indicating that, in mouse embryos, such precursors arise directly from the primitive streak. 13 The majority of progenitors at this time were hemangioblasts, precursors with both hematopoietic and endothelial potential which, in the embryo, contribute to the primary vascular plexus and primitive erythropoiesis of the yolk sac. Thus, these progenitors as well as lineage restricted primitive erythroid precursors represent the first differentiated mesodermal derivatives that arise after the onset of gastrulation at embryonic day (E) 6.5. 9Because of the scarcity of examples, events surrounding gastrulation in the human have largely been inferred from comparative embryology, 8 a situation that has led to uncertainty surrounding the relationship between the first mesodermal like cells (mesoblasts) documented from postovulation day 13 onward, the appearance of hematopoietic cells, and the overt manifestation of the primitive streak, a structure that is first visible in embryos representing embryonic day 15 (E15). 8,[14][15][16] Differentiating human embryonic stem cells (HESCs) represent an experimental platform for dissecting the relationship between specific lineages and the early differentiation events surrounding formation of the primary germ layers. To examine the correlation between mesoderm formation in the human and the emergence of hematopoietic precursors, we targeted sequences encoding GFP to the MIXL1 locus using homologous recombination. We demonstrate that GFP fluorescence faithfully reported expression of the endogenous MIXL1 gene and that a mesodermal cell population defined by coexpression of GFP (MIXL1) and the platelet-derived growth factor receptor alpha (PDGFR␣) wa...

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.