title: Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors.
abstract: Differentiating human embryonic stem cells (HESCs) represent an experimental platform for establishing the relationships between the earliest lineages that emerge during human development. Here we report the targeted insertion in HESCs of sequences encoding green fluorescent protein (GFP) into the locus of MIXL1, a gene transiently expressed in the primitive streak during embryogenesis.(1,2) GFP fluorescence in MIXL1(GFP/)(w) HESCs differentiated in the presence of BMP4 reported the expression of MIXL1, permitting the identification of viable human primitive streak-like cells. The use of GFP as a reporter for MIXL1 combined with cell surface staining for platelet-derived growth factor receptor alpha (PDGFRalpha) enabled the isolation of a cell population that was highly enriched in primitive hematopoietic precursors, the earliest derivatives of the primitive streak. These experiments demonstrate the utility of MIXL1(GFP/w) HESCs for analyzing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to hematopoiesis.
authors.affiliation: Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Davis
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Ng
authors.forename: Magdaline
authors.initials: M
authors.lastname: Costa
authors.forename: Anna K
authors.initials: AK
authors.lastname: Mossman
authors.forename: Koula
authors.initials: K
authors.lastname: Sourris
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Elefanty
authors.forename: Edouard G
authors.initials: EG
authors.lastname: Stanley
mesh_terms: D000818:Animals; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D023421:Models, Animal
chemical_list: D018398:Homeodomain Proteins; C499230:MIXL1 protein, human; D049452:Green Fluorescent Proteins