Mixl1 and oct4 proteins are transiently co-expressed in differentiating mouse and human embryonic stem cells.

Embryonic stem cells (ESCs) have the capacity to form all the tissues in the body and hence, directed differentiation of ESCs along specific lineages represents a means to generate therapeutically useful cell types. It has been postulated that, during in vitro differentiation, ES cells sequentially pass through similar developmental stages as cells in the embryo. The availability of reagents that identify these stages would facilitate the monitoring and optimization of ESC differentiation. One key stage, the development of endodermal and mesodermal precursors in the early embryo, is marked by the transient expression of the transcription factor, Mixl1 and the stem cell gene, Oct4. In order to identify corresponding cells during ESC differentiation, we generated monoclonal antibodies to the Mixl1 protein that robustly detected both mouse and human proteins. Intracellular flow cytometry was used to show that approximately 90% of differentiating mouse ESCs transiently co-expressed Oct4 and Mixl1 proteins and that a subset of differentiating human ES cells also coexpressed MIXL1 and OCT4 proteins. These experiments have demonstrated for the first time by protein expression that both human and mouse ESCs passed through developmental stages during in vitro differentiation that corresponded to those observed in early mammalian development. Furthermore, these studies confirmed that anti-Mixl1 antibodies are a valuable reagent for monitoring ESC differentiation and will facilitate the efficient generation of clinically relevant cell types.

Pdgfrα and Flk1 are direct target genes of Mixl1 in differentiating embryonic stem cells.

The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.

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.

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.