Submitted June 4, 2007
Accepted November 3, 2007
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
Richard P Davis, Elizabeth S Ng, Magdaline Costa, Anna K Mossman, Koula Sourris, Andrew G Elefanty, and Edouard G Stanley*
Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia
* Corresponding author; email: ed.stanley{at}med.monash.edu.au.
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 GFP into the locus of MIXL1, a gene transiently expressed in the primitive streak during embryogenesis1,2. GFP fluorescence in MIXL1GFP/w HESCs differentiated in the presence of BMP4 faithfully 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 PDGFR
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 MIXL1GFP/w HESCs for analysing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to hematopoiesis.
