Figure 1.

HOXB4 Does Not Promote Early Mesoderm Specification, In Vitro

(A) Scheme depicting the gammaretroviral, FMEV-based expression vectors (Lesinski et al., 2012, Schiedlmeier et al., 2007). The vectors co-express a fluorescent protein (eGFP, mPlum, or tdTomato) together with HOXB4 or a 4-hydroxytamoxifen (Tam) inducible form, HOXB4^ERT2. Co-translational separation of the proteins is mediated by the TAV-2A esterase. LTR, long terminal repeat; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

(B) Vector-transduced GFP-Bry ESCs (mPlum +/− HOXB4) were differentiated as embryoid bodies (EBs). At the indicated days, eGFP fluorescence as well as FLK-1 expression were determined by flow cytometry. The percentages of eGFP⁺FLK-1⁺ cells are shown. CCE ESCs were used as eGFP-negative controls (top row).

(C) After 6 days of differentiation, GFP-Bry EBs were dissociated and 10⁵ cells each co-cultured on OP9 stoma cells for further 8 days. Contour plots of flow cytometry analysis are shown, the percentages of CD41⁺ and CD45⁺ cells are indicated. For (B) and (C), representative results of n = 3 independent experiments are shown.