Fig. 1.

General outline of this protocol. ES cells were differentiated into EBs in the absence of murine leukemia inhibitory factor. CD34⁺ cell populations were isolated from whole EB cell suspension by magnetic associated cell sorting magnetic bead cell separation. Cells were then transfected with Pu.1 siRNA and maintained with mSCF in culture, or secondary differentiation was initiated in myeloid differentiation conditions; i.e., mGM-CSF and mIL-3 were added in the medium. Pro-B cell formation was examined in the CD19⁺CD43⁺CD45R⁺ phenotype. To test whether the B progenitors were functional, cells were matured and examined for CD21 expression, and further examined for their LPS stimulation response, such as CD80 expression and IgM secretion.