Skip to main content
. 2012 Jun 14;7(6):e39091. doi: 10.1371/journal.pone.0039091

Figure 2. Specific expression of WAS-promoter driven LVs in hESCs-derived hematopoietic cells.

Figure 2

(A) Representation of the SIN LVs used to transduce hESCs. WE vector [31] contains a 500-bp fragment of the human WAS proximal promoter driving the expression of the reporter gene eGFP. The AWE vector [33] contains an aditional 387-bp fragment of the WAS alternative promoter. The CE and pLVTHM are control lentiviral vectors expressing constitutively eGFP through the CMV and the EF1α promoters, respectively. (B) Constitutive LVs (CE and pLVTHM) express eGFP in hESC undifferentiated cells (AND-1) while the hematopoietic-specific AWE and WE LVs are silent. AND-1 cells were transduced with the LVs in order to obtain 0.5–3 vg/c (see materials and methods). (C) Hematopoietic differentiation induces eGFP expression in AWE and WE-transduced hESCs. AWE-, WE- and pLVTHM- transduced H9 cells were prompt to differentiate and 22 days later analyzed for eGFP expression (Middle plots). eGFP+ (right) and eGFP− (left) populations were analyzed for expression of mature hematopoietic markers CD45 and CD33. Note that WE and AWE-transduced cells (top and middle plots) mark specifically CD45+CD33+ cells while pLVTHM (bottom plots) express eGFP equally in hematopoietic and non-hematopoietic cells. (D) Graph showing the percentage of CD45+ cells within the eGFP+ population from AWE-, WE- and pLVTHM-transduced hESCs at day 22. AWE and WE- transduced cells did not show statistics differences. pLVTHM-transduced hESCs were used as controls. Data are average of at least three independent experiments +/− SEM *** P = 0.0002; ** P = 0.0023 (E) Neuronal and endothelial differentiation of AWE-transduced hESCs does not induce eGFP expression. AWE-transduced H9 cells (AWE) were differentiated into neural progenitors (Top plots) and endothelium (middle plots) and analyzed for eGFP, A2B5 (an early neuroectodermal marker) and VE-Cadherine (an endothelial marker) expression. The AWE-transduced H9 cells were also used for EB-mediated hematopoietic differentiation and analyzed after 22 days for eGFP and CD45 (Right plot). Untransduced (NT) H9 were used as a negative control for eGFP expression.