Figure 1.
Transduction efficiency in hESCs SA121.
A. Schematic representation of pTRIP lentiviral vectors, in which eGFP are under the control of ACTB-, CMV-, EF1α-, PGK- or UbC promoters. The hESC line SA121 was transduced with lentiviral vectors; pTRIP-ACTB-, CMV-, EF1α-, PGK-or UbC-eGFP. Ten days after transduction, cell populations were analyzed by FACS to determine the percentage of cells that expressed GFP. B. Percentage of transduced hESCs expressing eGFP. C. Determination of number of transgenic inserts in the GFP positive cells by qPCR at time of FACS isolation (day 0) and after 50 days of culture. Data in A and B are shown as mean of three independent experiments. Error bars represent standard deviation of the mean (±s.d.).
Figure 2.
Constitutive promoter activity in long term culture of undifferentiated hESCs.
The hESC line SA121 was transduced with lentiviral vectors containg the pTRIP-ACTB-, CMV-, EF1α-, PGK-or UbC-eGFP lentiviral vectors. 10 days after transduction, eGFP+ and eGFP− cells were separated by FACS sorting, referred to as day 0. Isolated eGFP+ cells were thereafter cultured for 50 days under self-renewing conditions and promoter activities were measured by FACS analysis at day 0, 15, 30, and 50. A. Promoter activities as percentage of eGFP+ cells at day 0, 15, 30 and 50. B. Intensity of fluorescent signal of eGFP expression from the same eGFP positive cells that were FACS analysed day 0 and 50. Intensity was measured by FACS analysis and EF1α promoter showed a significant decrease in intensity of eGFP expression from day 0 to day 50 (*p<0,014 students t'test). Decrease of ACTB-, CMV-, PGK- and UbC-eGFP intensity from day 0 to day 50 is not significant. A–C. Data are shown as mean of three independent experiments. Error bars represent standard deviation of the mean (± s.d.). C–L. Immunofluorescence stainings of ACTB-eGFP+ cells 30 days after FACS sorting. eGFP expressing cells show uniform expression of pluripotency markers; hES-Cellect (C), inset in C shows low and high intensity eGFP expressing cells, Nanog (D–F) and Oct3/4 (G–I). L. Merged image of colony morphology of human ES cells cultured on Matrigel (K) and eGFP expression within the colony (J). Scale bar in C represent 100 µM, inset and D–L 200 µM. Cells representative of high eGFP expressing is indicated by arrowhead and low eGFP expression by arrow.
Table 1.
% eGFP+ cells of hESC line SA121 transduced with pTRIP-ACTB-, CMV-, EF1α-, PGK or UbC-eGFP lentiviral vectors.
Figure 3.
Promoter activity during differentiation of hESCs.
A. Gene expression analysis of undifferentiated ACTB-, CMV-, EF1α-, PGK- or UbC-eGFP transduced hESCs and after EB differentiation, plotted as relative to reference gene GAPDH. SOX17, ALBUMIN, PAX6, NESTIN, PPARγ and, CD31 were used as marker genes for endodermal, ectodermal and mesodermal cell lineages and OCT3/4 as pluripotency marker. Results in A–C are plotted as mean of three independent experiments and error bars indicate ± s.d, B. hESC line SA121 was differentiated as embryonic bodies for 22 days and promoter activities in ACTB-, CMV-, EF1α-, PGK- or UbC-eGFP transduced cells were measured by FACS analysis as % eGFP+ cells. In parallel, % eGFP+ cells were measured on ACTB-, CMV-, EF1α-, PGK- or UbC-eGFP transduced cells that were maintained in their undifferentiated state for 22 days. Statistical analysis of EB day 22 as compared to undifferentiated day 22 (**p≤0.0039 students t'test). C. Average level of intensity of eGFP fluorescent signal of the eGFP+ population, detected by FACS analysis at start of differentiation, day 0 and after 22 days, measured as average mean fluorescence intensity. Statistical analysis of EB d22 as compared to undifferentiated cells day 0 (**p<0.001,***p<0.0001 students t'test).
Figure 4.
Activity of promoters in cell types representing all three embryonic germ layers.
hESCs were spontaneously differentiated for 22 days and thereafter separated by FACS sorting into the eGFP+ and eGFP− cell populations. Relative gene expression was performed by qPCR on the eGFP+ and eGFP− populations. A–F. Expression analysis of genes representative for differentiation to the three embryonic germ layers; endoderm, mesoderm and ectoderm. SOX17(A) and ALBUMIN(B) originates from endoderm. Neural progenitors PAX6 (C) and NESTIN (D) originates from ectoderm. E–F. Mesodermal cells; PPARγ (E) and CD31 (F). Expression levels for each gene in eGFP+ and eGFP− populations are plotted as relative to expression levels in undifferentiated hESCs. Results are plotted as mean of three independent experiments and error bars indicate ± s.d. Statistical analysis of GFP+cells compared to GFP− cells determined by students t'test (p*≤0,0392).