Figure 1.
Characterisation of Human Fetal Liver HSC.
A & B) Human fetal liver haematopoietic stem cell (hflHSC) populations contain a higher proportion of CD34+ cells than that found in umbilical cord blood (UBC) (1.8×107 versus 8×105; p = 0.0002 and 8.1% versus 0.4%; p = 0.0001). (C) Representative images of multilineage differentiation of hflHSC in methylcellulose culture assays.
Figure 2.
More efficient transduction of fetal liver-derived than cord blood-derived HSC.
(A) Human fetal liver HSC are transduced more efficiently with the A2UCOE-eGFP LV than UCB-HSC, with higher efficiencies achieved with increasing gestational age from 13 through 22 weeks. (B) Higher levels of transduction of hflHSC were achieved with the A2UCOE-eGFP LV than with the PGK-eGFP and EF1α-eGFP vectors at differing multiplicities of infection (MOI).
Figure 3.
Sustained expression of A2UCOE-eGFP in in vitro.
(A) hflHSC transduced with A2UCOE-eGFP, PGK-eGFP and EF1α-GFP LVs were maintained in culture for 24 days and analyzed at 3, 10, 17 and 24 days post-transduction. Expression from A2UCOE-eGFP was sustained over the 24 day period of culture. A rapid decline in PGK-eGFP (58% to 22%) and EF1α-eGFP (71% to 23%) expression was seen by Day 10. Average vector copy number per cell (VCN) at Day 17 of culture is given in italic text above the histogram lines. (B) A2UCOE-eGFP LV transduced cells showing maintenance of multilineage differentiation capability in methylcellulose culture assays at 17 days post-transduction. (C) Quantification of proportion of eGFP expressing colonies following differentiation of LV transduced hflHSC in semi-solid methylcellulose culture assays. Note comparable levels of eGFP-positive colonies with all three (A2UCOE, EF1α, PGK) LV types.
Figure 4.
A2UCOE confers sustained transgene expression in vivo.
(A) hflHSCs were transduced with either A2UCOE-eGFP, EF1α-eGFP or PGK-eGFP LVs and transplanted into sub-lethally irradiated NSG mice. A2UCOE-eGFP transduced hflHSC maintained transgene expression levels over 3 and 10 months post-transplantation whereas EF1α-eGFP and PGK-eGFP transduced cells lost eGFP expression rapidly. (B) Multilineage engraftment at 10-months post-transplantation from LV transduced hflHSC with higher percentage of eGFP-positive cells in A2UCOE-eGFP compared to EF1α-eGFP and PGK-eGFP. (C) Immunoflourescent staining of of bone marrow cells at 10 months post-transplantation showing sustained eGFP expression for A2UCOE-eGFP in comparison to PGK-eGFP and EF1α-eGFP.