1. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice
by Wanda Piacibello, Stefania Bruno, Fiorella Sanavio, Sara Droetto, Monica Gunetti, Laurie Ailles, Francesca Santoni de Sio, Andrea Viale, Loretta Gammaitoni, Angelo Lombardo, Luigi Naldini, and Massimo Aglietta
Blood
Volume 100(13):
December 15, 2002
©2002 by American Society of Hematology

2. Long-term expansion of CD34+ CB cells transduced with lentiviral vectors.Twenty thousand CB CD34+ cells were prestimulated for 96 hours with FL, TPO, SCF, and IL6; then 1 × 105CD34+ prestimulated cells in 100 μL were transduced overnight with 2 × 106 lentiv...
Long-term expansion of CD34+ CB cells transduced with lentiviral vectors.Twenty thousand CB CD34+ cells were prestimulated for 96 hours with FL, TPO, SCF, and IL6; then 1 × 105CD34+ prestimulated cells in 100 μL were transduced overnight with 2 × 106 lentiviral particles (MOI of 20) or with control medium. Cells were then washed and seeded in new 24-well plates and cultured in the presence of the same growth factors for up to 30 weeks. Aliquots of weekly demidepopulated wells were counted, analyzed for CD34/GFP expression (A), and plated in semisolid cultures for assessment of clonogenic progenitor output (B). Values represent the fold expansion, compared with input cells and colonies present in a single well (input CD34+/GFP+cells are those evaluated by FACS on the third day of liquid culture after transduction procedure; input GFP+ CFCs are the fluorescent colonies present in clonal cultures prepared soon after transduction). Representative experiment performed in triplicate wells.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology

3. Multilineage repopulation capacity of transduced and expanded SRCs in primary and secondary mice.(A) Isotype controls.
Multilineage repopulation capacity of transduced and expanded SRCs in primary and secondary mice.(A) Isotype controls. (B) FACS analysis of BM marrow cells from a primary mouse that had received a transplant 8 weeks previously of 2 × 105 transduced CB CD34+ cells expanded for 4 weeks with FL, TPO, SCF, and IL6. BM cells were analyzed as described in “Materials and methods.” Analysis of lineage markers (CD45/CD19, CD45/CD41, CD45/CD34, CD45/CD14) was performed on cells comprised within the human CD45 gate; analysis of GpA+cells was performed on total BM cells. The numbers in the top right quadrants show the percentages of GFP+ cells within the CD45+ population or within CD34+, CD19+, CD14+, CD41+, and GpA+ cells. (C) Representative FACS profiles of marrow cells from an individual NOD/SCID mouse that had received a transplant 8 weeks previously of unfractionated BM cells of a primary mouse injected with transduced and expanded CB CD34+ cells. GFP positivity and multilineage engraftment were evaluated as described for panel A.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology

4. Clonal analysis of the provirus in lentiviral-transduced bone marrow cells of primary NOD/SCID recipient mice.(Panel A) Southern blot on EcoRI-digested genomic DNA from the BM of mouse A (corresponding to the primary mouse in Figure 4) and mouse B (Table 3,...
Clonal analysis of the provirus in lentiviral-transduced bone marrow cells of primary NOD/SCID recipient mice.(Panel A) Southern blot on EcoRI-digested genomic DNA from the BM of mouse A (corresponding to the primary mouse in Figure 4) and mouse B (Table 3, A4), which 8 weeks earlier had received a transplant of 2 × 105 infected CB CD34+cells expanded for 4 weeks. The probed bands are the EcoRI fragments released from an internal region to vector LTRs; thus they represent the total transduced cell population. (Panel B) Southern blot onBamHI-digested genomic DNA from the BM of mice A and B. The probed bands are the BamHI fragments released from the vector LTR and the flanking gDNA; thus each of them should represent a single integrant population, as shown in the positive control. In samples A and B, distinct bands are not detectable. (Panel C) Genomic DNA from mice A and B was assessed by inverse PCR for the presence of clonal vector insertions. At least 4 bands were seen in mouse A and 2 in mouse B, each indicating a single integrant. + indicates GFP-transduced HeLa cells were cultured in limiting dilution and oligoclonal derived DNA was used as a positive control; − indicates nontransduced genomic DNA was used as a negative control.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Serial transplantations in NOD/SCID mice
Serial transplantations in NOD/SCID mice.(Panel A) FACS profile of marrow cells from a representative NOD/SCID mouse that 8 weeks earlier had received a transplant of 2 × 105 infected CB CD34+ cells that had been expanded for 4 weeks.
Serial transplantations in NOD/SCID mice.(Panel A) FACS profile of marrow cells from a representative NOD/SCID mouse that 8 weeks earlier had received a transplant of 2 × 105 infected CB CD34+ cells that had been expanded for 4 weeks. The BM of the primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse; the BM of this mouse was injected into a tertiary mouse. FACS analysis of human CD45 expression in the BM of primary, secondary, and tertiary mice was performed on total BM cells. The numbers in the top right quadrants show the percentages of GFP+ cells within the CD45+ population (panel B) FACS histograms representing human GFP+ cells on total BM population of the same primary, secondary, and tertiary recipients shown in panel A. (Panel C) GFP transgene amplification curves, obtained by real-time quantitative PCR analysis, of 100 ng DNA from the same primary, secondary, and tertiary mice shown in panels A and B (shown in Table 3 as mice A5, A5.1, and A5.1.1). A standard curve was obtained by using increasing amounts of vector plasmid as follows: ng (Ct: 26.1), ng (Ct: 22.9), 0.86 ng (Ct: 19.5), 8.6 ng (Ct: 16.4), and 86 ng (Ct: 14.2). To calculate the vector copy number per genome (human plus murine) we used HeLa cell clone (C3) containing one copy of GFP vector, as previously assessed by Southern blot analyses. The amplification curve obtained with a 10-fold dilution of C3 DNA, corresponding to 0.1 copy of vector per genome, is shown (Ct: 25.5). The analyses indicate that the BM of primary (Ct: 25), secondary (Ct: 27), and tertiary (Ct: 28.22) mice contained 0.10 (corresponding to 10% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A), 0.02 (corresponding to 2% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A), and (corresponding to 0.8% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A) copies of vector per genome (human plus murine), respectively. The corresponding Southern blot analysis for human engraftment is shown in Figure 5 (sample A). One of 2 analyses giving similar results is shown. NT indicates untreated mouse; Ct, the cycle number at which the fluorescence signal was more than 10 SD of the mean background noise collected from the 3rd to the 15th cycle.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Representative Southern blot analysis of 2 NOD/SCID mice that had received transplants of 2 × 105 infected CB CD34+ cells that had been expanded for 4 weeks.The BM from 2 primary mice (A and B) was injected into 2 different secondary sublethally irradiated ...
Representative Southern blot analysis of 2 NOD/SCID mice that had received transplants of 2 × 105 infected CB CD34+ cells that had been expanded for 4 weeks.The BM from 2 primary mice (A and B) was injected into 2 different secondary sublethally irradiated NOD/SCID mice (A and B); the BM cells of those mice were injected into 2 different tertiary recipients (A and B). DNA was extracted from the murine BM at week 8 after transplantation and hybridized with a human chromosome 17–specific α-satellite probe. Human-mouse controls are given as percentage of human DNA.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Quantitative real-time PCR analysis of BM from a tertiary NOD/SCID mouse .GFP transgene amplification curves, obtained by real-time quantitative PCR analysis of 100 ng of DNA from the BM of tertiary mice (Ct: 30.1).The analysis was performed as described fo...
Quantitative real-time PCR analysis of BM from a tertiary NOD/SCID mouse .GFP transgene amplification curves, obtained by real-time quantitative PCR analysis of 100 ng of DNA from the BM of tertiary mice (Ct: 30.1).The analysis was performed as described for Figure 4C and indicates that BM of this mouse contained copies of vector per total genome analyzed (human plus murine). The corresponding FACS analysis (0.43% GFP+ cells on total BM cells) is represented in Table 3 (mouse A2.1.1). The Southern blot analysis of this particular tertiary mouse is also represented in Figure 5B. One of 2 analyses giving similar results is shown. NT indicates untreated mouse.
Wanda Piacibello et al. Blood 2002;100:
©2002 by American Society of Hematology