Figure 1.
Decitabine reduced GFP-positive rates (%GFP) in transduced human CD34+ cells.
(a). Human CD34+ cells were cultured in serum-free media containing cytokines (SCF, FLT3L, and TPO). After 24 hours of prestimulation, the cells were transduced with GFP-expressing HIV-1 based lentiviral vectors for 24 hours. Decitabine (0.5 µM) was added into media during either prestimulation (24 hours) or transduction (24 hours). After transduction, media were changed to fresh media containing cytokines and decitabine, and 3 days later, both CD34 expression and GFP expression were evaluated by flow cytometry. In addition, we investigated supplement of IL3 into media during prestimulation and transduction. (b). The %GFP in CD34+ cells was decreased by decitabine exposure in both prestimulation and transduction. Decitabine treatment led to higher CD34-positive rates (%CD34) when IL3 was added. We tested statistical significance of data in both decitabine exposure groups, compared to no decitabine exposure. DAC: decitabine, SCF: stem cell factor, FLT3L: fms-like tyrosine kinase 3 ligand, TPO: thrombopoietin, IL3: interleukin-3, MFI: mean fluorescence intensity, and MOI: multiplicity of infection.
Figure 2.
Decitabine increased CD34 expression and reduced cell proliferation in transduced human CD34+ cells.
(a). To further investigate the decitabine effects on transduction efficiency for human CD34+ cells, we added increasing dose of decitabine (0.05, 0.1, 0.2, and 0.5 µM) into media during 24-hour prestimulation (SCF, FLT3L, TPO, and IL3). The GFP expression, CD34 expression, and cell counts were evaluated 4 days after transduction. (b). We observed dose-dependent effects of decitabine exposure, which resulted in higher %CD34, lower %GFP, and lower cell counts. (c). We evaluated cell counts and viability of human CD34+ cells at 24, 48, and 72 hours after decitabine exposure (0.5 µM) with cytokine stimulation (SCF, FLT3L, TPO, and IL3) without transduction. Decitabine exposure resulted in lower cell counts at 48 hours after exposure; however, equivalent cell counts were observed at 24 hours and 72 hours after exposure. Decitabine exposure did not decrease viability at any time points. We tested statistical significance of data in all decitabine exposure groups, compared to no decitabine exposure.
Figure 3.
48-hour decitabine exposure resulted in equivalent %GFP in transduced human CD34+ cells.
(a). To evaluate whether transduction efficiency is changed by interval of time between decitabine exposure and transduction, we compared %GFP between 24-hour and 48-hour prestimulation. Human CD34+ cells were cultured in serum-free media containing cytokines (SCF, FLT3L, and TPO) supplemented with decitabine (0.5 µM). At 24 or 48 hours after starting exposure with or without decitabine, the cells were transduced with GFP-expressing HIV-1 based lentiviral vectors without decitabine for 24 hours. In addition, we investigated supplement of IL3 into media during prestimulation and transduction. (b). After 48-hour prestimulation, decitabine exposure resulted in higher %CD34, equivalent %GFP, and lower coefficient of variation (CV) of GFP intensity, while %GFP decreased in 24-hour decitabine exposure. (c). In transduction of CD34+ cells obtained from six healthy donors, average data revealed similar trends of higher %CD34 and equivalent %GFP after 48-hour decitabine exposure (0.5 µM). In addition, the decitabine exposure reduced standard deviations (SD) of GFP intensity among transduced CD34+ cells from six individuals (right panel). We tested statistical significance of data in all decitabine exposure groups, compared to no decitabine exposure.
Figure 4.
Post-transduction decitabine exposure reduced variability of GFP intensity in transduced CD34+ cells.
(a). To evaluate whether decitabine exposure affects internal promoter activity in lentiviral vectors to drive GFP expression, human CD34+ cells were exposed to decitabine (0.5 µM) following lentiviral transduction (Post DAC) with 48-hour prestimulation (SCF, FLT3L, TPO, and IL3), and we analyzed GFP expression by flow cytometry and average vector copy number per cell by real time polymerase chain reaction (PCR). (b). Post-transduction decitabine exposure resulted in equivalent %GFP, GFP intensity, and vector copy number, while lower CV of GFP intensity was observed in post-transduction decitabine exposure, compared to no decitabine exposure. The similar decitabine effects were detected when CD34+ cells were exposed to decitabine before and after transduction. In addition, higher %CD34, lower cell counts, and equivalent viability were observed in both decitabine exposure groups. We tested statistical significance of data in all decitabine exposure groups, compared to no decitabine exposure.
Figure 5.
Decitabine reduced subclonal expansion of transduced CD34+ cells.
(a). We performed a limiting dilution of human CD34+ cells which were transduced with lentiviral vectors after 48-hour decitabine exposure (0.5 µM), and then cultured in 96-well plates for 3–4 weeks to assay the expansion ability of transduced cells. (b). We detected subclonal expansion of GFP-positive cells that had originated from the transduced CD34+ cells. (c). The expansion rates were reduced by decitabine exposure under cytokine prestimulation without IL3 (2.1% vs. 9.4%) and with IL3 (0.0% vs. 7.3%). FBS: fetal bovine serum, NA: not applicable.
Figure 6.
Decitabine promotes efficient erythroid differentiation from transduced CD34+ cells.
(a) To evaluate decitabine effects on erythroid differentiation and transduction efficiency, we transduced human CD34+ cells with a GFP-expressing HIV-1 vector following 48-hour prestimulation with 0.5 µM decitabine. After 24-hour transduction (without decitabine), the cells were differentiated into erythroid cells. (b) Both %GFP and GFP intensity in GPA-positive cells gradually decreased during the erythroid differentiation. The glycophorin A-positive rates (%GPA) were increased by decitabine exposure, while equivalent %GFP was observed. We analyzed data in decitabine exposure groups, compared to no decitabine exposure.
Figure 7.
Decitabine modestly increased human cell engraftment in humanized xenograft mice.
(a). To evaluate decitabine effects on long-term progenitor cells in vivo, we transduced human CD34+ cells after 48-hour prestimulation with and without decitabine (0.5 µM), and injected into sublethally-conditioned immunodeficient mice. (b). In 48-hour ex vivo decitabine exposure either with or without IL3, we observed equivalent %GFP and a tendency towards higher human cell engraftment at 20–24 weeks after transplantation, compared to no decitabine exposure (48-hour prestimulation without IL3).