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M.S. Talkhoncheh et al.
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ical NF-κB signaling, we decided to target IKKβ which is a central molecule in integrating upstream signals in the canonical pathway.13 Thus, we treated CD34+ cells with two specific inhibitors of the IKKβ subunit: PF184 and TPCA1.14,15 IκBa is the canonical target of IKKβ and both inhibitors induced a strong reduction of the phosphorylat- ed-to-total IκBa ratio after four hours treatment (Figure 2A). After seven days of culture, we observed a marked increase in both the frequency and number of CD34+CD90+ cells (Figure 2B).
However, the total cell number remained unaffected (Figure 2C), suggesting that the increase in CD34+CD90+ cells number is not due to a generally higher proliferation rate of cells treated with NF-κB inhibitors. Since NF-κB has well described effects on apoptosis16 and prolifera- tion,17 we assayed cell cycle and apoptosis parameters in CD34+ cells treated with PF184 and TPCA1 in an attempt to understand the basis for the increased numbers of CD34+CD90+ cells. However, we could not detect any sig- nificant difference in cell cycle status (Figure 2D) or apop- totic cell levels (Figure 2E) of CD34+90+ cells.
NF-κB pathway inhibition enhances the ex vivo propagation of transplantable HSCs
To assess in more detail the functional consequences of growing HSPCs in the presence of NF-κB inhibition, we cultured CD34+ cells for seven days with or without IKKβ inhibitors, and then assayed the cells for colony forming ability in methylcellulose medium. Cells cultured in the presence of inhibitors produced a significantly higher number of colonies compared to the control, and also gen-
erated a higher proportion of primitive colonies with a mixed phenotype (Figure 3A), indicating improved func- tional integrity of the cultured progenitor cells. Next, to test the in vivo regenerative capacity of HSCs cultured with IKKβ inhibitors, we performed transplantation experi- ments. Fresh CB CD34+ (30,000) cells or 7-day cultured equivalents of 30,000 CB CD34+ were transplanted into sub-lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (Figure 3B). Cells treated with PF184 and TPCA1 yielded 53% and 64% human engraftment, respectively, while control cells generated lower (38%) human engraftment in bone marrow 16 weeks post trans- plantation (Figure 3C). The engrafted cells contributed to both myeloid and lymphoid lineages (Figure 3D and E). Finally, the frequency of the HSC enriched CD34+CD38- population among the engrafted human cells was similar to that of control cells (Online Supplementary Figure S1), demonstrating an overall higher long-term engraftment of also the most immature cells from cultures with PF184 and TPCA1. Although IKKβ inhibitor treated cells showed slightly lower long-term engraftment compared to non- cultured cells, our data demonstrate a strong benefit of NF-κB inhibition in preserving functional HSPCs during ex vivo expansion cultures.
NF-κB inhibition is most beneficial during an early time window where it down-regulates several pro-inflammatory cytokines
We had previously observed a peak in p-IκBa levels dur- ing the first 24 hours of culture (Figure 1D), and therefore next assessed if there is a critical time window for addi-
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haematologica | 2018; 103(9)
Figure 1. The sh758-depen- dent ex vivo expansion effect is associated with a downmodulation of the NF- κB pathway. (A) Growth of GFP+ cells was assessed by FACS and normalized to day 2-transduction efficiency. (B) The percentage of CD34+90+ cells in the GFP+ fraction was quantified by FACS seven days after transduction of CD34+CD38-CD90+CD45RA- cells (n=2). (C) Gene expres- sion data mining showed modulation of members of NF-κB signaling in sh758 expressing CD34+CD90+ cells. (D) The expression of total and phosphorylated ΙκΒa was quantified by west- ern blot in CD34+ cells at
indicated culture
points. Actin expression is shown as loading control.
time
B