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NF-κB inhibition promotes human HSCs in culture
Figure 2. Targeting of the NF-κB regulator IKKβ increases the number of human hematopoietic stem and progenitor cells (HSPCs) in vitro without affecting cell proliferation and survival. (A) The protein level of total and phosphorylated IκBa were quantified in CD34+ cells after 4-hour treatment with STF/PF/TP in culture. Actin expression is shown as loading control (n=2). (B) FACS plots and graph show cell surface expression of CD34 and CD90 and number of CD34+CD90+ cells respectively at day 7 (n=4). (C) Graph displays total live (7AAD-) cell number at day 7 (n=4). (D) Cell cycle distribution, and (E) apoptosis (7AAD-/ AnnexinV+) of CD34+CD90+ cells were assessed by FACS at days 4 and 7, respectively (n=3). p-IκBa: phosphorylated IκBa; t-IκBa: total IκBa; PF: PF184; TP: TPCA1.
role in other processes such as cell growth, survival and development.13 To further identify which NF-κB associat- ed functions were targeted in our cultured HSPCs, we sub- jected bulk CD34+ and sorted CD34+CD90+ cells to global gene expression profiling using microarrays (Figure 4D and E, Online Supplementary Table S1 and Online Supplementary Figure S3). We used 6-hour culture time for the transcriptome analysis as this time point showed a clear increase in NF-κB activity (Figure 1D). Although rel- atively few transcriptional changes were detected within the more heterogeneous bulk of CD34+ cells (Online Supplementary Figure S3), 51 annotated genes were modu- lated in the CD34+CD90+ population upon exposure to the IKKβ inhibitor (Figure 4D and E, and Online Supplementary Table S1). Out of these, all coding genes (23 genes) were down-regulated and mainly related to inflammatory processes (15 genes), for example, IL1a, TNF-a, GM-CSF (CSF2), MCP-1 (CCL2), TCA3 (CCL1), and MIP-1β (CCL4) (Online Supplementary Table S1). Using Bioplex bead arrays, we confirmed the reduction of several pro- inflammatory factors, including TNF-a, IL-6, IP-10 (CXCL10) and IL-8 in supernatants collected from cultures of CB CD34+ cell after a 6-hour exposure to the IKKβ inhibitor (Figure 4F and G). Taken together, these findings indicate that the beneficial effect of IKKβ inhibitors on cul- tured human HSPCs is associated with the downregula- tion of NF-κB-dependent pro-inflammatory molecules.
Discussion
In this study, we demonstrate that the NF-κB signaling has a negative impact on ex vivo cultured human CB HSPCs, and that pharmacological inhibition of the path- way improves their propagation and regenerative poten-
tion of IKKβ inhibitors. We found that delaying IKKβ inhi- bition for 24 hours abolished the beneficial effects and resulted in lower numbers of CD34+CD90+ cells after five days compared to cultures where the inhibitor was added directly. Similarly, treating cells for only the first 24 hours with IKKβ inhibitor resulted in similarly elevated numbers of CD34+CD90+ cells compared to 5-day treatment (Figure 4A). Collectively, these findings suggest that NF-κB activa- tion is most detrimental during the first 24 hours of culture and define a time window when NF-κB inhibition is both necessary and sufficient to execute protective effects on HSPCs.
In order to further characterize the potential of NF-κB inhibition in improving expansion protocols, we com- pared TPCA1 to two other known expansion molecules for human HSCs: SR1 and UM171.18,19 We evaluated the output of CD34+90+ cells as well as CD34+CD90+EPCR+ cells, as EPCR expression recently has been associated with the regenerative activity of ex vivo cultured HSPCs in the context of UM171 mediated expansion.20 All three compounds showed a clear increase in both CD34+CD90+ and CD34+CD90+EPCR+ cell numbers compared to control cultures (Figure 4B and C, and Online Supplementary Figure S2). However, TPCA1 treatment resulted in larger num- bers of these cell populations compared to SR1, while UM171 showed a significant increase compared to both TPCA1 and SR1 (Figure 4B and C). Interestingly, the com- bination of the IKKβ inhibitor with either SR1 and UM171 led to a marked increase in CD34+90+ and CD34+CD90+EPCR+ cell numbers, compared to each com- pound alone (Figure 4B and C), indicating that targeting NF-κB signaling may enhance HSPC expansion protocols driven by other compounds.
NF-κB proteins are considered key players in inflamma- tion and immunity. However, they also play an important
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