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NF-κB inhibition promotes human HSCs in culture
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Figure 4. The benefit of NF-κB pathway inhibition is limited to a short time window, and results in downregulation of inflammatory signaling. (A) Number of CD34+CD90+ cells was assessed after five days by FACS, when TP/STF was added directly or 24 hours after culture start or removed after 24 hours (n=3). (B) Graph shows number of CD34+CD90+ cells after treatment with STF, TP, SR1, UM171 and combination of TP with either SR1 or UM171 at day 6 (representative data from 2 experiments). (C) Graph shows number of CD34+CD90+EPCR+ cells after treatment with STF, TP, SR1, UM171 and combination of TP with either SR1 or UM171 at day 6 (representative data from 2 experiments). (D and E) CD34+ cells were treated with or without TP for six hours and subjected to transcriptome analysis by microarrays after sorting for CD34+90+ population. Volcano plot and GO classification of 2-fold modulated (down-regulated) genes are shown for each condition. See also Online Supplementary Figure S3 and Online Supplementary Table S1. (F) The log ratio (TP/STF) of factors present in supernatant after six hours of culture of CB-derived CD34+ cells is shown (n=3). (G) Each graph displays individual data point for significantly modulated cytokines (IL6, TNFa, IL8, and IP10), extracted from the experiments presented in (F). The stars in (F) indicate that those factors were not detectable. PF: PF184; TP: TPCA1.
to negatively influence self-renewal and lineage commit- ment of HSCs.32,33 Therefore, considering the lack of effect on cell proliferation, the increased HSC output after NF-κB pathway inhibition may rather be associated with pre- served stem cell integrity by restricting differentiation. It is likely that the most immature HSPC are particularly sen- sitive to the early culture shock as they showed the most profound transcriptional changes upon NF-κB pathway inhibition.
It could be argued that pro-inflammatory stress could be relieved by simply exchanging the media similar to the fed-batch system.8 However, during the critical first 24 hours, the cell density is very low and a media exchange system is not likely to have an impact during this short time frame. It is possible that restricting inhibitory signals in expansion cultures have a bi-phasic pattern; one pulse that is induced immediately upon culture initiation and one that accumulates over time and that is dependent on cell density and media changes. The latter can be relieved
not significantly altered during the early phase of the cul- ture (data not shown), when the effects of NF-κB pathway inhibition were seen to enhance HSPC activity. This sug- gests that the preserved stem cell output of cultured HSPCs upon NF-κB pathway inhibition is not related to ROS modulation.
Based on the IκBa phosphorylation pattern, we showed that there is a critical 6-24-hour time frame necessary and sufficient for NF-κB pathway inhibition to benefit the propagation of the CD34+CD90+ population. In agreement with this, previous studies also reported on an immediate variation in gene expression profile of inhibitory HSPC regulators as the cultured cells experience an initial culture shock.7,8 In fact, we showed downregulation of several pro-inflammatory genes such as MCP-1 (CCL2), TCA3 (CCL1), IL1a, TNF-a and MIP-1β (CCL4) upon NF-κB inhibition, which are known to inhibit HSPC function.31 Although these pro-inflammatory cytokines are essential for a proper defense mechanism,24 they have been shown
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