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haematologica | 2018; 103(9)
Results
NF-κB inhibition promotes human HSCs in culture
involved the downmodulation of genes involved in NF-κB signaling pathway. We found that pharmacological inhibi- tion of the NF-κB signaling leads to a significant improve- ment in HSC function from ex vivo cultured CB-derived CD34+ cells, as assessed by transplantation to NSG mice. The effect of NF-κB pathway inhibition was most critical early during the culture where it reduced the levels of sev- eral pro-inflammatory cytokines induced as an immediate response to culture initiation.
Methods
shRNA experiments
The RNAi screening strategy has been thoroughly described previously.9,10 The target sequence for the candidate shRNA sh758 is GATATGCAAGTCTGTGAATTT. CD34+ cells were trans- duced with a pLKO1-GFP lentiviral vector harboring either sh758 or control (scrambled) shRNA and subsequently cultured for sev- eral weeks according to previously described protocol.9,10
Cord blood CD34+ isolation and culture
Umbilical CB samples were collected from full-term deliveries at maternity wards of Lund, Malmö and Helsingborg Hospitals. CB unit collection, mononuclear cell isolation, and CD34+ cell enrichment and culture were carried out as previously described.10 IKKβ inhibitors, PF184 and TPCA1 (Tocris Bioscience), kept in DMSO, were added at a final concentration of 400nM. Control wells were supplemented with DMSO at a matching concentra- tion. Cultures were kept at 37°C and 5% CO2 and the medium (including inhibitors) was refreshed after four days.
Flow cytometry and cell sorting
For cell surface marker staining, cells were collected, washed once with PBS supplemented with 2% FCS (FACS buffer). Cells were incubated with anti CD34 (#343516581), CD90 (#3281145E10) and EPCR (#351906) (BioLegend) antibodies for 30 minutes (min) at 4°C, and washed once with cold FACS buffer. For cell sorting, CD34+ cells were quickly thawed and stained for CD34, CD38 (#345806), CD45RA (#560362) (BD Bioscience) and CD90 following the same procedure as above. When specified, cells were stained with the Annexin V Apoptosis Detection Kit, according to the manufacturer’s protocol (BD Bioscience). All data were collected on FACS Canto II or LSRII analyzer (Becton Dickinson), and analyzed with FlowJo software. Cells were sort- ed on a FACS Aria II or III (Becton Dickinson).
Human engraftment assay
All experiments with mice were reviewed and conducted under approved protocol from the Lund/Malmö Local Ethical Committee. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (NSG; Jackson Laboratory) were sublethally irradiated (300 cGy) before trans- plantation. Fresh cells or the cultured equivalent of 30,000 input CD34+ cells were injected intravenously into 10-12-week old NSG mice. Human cell contribution in peripheral blood (PB) and bone marrow (BM) of NSG was assessed 16 weeks post transplanta- tion.
Cytokine secretion and Bioplex assay
Supernatants were collected from duplicate samples after six hours treatment of CB CD34+ cells with TPCA1 or STF. The secreted cytokines in the supernatants were measured by using human 27-plex panel (M500KCAF0Y, Bio-Rad) in the Bio-Rad Luminex instrument. Samples were prepared and analyzed as per the manufacturer’s protocol.
Statistical analysis
Statistical significance was calculated using a two-tailed Student t-test with GraphPad Prism software unless otherwise stated. Statistical significance in the figures are indicated: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars indicate Standard Error of the Mean (SEM) unless otherwise stated and n represents the number of independent experiments.
Methods describing microarray analysis, CFC assays, western blot and cell cycle analysis can be found in the Online Supplementary Appendix.
From RNAi-based screens conducted in our laboratory aimed at identifying novel modifiers of HSPC expan- sion,10,11 we have identified several off-target hits: shRNAs that display profound effects on HSPC expansion but do not affect the expression of their predicted target. One such shRNA, TRC00000758 (sh758), predicted to specifi- cally knock-down (CLK1), induced an especially strong proliferative advantage of CB CD34+ cells, as the trans- duced, GFP+ population eventually dominated the culture (Figure 1A). Moreover, when cultured for seven days, CD34+CD38-CD90+CD45RA- cells expressing sh758 main- tained a substantially higher percentage of immature CD34hiCD90+ cells, which is known to contain the engraftable HSCs (Figure 1B). Given the dramatic increase of CD34hiCD90+ cells induced by sh758, we reasoned that the molecular signature of cells expressing sh758 could provide valuable clues about the genes and pathways that modulate HSC expansion ex vivo. We performed global transcriptional profiling of CD34hiCD90+ cells expressing either sh758 or a control shRNA (shCTL) after seven days of culture. The gene expression profiles revealed a down- modulation of cellular stress-related genes and pathways, such as MAPK14 and genes involved in NF-κB signaling, in sh758 transduced cells (Figure 1C). Of note, the expression levels of CLK1, as well as other predicted targets of sh758 (based on sequence homology), were not repressed by the shRNA. We have previously reported on the role of MAPK14 (p38) in HSPC expansion10 and therefore decided to further investigate the role of NF-κB signaling in this context. We first addressed whether the NF-κB pathway is activated upon ex vivo culture of CB CD34+ cells and found that the key regulator of NF-κB signaling, IkBa,12 was read- ily detected and also showed an increased expression dur- ing culture (Figure 1D). We further measured the phospho- rylated form of the IκBa protein (p-IκBa), as an indicator of NF-κB activity, at different time points during culture of CB CD34+ cells. We observed that the p-IκBa signal was detected throughout the culture and with particularly high levels during the first 24 hours (Figure 1D). Collectively, this suggests that the NF-κB pathway is activated in cul- tured HSPCs and may negatively influence their expan- sion.
The NF-κB pathway modulates ex vivo cultured human HSPCs
NF-κB pathway inhibition mediated by targeting IKKβ increases expansion of CD34+CD90+ cells ex vivo
To further explore the role of NF-κB signaling in cul- tured HSPCs, we next blocked the pathway pharmacolog- ically. As the down-regulated genes detected in the gene expression profiling (Figure 1C) are acting through canon-
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