Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

Rapid activation of hematopoietic stem cells.

Adult hematopoietic stem cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation. Surprisingly, little is known about the earliest stages of HSCs activation. We utilize surface markers of HSCs activation, CD69 and CD317, revealing a response as early as 2 h after stimulation. The dynamic expression of HSCs activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify dose response, revealing a low threshold, and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence. Our data show that the response of adult stem cells to immune stimulation is rapid and sensitive, rapidly leading HSCs out of quiescence.

Isolation of Murine Myeloid Progenitor Populations by CD34/CD150 Surface Markers.

Myeloid progenitors are intermediates between Hematopoietic Stem Cells (HSCs) and Myeloid effector progeny. In mouse bone marrow, they are part of the Lineage- cKit+ Sca1- (LK) compartment. To date, most researchers used CD34 and FcγR surface markers for the dissection of this compartment into various populations. Surprisingly, however, this approach does not provide distinct separation by fluorescence-activated cell sorting (FACS). In this study, we suggest using CD150 instead of FcγR. We re-analyzed published single-cell RNA-Seq data and found that CD34/CD150 provides better sub-populations separation, compared to the "classical" CD34/FcγR-based approach. We confirm our findings by independent FACS analysis. We demonstrate comparable differentiation potential of the newly-obtained LK sub-populations, like previous "classical" ones. Therefore, we suggest the CD34/CD150 gating strategy, utilizing commonly-used surface markers, as a robust and reproducible separation of the LK compartment into distinct sub-populations.

High throughput screen for the improvement of inducible promoters for tumor microenvironment cues.

Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.

Method for the Generation of Induced Hematopoietic Stem Cells.

Reprogramming proved the possibility to change cell identity by transient overexpression of defined transcription factors. However, the efficiencies of pioneer protocols are extremely low, and mechanistic understanding is still under intensive research. Hematopoietic stem cells (HSCs) are prototypic adult stem cells, leading scientific research and clinical applications. We had reported the possibility of direct reprogramming of blood cells into induced-HSCs. In this chapter, we detail the protocol and elaborate information on critical steps. From the identification of candidate factors, through cloning and lentiviral production, this protocol can help anyone interested in reprogramming toward the adult stem cell state. A detailed protocol should enable new ideas to realize and further open new frontiers for adult stem cell research.

Hypersensitivity response has negligible impact on Hematopoietic Stem Cells.

Immune cells are generated from hematopoietic stem cells (HSCs) in the bone marrow (BM). Immune stimulation can rapidly activate HSCs out of their quiescent state to accelerate the generation of immune cells. HSCs' activation follows various viral or bacterial stimuli, and we sought to investigate the hypersensitivity immune response. Surprisingly, the Ova-induced hypersensitivity peritonitis model finds no significant changes in BM HSCs. HSC markers cKIT, SCA1, CD48, CD150, and the Fgd5-mCherry reporter showed no significant difference from control. Functionally, hypersensitivity did not alter HSCs' potency, as assayed by transplantation. We further characterized the possible impact of hypersensitivity using RNA-sequencing of HSCs, finding minor changes at the transcriptome level. Moreover, hypersensitivity induced no significant change in the proliferative state of HSCs. Therefore, this study suggests that, in contrast to other immune stimuli, hypersensitivity has no impact on HSCs.

Cyclosporine H Improves the Multi-Vector Lentiviral Transduction of Murine Haematopoietic Progenitors and Stem Cells.

Haematopoietic stem cells (HSCs) have the potential for lifetime production of blood and immune cells. The introduction of transgenes into HSCs is important for basic research, as well as for multiple clinical applications, because HSC transplantation is an already established procedure. Recently, a major advancement has been reported in the use of cyclosporine H (CsH), which can significantly enhance the lentivirus (LV) transduction of human haematopoietic stem and progenitor cells (HSPCs). In this study, we employed CsH for LV transduction of murine HSCs and defined haematopoietic progenitors, confirming previous findings in more specific subsets of primitive haematopoietic cells. Our data confirm increased efficiencies, in agreement with the published data. We further experimented with the transduction with the simultaneous use of several vectors. The use of CsH yielded an even more robust increase in rates of multi-vector infection than the increase for a single-vector. CsH was reported to reduce the innate resistance mechanism against LV infection. We indeed found that additional pretreatment could increase the efficiency of transduction, in agreement with the originally reported results. Our data also suggest that CsH does not reduce the efficiency of transplantation into immune-competent hosts or the differentiation of HSCs while enhancing stable long-term expression in vivo. This new additive will surely help many studies in animal models and might be very useful for the development of novel HSC gene therapy approaches.

Attenuated DNA damage responses and increased apoptosis characterize human hematopoietic stem cells exposed to irradiation.

Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.