ALBERT

Description: During ageing, the haematopoietic stem cell (HSC) pool expands numerically, but declines functionally. This functional decline is characterised by myeloid skewing and decreased long-term reconstitution potential and clinically manifests as anaemia, immune compromise and increased risk of clonal malignancy. We postulate that HSCs do not synchronously functionally decline with age, but instead represent a spectrum in which physiologically aged HSCs become dominant. In this study, we aimed to reveal properties that might identify physiologically young HSCs during chronological ageing and to exploit these in an attempt to rescue haematopoietic ageing. Young adult ((y), 2-3 months) and aged (old (o), 〉18 months) mouse HSCs were profiled and we observed a significant decrease (30-50%) in mitochondrial membrane potential (MMP) in oHSCs. Interestingly, a small fraction (15%) of oHSCs maintained a similar MMP to the bulk (70%) of yHSCs. We explored, initially at the transcriptional level, whether these MMPhigh HSCs are distinct from MMPlow HSCs. Strikingly, RNA sequencing of MMPhigh and MMPlow young and aged HSCs revealed that samples cluster by MMP over age. MMPhigh young and aged HSCs were characterised by upregulation of lymphoid and erythroid lineage markers, as well as RNA processing, MYC and E2F pathways. MMPlow young and aged HSCs were transcriptionally associated with ageing, inflammation and myeloid bias. Based on these results, we hypothesised that enhancing MMP in oHSCs might restore lineage-balanced peripheral blood (PB) output, used as a measure of functional improvement. To achieve this, we chemically enhanced MMP in oHSCs in vivo, using the mitochondrially-targeted drug mitoquinol (MQ). Interestingly, our initial experiments show that a 5-day treatment with MQ significantly shifted the B-cell/myeloid ratio in PB from 0.6 (aged) to 1.5 (MQ), in the direction of the ratio observed in young mice (2.9). This ratio change was not due to numerical depletion of myeloid cells, but due to restoration of B-cell numbers, including IgM+ B-cells commonly reduced with age. To assess whether the effect of MQ was due to HSC-intrinsic changes and could be sustained over time, HSCs were isolated from MQ-treated or untreated aged mice and transplanted into lethally irradiated recipients. Our experiments to date show that HSCs isolated from MQ-treated aged mice show superior engraftment and faster and greater B-cell reconstitution than HSCs from age-matched untreated animals, and that these improvements are stable over the 16-week assay. Based on our sequencing results indicating that RNA processing, MYC and E2F pathways are associated with MMPhigh HSCs, and previous work reporting a relationship between MMP and rate of mRNA transcription (das Neves et al., PLoS Biol. 2010; Johnston et al., PLoS Comput. Biol. 2012), we explored the possibility that MMP might orchestrate the observed changes by altering transcriptional rate of HSCs. We tested transcription rate in HSCs in vivo and found that yHSCs display a particularly high rate of mRNA transcription. Rate of transcription was significantly reduced in oHSCs compared to yHSCs, equivalent to their quantitative reduction in MMP. We could demonstrate a direct correlation between MMP and transcription rate in HSCs, by showing that MMPhigh sorted HSCs were transcribing nearly twice as fast as MMPlow sorted HSCs. Furthermore, in vivo injection with mitochondrial uncoupler CCCP caused a similar reduction in transcription rate of HSCs (〉50%) as did conventional RNA Pol-II inhibitors (DRB, Flavopiridol), hereby demonstrating that transcription rate directly depends on MMP. This work indicates that mitochondrial state can separate HSCs with distinct transcriptional profiles linked to different cell fates. We speculate that changes in transcriptional profile arise from MMP-driven regulation of transcriptional rate in HSCs. This would open up the possibility that pharmacological manipulation of mitochondrial activity can alter transcriptional programs of HSCs with consequences for functionality. Disclosures No relevant conflicts of interest to declare.

Description: We have used a combination of hematopoietic growth factors to induce in vitro granulocytic maturation. A fraction of marrow cells enriched for hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from normal human bone marrow by monoclonal antibody staining and fluorescence-activated cell sorting. Cells were cultured in a suspension system for 3 days in the presence of stem cell factor and interleukin-3 (IL-3), after which granulocyte colony-stimulating factor (G-CSF) was added. Cells were harvested daily and analyzed for phenotypic maturation by morphologic criteria, and total RNA was obtained for analysis of myeloid gene expression. Maturation was observed to progress to the late metamyelocyte and band stage over a period of 10 to 12 days. Neutrophil-specific gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Induction with G-CSF resulted in sequential expression of primary and secondary granule proteins, with asynchronous expression of primary granule proteins starting from days 1 to 5, and synchronous expression of lactoferrin and transcobalamin I (secondary granule proteins) from days 7 to 8. Interestingly, myeloperoxidase (MPO) mRNA expression was easily detected in both the freshly isolated CD34+, HLA-DR+ cells and cells at all subsequent stages of induction. This suggests that MPO mRNA is expressed very early during neutrophil development, perhaps before the development of significant numbers of phenotypically recognizable granules. This recapitulation of a program of sequential expression of primary and secondary granule protein genes suggests that in vitro marrow culture suspensions to which appropriate growth factors are added can mimic normal granulocytic maturation. This system should provide an important model for the study of neutrophil-specific gene expression.

Description: Hematopoietic stem cells (HSCs) are effectively expanded in fetal liver (FL), while they are maintained in a dormant state in adult bone marrow (BM). However, developmental mechanisms allowing this have not been fully explained. BM-HSCs have the lowest protein synthesis rate within the blood hierarchy, even under forced self-renewal divisions. In addition, HSCs are vulnerable to and quickly activate endoplasmic reticulum (ER) stress responses fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). Of note, we have seen that FL-HSCs have low levels of ER stress related genes despite their high proliferation status without an increase in heat shock protein levels, strongly indicating that other factor(s) block ER stress elevation. This raises the question how HSCs deal with the higher protein-folding requirement during expansion in the FL. Here we demonstrate that bile acids (BAs) are required to eliminate ER stress in the FL and are essential for proper expansion of FL-HSCs. Measurement of protein synthesis rate using OP-puro incorporation revealed that protein synthesis was enhanced in FL-HSCs, whereas BM-HSCs have half the rate of other populations in BM. Mass spectrometry analyses showed that BAs in the FL were all taurine conjugated while 30% of BA in the adult liver was taurine-conjugated, and the main proportion was taurocholic acid (TCA) that is known for its low toxicity. In the FL we also detected secondary BAs (e.g. TDCA), requiring intestinal bacteria in the production process, suggesting that FL BAs are a mixture of fetal and maternal BAs. Reduction of BA levels using GW4064, a chemical inhibitor of BA synthesis, significantly decreased the number of HSCs (6.6 fold decrease compared to vehicle treatment). This decrease was due to increased apoptosis caused by elevated ER stress levels. Similarly, dual deletion of Cyp27a1, a key BA synthetic enzyme, in both mother and fetus severely decreased total cellularity (2.0 fold decrease compared to littermate heterozygotes) and number of HSCs (6.8 fold decrease) in FL due to increased ER stress and subsequent apoptosis. Interestingly, FL of homozygotes grown in heterozygous mothers did not show any significant differences compared to littermate heterozygotes, suggesting that the contribution of maternal BA in FL is critical for HSCs. In both models, ER stress-oriented apoptosis and reduction in cellularity were most pronounced within the HSC population, indicating that stem cells are particularly sensitive to BA levels during development in FL. Importantly, injection of TCA or Salubrinal, an ER stress inhibitor, rescued the effects of BA reduction in both models. These data strongly suggest that BAs are required to block ER stress elevation in expanding FL-HSCs. ER stress and protein aggregation are closely linked together in number of pathological diseases like AlzheimerÕs- and HuntingtonÕs disease. Quantification of aggregated proteins (aggresomes) revealed that Cyp27a1 KO FL-HSCs from homozygote mothers contained significantly higher amount of aggresomes (2.0 fold), while KO FL-HSCs from heterozygote mothers showed no increase. Higher levels of aggregated proteins were most pronounced within the HSC population and BA suppressed formation of aggresomes during in vitro culture. This leads to reduction of ER stress and the maintenance of functional HSCs. Finally, transplantation assay showed that TCA can support functional HSCs ex vivo for up to 14 days. These findings propose a novel role for BA as a critical part of fetal hematopoiesis supporting expansion of HSC. Maternal and fetal BA coordinately contribute to this natural chaperone regulation. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells (HSCs) give rise to all lineages of hematopoietic cells in the body for entire life span and are thus protected from risk factors by multiple defense systems. We have recently discovered that HSCs are highly susceptible to stress caused by accumulation of mis-/un-folded proteins, so called endoplasmic reticulum (ER) stress upon enhanced growth conditions, and addition of a specific type of bile acid (BA), Tauroursodeoxycholic acid (TUDCA), known as a chemical chaperone can maintain functional murine HSCs for 2 weeks in vitro, by reducing ER stress (Miharada et al., Cell Rep. 2014). This work depicts the importance of proper protein quality control in HSC maintenance, particularly during the expansion. HSCs are kept in dormant state in the adult body, but actively expanding in the fetal liver. BAs are synthesized from cholesterol in the liver. Interestingly, bile acid synthesis is highly up-regulated in the fetal liver during embryogenesis and the composition of fetal BAs gradually reduces after birth. In addition, composition of bile acids in the fetus is different from adult liver, with the vast majority of fetal BAs are of Taurine-conjugated form that is more stable and non-toxic. Of note, hematopoietic cells and hepatocytes producing BAs are in close contact in the fetal liver and HSCs are therefore exposed to BAs, whereas the adult liver has anatomically isolated bile duct structures that separate blood flow and bile flow. However the role for these fetal BAs has been unknown. Here we report that bile acids support expansion of hematopoietic stem and progenitor cells (HSPCs) in the fetal liver and ex vivo. Since TUDCA is a rare component in human and mouse BAs, even in the fetal liver, we sought analogue(s) that similarly function as ER stress inhibitors. We identified that Taurocholic acid (TCA), one of the main components of fetal BA, and Tauro-alpha-muricholic acid (TαMCA) that is a rodent specific BA have a potential to reduce ER stress, similar to TUDCA. Mouse HSCs cultured with TCA or TαMCA in vitro for 2 weeks showed a robust increase in the reconstitution level compared to non-treated cells (14-fold, n=14, p

Description: The majority of adult hematopoietic stem cells (HSCs) are maintained in a dormant state under homeostatic conditions. In contrast, under stressed conditions such as myeloablation and infection, HSCs are known to proliferate and rapidly give rise to downstream progeny. However, it is unclear whether and how HSCs respond to severe anemic conditions. Here we report that HSCs rapidly expand with a biased differentiation towards erythroid cells upon the induction of acute anemia. Injection of 60 mg/kg of phenylhydrazine (PHZ) was used to induce hemolytic anemia, after which the peripheral blood (PB), bone marrow (BM) and spleen of the mice were analyzed for the blood profiles and stem/progenitor cell content. The red blood cell (RBC) count of the PHZ treated mice was at its lowest at day 6 post injection. BM analysis showed that the number of HSCs (CD150+CD34-c-kit+Sca-I+Lineage-) immediately started increasing, as well as megakaryocyte-erythroid progenitors (MEP, CD34-FcγIII/IIR-c-kit+Sca-I-Lineage-) with a peak at day 3-4 (3.0 and 3.4 fold increase, respectively). Interestingly, the number of common myeloid progenitors (CMP, CD34+FcγIII/IIR-c-kit+Sca-I-Lineage-) did not show a clear increase over time and the number of erythroid progenitors (Ter119+) started increasing at a later time point than the HSC/MEP expansion, suggesting that the expansion of primitive cells is a primary response to the anemic condition that possibly skips some of the regular stages that are observed in the normal differentiation towards erythrocytes. In contrast to the BM, in the spleen HSC expansion was modest while MEP and CMP were robustly expanded (5.7 and 6.6 fold increase, respectively). These findings indicate that the BM and spleen have distinct roles in the response to the anemic conditions. In order to accurately evaluate the lineage potential of HSCs in vitro, we developed a combined assay utilizing colony formation and flow cytometry analysis (CFU-FACS), with which all generated colonies were analyzed for the morphology and the frequency of each lineage. The result showed that HSCs isolated from control mice had a balanced differentiation towards megakaryocyte and erythroid cells with 20-25% of the colonies containing only granulocytes/macrophages and megakaryocytes, but not erythroid cells (GMMk colonies). In contrast, HSCs isolated from PHZ treated mice showed significantly increased the number of colonies containing a higher content of erythroid cells, whereas the ratio of GMMk colonies was decreased. Furthermore, 3-dimensional analysis of the three lineage potentials (myeloid, megakaryocyte and erythroid) in the colonies revealed an imbalanced lineage potential of HSCs from anemic mice, showing higher erythroid potential instead of the megakaryocyte potential. As an alternative method, phlebotomy was performed to induce acute anemia. Although phlebotomized mice did not display a clear expansion of the HSC population, CFU-FACS analysis showed an erythroid-biased lineage potential of the HSCs, indicating that the HSC expansion and the lineage bias may be caused by independent mechanisms. To demonstrate if the alterations in the HSCs affect the in vivo function of these cells, 50 HSCs isolated from control or PHZ injected Kusabira Orange (KuO) mice were transplanted into lethally irradiated mice. Two weeks after the transplantation, the ratio of KuO+ RBCs against KuO+ platelets was higher in the PHZ-HSC transplanted mice than control-HSC transplanted mice. This difference was not seen four weeks after transplantation and the long-term reconstitution (〉12 weeks) levels did not differ between both groups, suggesting that the enhanced erythropoiesis is a transient event that does not reduce the stem cell capacity. In summary, we demonstrated that not only progenitor cells but also HSCs respond to severe anemic conditions and contribute to erythropoiesis through rapid expansion and a transient fate change, depicting a novel model of stress response. Disclosures No relevant conflicts of interest to declare.

Description: Phenotypically well-characterized hematopoietic stem cells (HSCs) still represent a heterogeneous pool of primitive cells regarding to their functionality. In particular, different lineage potential of HSCs have been considered as one of key features of the HSC heterogeneity. The lineage output of HSCs is often coupled with cell cycle status or long-term reconstitution potential, however molecular mechanisms of the mutuality are unclear and other type of the regulation may exist. In addition, prospective isolation of such HSCs biased towards specific lineage(s) is still problematic, as many of categorizations highly rely on retrospective information, e.g. transplantation assay. Although several markers have been reported to be able to subdivide HSCs into subcategories, exploration of additional markers will allow us understanding further molecular mechanisms of HSC regulations including activation and lineage choice. Here, we show that cell surface expression of Junctional adhesion molecule 2 (Jam2) represents higher reconstitution capacity of HSCs and the T cell potential. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0 %), while other Jam family member Jam1 (F11r) was expressed on all HSCs and Jam3 was not detected. To examine functional differences of Jam2+ and Jam2-HSCs, 30 cells were separately transplanted into lethally irradiated mice. Peripheral blood analyses revealed that Jam2+HSCs reconstituted more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3 %, respectively). In case of transplantation using 5 cells, the frequency of reconstituted mice was higher in Jam2+HSCs (7 in 11) compared to Jam2-HSCs (4 in 11), indicating that Jam2+ population is more enriched for functional HSCs. The expression of Jam2 on HSC is reversible, but not hierarchical, as both Jam2+ and Jam2-HSCs reconstituted opposite population in the BM.Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100 %. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell (or lymphoid) potential of Jam2+HSCs is independent of cell cycle progression. Jam2 has been reported to interact with Jam1, which mediates the Notch signaling (Kobayashi et al., Nature, 2014). Competitive co-culture of Jam2+ vs Jam2-HSCs on OP9-DL1 showed that Jam2+HSCs dominated the T cell production, whereas no difference was seen in B cell production upon OP9 co-culture. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoiesis environment affects the cell surface Jam2 expression. Comparison of C57BL/6, NOD, NOD-Scid and NOD-Scid Il2rγ KO (NSG) mice showed that HSCs of NSG mice have significantly higher frequency of Jam2+HSCs, suggesting that cell surface Jam2 expression might be regulated by specific cytokine(s) binding to IL2Rγ. Our findings suggest Jam2 is a new marker for a subset of HSCs that preferentially generate T cells. In addition, this work uncouples the lineage choice and cell cycle status, which proposes a novel model to the lineage-determining machineries. Since efficient and immediate generation of T cells in transplantation therapy is important to minimize infectious risks, understanding the molecular basis of the Jam-Notch cooperation would contribute to establish safer and more efficient treatment. Disclosures No relevant conflicts of interest to declare.