Fraction Of Hematopoietic Stem Cells Research Articles

7574 Diabetes Stem Cells: A Common Target for Abnormal Insulin Secretion and Insulin Resistance

Abstract Disclosure: H. Kojima: None. M. Katagi: None. J. Okano: None. T. Nakagawa: None. Diabetes is based on chronic inflammation and cannot be cured using only drugs that lower blood glucose levels. A culprit was lurking there that was hindering healing. We discovered abnormal hematopoietic stem cells that caused persistent chronic inflammation in the bone marrow of diabetic model animals. The real culprit is born after several days of hyperglycemia, never dies, and each time glycemic control deteriorates, it moves its progeny in large numbers throughout the body, fusing with unique cells in various organs and producing TNF-α from the organ cells. We have named these worst stem cells, which resemble cancer stem cells, "diabetes stem cells," and attempted to induce complete remission in diabetic model animals by eliminating them with drugs. In this study, we used streptozotocin (STZ)-induced insulin-deficient diabetes model mice. "Diabetes stem cells" were included in the CD106-positive short-term hematopoietic stem cell fraction, in which progenitor cells that play an important role in the regeneration of the thymus and peripheral tissues, and were characterized by abnormal expression of proinsulin and TNF-α. Furthermore, like cancer cells, the expression of histone deacetylases (HDACs) increased, and once the cells were born, they would not disappear. Therefore, we aimed to eliminate diabetes by administering an HDAC inhibitor and subcutaneously injecting insulin pellets to temporarily correct blood glucose levels. We treated diabetic mice with HDAC inhibitor for 8 weeks, along with several weeks of insulin therapy. Insulin aims to prevent the formation of new abnormal cells in the bone marrow and to prevent the inhibition of pancreatic islet wasting due to the forced insulin secretion caused by hyperglycemia. The combination therapy allowed the beta cells to regenerate and produce enough insulin to normalize blood glucose levels and maintain them even after treatment was discontinued. It also solved the mystery of how the abnormal cell population evades attacks from the T cells. In STZ diabetes, the supply of normal CD106-positive progenitor cells from the bone marrow for thymic regeneration was completely blocked, resulting in marked thymic atrophy. As a result, the thymus, which had lost its progenitor cells, was unable to establish an immune surveillance system to eliminate mutated cells throughout the body. In other words, the combination therapy completely broke the vicious cycle caused by atrophies in both the islet and the thymus. Moreover, when the thymus was surgically removed along with the adventitia to prevent regeneration, diabetes remission was completely inhibited, supporting the importance of thymic normalization. Diabetes is a hematopoietic stem cell disease accompanied by thymic dysfunction, and the removal of "diabetes stem cells" paves the way for the cure of diabetes. Presentation: 6/2/2024

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

Distinct Cellular, Transcriptional, and Mechanistic Properties Determine HSC Fate during Emergency Granulopoiesis

Emergency granulopoiesis (EG) is the process responsible for the rapid and enhanced production of granulocytes during acute infections. Traditionally, EG was understood as a cellular mechanism mostly initiated and supported by myeloid progenitors, however, recent data suggest that hematopoietic stem cells (HSCs) might also participate in the process. To investigate whether and how HSCs contribute to the initial steps of EG, we performed scRNA-seq and ATAC-seq analysis of sorted murine HSCs 4 hours after in vivo lipopolysaccharide (LPS) administration, mimicking an acute bacterial infection. Strikingly, we observed radical transcriptional changes between HSCs isolated from PBS control and LPS treated mice, mostly marked by alterations in HSC lineage bias. We identified a steady state lymphoid-biased subpopulation of HSCs, marked by expression of Procr (CD201). Following LPS stimulation, the CD201 expression was lost and this population was transcriptionally rewired to a myeloid-biased HSC population. Accordingly, ATACseq data corroborated opening of myeloid-bias loci upon LPS administration rather than steady-state lymphoid-biased loci. Further, we confirmed the loss of CD201 expression in HSCs by flow cytometry in mice after LPS and G-CSF treatment, as well as Candida albicans infection, suggesting that the loss of CD201 expression is a general event during EG induced by different stimuli. Interestingly, the CD201 downregulation in HSCs was independent of the master regulator of EG C/EBPβ, while we observed the contribution of the TLR4-MyD88 signaling axis. Next, we functionally validated our scRNA-seq results in vitro and in vivo. When cultivated under myeloid differentiation conditions, the CD201- HSCs gave rise to mature granulocytes, whereas CD201+ HSCs remained rather immature. In transplantation settings, CD201+ HSCs showed increased engraftment and preferential bias to produce lymphoid cells, while CD201- HSC showed decreased engraftment ability and a bias towards myeloid production. Surprisingly, LPS challenge of BM chimeras transplanted with lymphoid-biased CD201+ HSCs led to EG response marked by rapid loss of lymphoid-biased multipotent progenitors (MPPs) and the expansion of myeloid-biased MPPs, thus confirming the lympho-myeloid switch observed in the scRNA-seq data. Interestingly, the treatment of WT mice with CD201 blocking antibody was able to partially impair the lympho-myeloid switch, suggesting that CD201 plays an active role in HSCs during EG. Mechanistically, we observed that both CD201+ and CD201- HSCs rely on different signaling pathways under EG. While CD201+ HSCs express higher levels of TLR4 and have higher activation of NF-κB signaling upon in vitro LPS stimulation, the CD201- HSCs express higher levels of G-CSF-R and have higher activation of pSTAT3 signaling upon G-CSF-stimulation. Moreover, while the LIP isoform of C/EBPβ, important for cell proliferation, is present in both CD201+ and CD201- HSCs, the LAP/LAP* isoform, important for myeloid differentiation, is present only in the CD201- HSCs fraction. Altogether, our data suggest that EG is supported by a population of HSCs which upon pathogen sensing undergo a radical transcriptional rewiring that promotes their myeloid output. Initially, the pathogen is directly sensed by TLR4 on the surface of a steady state lymphoid-biased CD201+ HSCs, causing a rapid activation of the downstream NF-κB signaling pathway. Subsequently, the lymphoid-myeloid transcriptional switch, marked by the loss of CD201 expression, occurs and EG is then supported by myeloid-biased CD201- HSCs. CD201- HSCs respond to the infection in an indirect manner through G-CSF-R on their surface and exhibit enhanced pSTAT3 activation and elevated LAP/LAP* C/EBPβ isoform, cellular mechanisms known to promote myeloid differentiation and granulocytic production. In conclusion, the switch from CD201+ to CD201- HSCs facilitates both fast and sustained EG leading to the supply of new granulocytes to fight the infection. This detailed understanding of the distinct cellular, transcriptional, and mechanistic properties that determine HSC fate during emergency granulopoiesis opens new venues to potentially modulate granulocytic production in certain clinical conditions such as sepsis. This work was partially supported by a GACR 22-18300S, GAUK 327722, and IMG institutional funding RVO 68378050.

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

Tetramer Formation of BCL11A Is Required for Both Protein Stability and Fetal Hemoglobin Silencing

The zinc-finger (ZnF) repressor protein BCL11A regulates the switch from fetal (HbF, α 2γ 2) to adult hemoglobin (HbA, α 2β 2) through direct binding at the γ-globin promoter. Clinical trials of gene-based reduction of BCL11A with ex vivo modified hematopoietic stem cells (HSCs) have yielded transformative outcomes in patients with sickle cell disease and β-thalassemia, establishing BCL11A as a therapeutic target. Unless in vivo methods for delivery of gene-based therapies become efficient enough to modify a substantial fraction of host HSCs, small molecule drugs will be required to reduce overall disease burden. With the goal of targeting BCL11A with small molecules, we have characterized its native state. The protein is largely unstructured, except for 7 ZnFs, numbered 0-6. Previously we showed that BCL11A protein appears stable in cells with a T 1/2 of ~ 24 hrs. Here we report the unexpected finding that the protein assembles and functions as a tetramer. Recombinant BCL11A behaves as a tetramer in biophysical assays. Gene editing of the endogenous BCL11A locus in CD34-derived, immortal HUDEP2 cells has been employed to correlate protein structure and expression with consequences of a given modification. We found that BCL11A lacking ZnF0 is unstable, leading to protein loss and derepression of γ-globin. Recombinant ZnF0 self-assembles into a stable tetramer, whose X-ray structure reveals a hydrophobic interface between subunits. Mutation of residues in the hydrophobic core disrupts tetramerization and markedly reduces steady-state protein in cells. Reciprocal immunoprecipitation in cells reveals that ZnF0 is both necessary and sufficient to mediate BCL11A mutlimer assembly. Collectively, these results indicate that BCL11A tetramerization is required for protein stability. In cells expressing BCL11A lacking ZnF0, the steady-state protein level is partially restored by addition of proteasome inhibitors, suggesting that mutant protein is actively degraded by the ubiquitin-proteasome system. In contrast to findings with ZnF0, discrete removal of ZnF1 within the context of full-length BCL11A results in a marked increase in steady-state protein in cells. In vitro purified ZnF1 forms a dimer and only dimeric ZnF1 can be stably expressed in cells. These findings re consistent with a model in which assembly of the BCL11A tetramer is required to mask a degron in ZnF1 that becomes obscured on its dimerization. Masking of a degron in ZnF1 predicts that discrete removal of both ZnF0 and ZnF1 within full-length BCL11A will lead to production of a monomer. Indeed, HUDEP2 cells expressing BCL11A lacking both ZnFs exhibit an elevated level of steady-state, monomeric protein, consistent with the degron masking model. However, despite an elevated level, cells expressing monomeric BCL11A are defective in HbF silencing, indicating that the tetramer structure is critical for γ-globin repression. Our findings provide novel insights into BCL11A action and suggest new strategies for therapeutic targeting. Combining structural, biochemical, and cellular approaches, we have shown that ZnF-mediated tetramer formation shields BCL11A from proteolytic degradation, and that the tetramer state itself is required for effective γ-globin repression. Of relevance to BCL11A as a therapeutic target, the tetramer of ZnF0 constitutes a structured, stable domain in an otherwise largely unstructured protein, and thereby offers possibilities for small molecule targeting. The multimer state of BCL11A is a vulnerability for HbF repression.

Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

RNA splicing factor Rbm25 underlies heterogeneous preleukemic clonal expansion in mice

AbstractClonal expansion sets the stage for cancer genesis by allowing for the accumulation of molecular alterations. Although genetic mutations such as Tet2 that induce clonal expansion and malignancy have been identified, these mutations are also frequently found in healthy individuals. Here, we tracked preleukemic clonal expansion using genetic barcoding in an inducible Tet2 knockout mouse model and found that only a small fraction of hematopoietic stem cells (HSCs) expanded excessively upon Tet2 knockout. These overexpanded HSCs expressed significantly lower levels of genes associated with leukemia and RNA splicing than nonoverexpanded Tet2 knockout HSCs. Knocking down Rbm25, an identified RNA splicing factor, accelerated the expansion of Tet2-knockout hematopoietic cells in vitro and in vivo. Our data suggest that mutations of an epigenetic factor Tet2 induce variability in the expression of an RNA splicing factor Rbm25, which subsequently drives heterogeneous preleukemic clonal expansion. This heterogeneous clonal expansion could contribute to the variable disease risks across individuals.

Validation of Kinetic Stem Cell (KSC) counting algorithms for rapid quantification of human hematopoietic stem cells

Specific quantification of therapeutic tissue stem cells (TSCs) is a major challenge. We recently described a computational simulation method for accurate and specific counting of TSCs. The method quantifies TSCs based on their unique asymmetric cell kinetics, which is rate-limiting for TSCs’ production of transiently-amplifying lineage-committed cells and terminally arrested cells during serial cell culture. Because of this basis, the new method is called kinetic stem cell (KSC) counting. Here, we report further validations of the specificity and clinical utility of KSC counting. First, we demonstrate its quantification of the expected increase in the hematopoietic stem cell (HSC) fraction of CD34+-selected preparations of human-mobilized peripheral blood cells, an approved treatment product routinely used for HSC transplantation therapies. Previously, we also used the KSC counting technology to define new mathematical algorithms with the potential for rapid determination of TSC-specific fractions without the need for serial culture. A second important HSC transplantation treatment, CD34+-selected umbilical cord blood (UCB) cells, was used to investigate this prediction. We show that, with an input of only simple population doubling time (PDT) data, the KSC counting-derived “Rabbit algorithms” can be used to rapidly determine the specific HSC fraction of CD34+-selected UCB cell preparations with a high degree of statistical confidence. The algorithms define the stem cell fraction half-life (SCFHL), a new parameter that projects stem cell numbers during expansion culture. These findings further validate KSC counting’s potential to meet the long-standing unmet need for a method to determine stem cell-specific dosage in stem cell medicine.

Inhibition of Hyper-ActivatedLysosomes Restores Youthful Properties and Functions in Old Hematopoietic Stem Cells

Aging is the primary risk factor for many late-onset diseases including myeloid malignancies and immune deficiencies of the elderly. Aging of hematopoietic stem cells (HSCs) is thought to be in part responsible for these blood disorders. As quiescence maintains HSCs' health and function throughout life, loss of quiescence with age is likely implicated in defects of old HSCs including their myeloid-biased lineage commitment. Despite major progress, mechanisms that promote aging of HSCs remain to be elucidated. We have identified dynamic lysosomal modulations as a switch in the precise control of HSC quiescence vs. priming/activation in young HSCs. Here we find that lysosomal properties and functions are compromised in old HSCs. We find that lysosomes are relatively depleted in old HSCs, as evaluated by high resolution confocal microscopy analyses of immunofluorescence (IF) staining of lysosomal-associated membrane protein (LAMP)-1 and the more specific LAMP-2 markers. Notably, lysosomal properties are altered in old HSCs beyond what is known of their function in autophagy. We find increased recruitment of damage-associated proteins Galectin 1 and 2 to lysosomes in old relative to young highly purified LSKCD150+CD48- HSCs indicating increased lysosomal damage in old HSCs. In addition, lysosomal LC3 flux that is an indication of lysosomal activity was increased by two-fold in highly purified old relative to young HSCs. This was in line with old HSCs' alteration of expression of lysosomal enzymes Cytochalasin C and D and the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase). The increased lysosomal activity is supported by greatly acidified lysosomes as well as enhanced lysosomal degradation capacity of cargo in old relative to young HSCs. An 18-hour inhibition of lysosomal acidification by repressing v-ATPase' activity using specific and high affinity v-ATPase inhibitor, ex vivo, led to a dramatic enrichment of old HSCs in large lysosomes, restoration of their properties and reducing their Galectin protein burden similar to what was observed in young quiescent and healthy HSCs. This was specific to lysosomes in the endo-lysosomal pathway as the effect on the Golgi and endoplasmic reticulum compartments in HSCs was relatively negligeable. The v-ATPase inhibitory treatment increased substantially the fraction of non-dividing old HSCs in culture, and the frequency of LTC-ICs recovered in limiting dilution analysis of old HSCs. Importantly, a 4-day ex vivo inhibition of lysosomal activity increased old HSCs' in vivo competitive repopulation ability by over 8-fold over 21 weeks and balanced the production of lineages downstream of old HSCs. Remarkably, only v-ATPase inhibitor- but not vehicle-treated old HSCs sustained survival in lethally irradiated secondarily transplanted mice and maintained self-renewal. v-ATPase inhibition normalized the expression of TFEB, master regulator of lysosomal biogenesis, suppressed CDK6 nuclear expression, normalized metabolic properties of old HSCs, including mTOR activity and glucose uptake, normalized expression of histone marks H4K16ac, H3K27ac and H3K56ac in old HSCs, as well as signs of intrinsic inflammation. Overall, our work indicates that inhibiting lysosomal hyper-activation restores optimum mitochondrial sequestration in old HSCs' lysosomes, leading to reduced inflammation and restitution of stem cell function in old HSCs. These findings may be exploited for improving HSC function in the elderly.

Tracking C-type lectin-like receptor positive leukemic stem cells to determine therapeutic efficacy in acute myeloid leukemia.

e19011 Background: Although the importance of leukemia stem cells (LSC) in disease course of acute myeloid leukemia (AML) is well established in basic research, detecting and tracking LSC is seldom adopted in routine clinical practice. This is partly due to difficulty in tracking LSC in routine clinical practice and the paucity of evidence to support the benefit of detecting LSC in guiding clinical practice. Recently, a functional LSC population has been methodically characterized immunophenotypically, and the C-type lectin-like receptor (CLL1; aka CD371) has been shown to be widely expressed on LSC but not on normal hematopoietic stem cells (HSC). We postulate that CLL1 can be used to track LSC in a clinical setting and by analyzing changes in the LSC Compartment, we may be better able to assess response to therapy. Methods: Bone marrow samples from non-M3 CD34+ AML patients that were positive for CLL1 expression were included. Flow cytometry data were retrospectively analyzed for CLL1 percent positivity in the HSC compartment, which was defined as the CD34+CD38low/- fraction. A chart review was conducted to determine correlation between CLL1 positive LSC compartment change and clinical outcome. Statistical analysis was performed using GraphPad Prism version 9. Results: From 12/2018 to 12/2021, 49 AML cases with both pre- and post-treatment data from a single institution, were identified for the study. Among them, fourteen patients with CLL1 positivity higher than 50% were analyzed for CLL1+/HSC fractional change between pre- and post-treatment. We found that 6 of these 14 patients with significant reduction in CLL1+/HSC achieved longer remission, while the other 8 patients with no significant decrease in the CLL1+/HSC fraction demonstrated no or only a temporary remission. Importantly, several cases of patients with significant reduction in CLL1 positive cells will be presented to highlight the fact that LSC fractional change was more accurate in evaluating therapeutic effects and more predictive of clinical outcome than traditional morphology and flow cytometric analysis. This may even alter interpretation of specific treatment outcomes by predicting, for example, the brief nature that can be expected from certain remissions. Lastly, the CLL1+/HSC compartment in the peripheral blood is highly correlated with that of the bone marrow, indicating future feasibility of tracking LSC fractional change non-invasively to guide therapeutic decisions. Conclusions: CLL1 as a LSC marker is readily applicable to the clinical setting. Tracking LSC before and after AML treatment provides critical information regarding therapeutic effects that can sometimes be missed by traditional morphological and flow cytometric analysis. Thus, measuring changes in the LSC compartment using CLL1 analysis should be incorporated routinely in clinical practice to guide therapy decisions.

Schlafen2 is a regulator of quiescence in adult murine hematopoietic stem cells.

Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.

Circulating hematopoietic stem cells (cHSC) in elite athletes: an antidoping perspective

Hematopoietic stem cells (HSC) are multipotent progenitor cells, resident primarily in human bone marrow. Their main function is the continuous renewal of all hematopoietic cells. A small fraction of HSC is also circulating in peripheral blood (cHSC) where they can be possibly detected thanks to the application of the new very sensitive flow cytometry methodologies.In recent times it has been debated whether the utilization of several different types of stem cells can be abused by athletes for doping purposes. Moreover, there is growing knowledge that the dynamic and turnover of circulating HSC may reflect changes affecting the physiologic processes at the erythropoietic level; also, cHSC and other circulating hematopoietic progenitors (cHP) resulted to give different responses in athletes engaged in endurance and maximal exercise sports. If it is true that the use of stem cells (mainly mesenchymal stem cells) to allow a rapid recover from an injury is not considered a doping practice, it is also true that the 2022 WADA list of prohibited substances and methods forbids “the use of normal or genetically modified cells” as well as “any form of intravascular manipulation of the blood or blood components by physical or chemical means”. In this context, HSC and hematopoietic erythroid progenitors may be a potential threat and the possibility of their abuse needs to be exploited in sport antidoping.In view of the above, we have preliminarily assessed the possibility of detecting circulating hematopoietic stem cells by flow cytofluorimetry. In details, we applied two flow cytofluorimetric protocols for the accurate and precise counting of circulating HSC, also discussing the potential applicability in a potential antidoping scenario. We concluded that i) flow cytofluorimetry is a pratical and rapid platform for the identification and the counting of cHSC and other erythroid progenitors in peripheral human whole blood, ii) the correlation between CD34+ circulating progenitors and standard haematological parameters may be an effective new tool to be exploited as in direct markers of blood doping and, iii) accurate baseline levels of HSC and erythroid progenitors need to be determined in order to identify abuses of HSC and/or conventional blood doping.

A FLI1-KLF6 Axis Regulates Aging in Human Hematopoietic Stem and Progenitor Cells and Normalization of KLF6 Levels in Aged Cells Leads to Their Rejuvenation

Aging causes a gradual decline in hematopoietic stem cell (HSC) function, which increases the risk for hematological malignancies. While much has been done in murine models, human HSC aging impairment is less understood. We recently showed that Krüppel-like transcription factor 6 (KLF6) is among the top downregulated genes during human HSC aging, which correlates with H3K27ac loss at several upstream putative enhancers. Moreover, loss of KLF6 in human CD34 + cells resulted in impaired in vitro differentiation, increased colony-forming potential and a transcriptional profile similar to that of aged CD34 + CD38 - cells. We hypothesized that age-acquired deregulation of KLF6 may be a key player in age-related HSC dysfunction and sought to fully characterize this. Thus, we isolated CD34 + cells from young (<32 y.o) and aged (>65 y.o.) healthy donors and performed CRISPR-Cas9 genome editing and transcriptional activation of KLF6, respectively, followed by epigenetic and transcriptional reprogramming, in vivo hematopoietic reconstitution, and analysis of DNA damage, apoptosis, and reactive oxygen species (ROS) levels.KLF6 knock-out (KO) and non-targeting control (NTC) cells from young healthy donors were engrafted into immunodeficient NSGS mice. Hematopoietic reconstitution analysis showed that KLF6 KO cells led to increased myeloid and reduced lymphoid reconstitution in peripheral blood (PB; p<1.62 -7) and an increase in immunophenotypically defined HSC and CD34 + CD38 - progenitor fractions in the bone marrow (BM; p=0.02, and p=0.04, respectively). H3K27ac analysis of KLF6 KO cells revealed a loss of 3,390 ChIP-seq peaks (FDR < 0.05) and 285 peaks gained. Functional annotation using ChIP-Enrich showed that H3K27ac loss associates with myeloid homeostasis, erythroid differentiation and oxidative stress (FDR < 0.05). Three putative enhancer (E) regions upstream of the KLF6 locus showed loss of H3K27ac with aging. Depletion of the E1 but not E2 or E3 regions phenocopied in vitro and in vivo findings of KLF6 KO. Transcription factor (TF) ChIP-seq data analysis revealed FLI1, ERG, and RUNX1 binding overlapping the E1 region. Knockdown of FLI1 but not ERG or RUNX1 led to an increase in KLF6. Notably, FLI1 mRNA levels, but not ERG or RUNX1, are increased during normal aging.We next performed in vitro KLF6 activation in aged CD34 + (KLF6a) cells using a dCas9-VP64 system to test if we could rejuvenate these cells. KLF6a cells exhibited a decrease in their in vitro myeloid differentiation potential, compared to aged NTC CD34 + cells (p<0.0041), and behaved instead similar to young controls. ChIP-seq analysis of KLF6a showed marked decrease of H3K4me1 (n=3,273 peaks) with relatively few regions with increased H3K4me1 (n=602) (FDR < 0.05). In contrast, we observed an increase in H3K27ac (n=3,361 peaks) with only 71 peaks lost compared to aged NTC (FDR < 0.05). Regions that gained H3K27ac in KLF6a were associated with platelet activation, cell junction and adhesion. In vivo analysis of KLF6a cells injected into NSGS mice revealed a significant reduction in the PB myeloid fraction compared to NTC (p<1.2-8), with a concomitant expansion in the lymphoid compartment (p<4.4 -11). BM composition analysis at week 16 showed a decrease in the HSC fraction in KLF6a cells (p=0.0029) as well as a reduction in CD34 +CD38 -, CD34 +CD38 + and MEPs (p=0.036, p<0.0001 and p=0.041, respectively). We next examined the impact of KLF6 modulation on DNA damage and observed that young human KLF6 KO cells had a significant increase in gH2AX and 53BP1 (p<0.0001, for both) whereas KLF6a in aged CD34 + cells exhibited reduced gH2AX and 53BP1 foci in comparison to aged NTC (p<0.0001, for both). In addition, apoptotic levels in KLF6 KO cells were higher than in NTC cells (p=0.006) whereas aged KLF6a cells showed a reduction in the incidence of apoptotic cells compared to NTC (p=0.019). Finally, ROS analysis in young KLF6 KO showed increased levels of total and mitochondrial ROS compared to NTC (p=0.0008 and p<0.0001, respectively) whereas both ROS fractions were reduced in KLF6a cells (p=0.0002 and p<0.0001, respectively).In summary, these results show that the FLI1-KLF6 axis plays a key role in regulating HSPC aging and that KLF6 is required for normal HSPC function and differentiation. In addition, normalization of KLF6 levels in aged HSPCs resulted in reprogramming and rejuvenation HSPCs, confirming the central role of this TF in aging HSPC biology. DisclosuresNo relevant conflicts of interest to declare.

EVI1-Positive AML Cells Show Distinct Features and High Dependency on ETS Transcription Factor ERG

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice.We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells.To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes.Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML.Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. DisclosuresMasamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.

Clonal Analysis of Acute Myeloid Leukemia Secondary to Myeloproliferative Neoplasms

[Background] Prognosis of patients with secondary acute myeloid leukemia (sAML) transformed from myeloproliferative neoplasms (MPNs) is extreamly poor. It has been demonstrated that several additional genetic alterations, which are engaged in epigenetic regulation, cell-growth signaling and RNA splicing machinery, cooperate with JAK2, CALR and MPL mutations for the development of sAML from MPN. The order of mutation acquisition also influences the clonal evolution in MPN. However, little is known about the mechanisms of its leukemic transformation. In previous studies, both JAK2 -mutated and -unmutated AML clones were identified in sAML cases transformed from JAK2 -mutated MPNs, suggesting the diversity of the clonal evolution during the leukemic transformation from MPNs. In this study, we investigated genetic alterations and clonal evolutions in sAML from MPN, especially in JAK2 -unmutated sAML, to elucidate the mechanisms of the leukemic transformation.[Methods] We performed targeted deep sequencing of 54 genes in six paired MPN and sAML samples. Whole-exome sequencing (WES) was also performed in a paired MPN, sAML and normal tissue cells. For a clonal analysis, bone marrow (BM) cells at complete remission (CR) stage after chemotherapy were sorted into hematopoietic stem cell (HSC) and hematopoietic progenitor fractions such as common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocytic/erythroid progenitor (MEP) fractions, and analyzed the mutations detected in targeted deep sequencing or WES. Furthermore, we sorted single cells from the HSC fraction and performed single-cell mutational analysis to clarify the clonal evolutions precisely during the development of sAML. In addition, we intravenously inoculated BM cells at the sAML stage into NOD/SCID/IL-2Rγnull (NOG) mice, and compared mutation status between primary AML cells and engrafted leukemia cells to evaluate the clonal hierarchy of leukemia cells.[Results] Four JAK2 -mutated and two CALR -mutated MPNs were analyzed in this study. Targeted deep sequencing identified TP53 mutations in five of six sAML samples with high variant allele frequencies (VAFs). In two of the four JAK2 -mutated MPNs, VAFs of JAK2 -V617F were significantly decreased in sAML cells compared with those in MPN cells, whereas VAFs of TP53 mutations were emerged or increased in sAML cells, suggesting that sAML clones with TP53 mutations were raised from JAK2-V617F negative hematopoietic cells in both cases. Moreover, we identified the mutations which was common to MPN and AML cells in both cases. These mutations formed common initiating clones prior to MPN and sAML, and additional mutation in JAK2 or TP53 was associated with their MPN or sAML development. The single-cell analysis in CD34+/CD38- HSC fractions revealed that a small part of the cells harbored TP53 -R248W and JAK2 -wild type (WT), half of the cells harbored homozygous JAK2 -V617F and TP53 -WT, and the other half cells harbored JAK2 - and TP53 -WT. Notably, three common mutations detected both in MPN and in sAML with the same VAFs were mainly found in JAK2 -WT cells. Thus, we validated that JAK2 and TP53 mutations were mutually exclusive HSC fractions, and confirmed that sAML clones were raised from JAK2 -unmutated clone. In patient derived xenotransplant models using JAK2 -WT sAML cells following JAK2 -mutated MPN or CALR -mutated sAML, engrafted leukemia cells showed the same mutational pattern as primary sAML cells. These results suggested that sAML clones but not MPN clones were able to engraft and proliferate in mice.[Conclusions] The genetic analysis in paired MPN and sAML samples revealed the diverse clonal evolution patterns during the development of sAMLs from MPNs. TP53 mutation was strongly associated with leukemic transformations both in JAK2 -V617F mutated and CALR -mutated MPNs. However, we demonstrated two cases in whom driver mutations in MPNs were not preserved in sAML clones. In those cases, there might be independent common initiating clones corresponding to MPN or sAML, and each initiating clone might be acquired JAK2 or TP53 mutation before the development of MPN or sAML, respectively. Our data also suggested the importance of single-cell analysis which enable us to clarify the clonal evolutions in leukemic transformation from MPN, that were not identified in targeted deep sequencing as well as WES. DisclosuresKiyoi:Phizer Japan Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Identification of a Hematopoietic Stem Cell Fraction That Retains Normal Function Following Chronic Inflammation

Chronic inflammation has been suggested to impair hematopoietic stem cell (HSC) function, resulting in decreased self-renewal and myeloid overproduction at the expense of other lineages. We previously identified the pro-inflammatory cytokine interleukin (IL)-1 as a key activator of precocious HSC differentiation that simultaneously impairs HSC self-renewal. However, recent identification of multiple HSC subsets with distinct functional and molecular characteristics calls into question whether the HSC compartment is uniformly affected by chronic inflammatory signals. Moreover, a growing body of work suggests that traditional markers in the mouse that define HSC potential under homeostatic conditions, such as Sca-1 and CD34, undergo expression changes that limit their utility in prospective identification of functional HSCs during non-homeostatic conditions such as inflammation. Here we use injection of IL-1β to model chronic inflammation in the mouse, and demonstrate that the rare EPCR+CD34- fraction contained within the phenotypic Lin-cKit+Sca-1+Flk2-CD48-CD150+ (SLAM) HSC compartment is uniquely able to maintain its function despite chronic IL-1 exposure.To assess the impact of chronic inflammation on HSC compartment dynamics, we injected wild-type (WT) C57BL/6 mice with recombinant murine IL-1β for 20 days, followed by analysis of HSC populations using multi-parametric flow-cytometry. Strikingly, t-Stochastic Neighbor Embedding (tSNE) analysis of our flow cytometry data identified changes in the relative abundance of several novel HSC sub-populations following IL-1 exposure, including a sharp decrease in the number of CD34- cells also expressing the long-term HSC (LT-HSC) marker EPCR (CD201). Consistent with the characteristics of a long-term HSC, CD34-EPCR+ HSCs exhibited slow growth and differentiation kinetics in liquid culture, and elevated colony forming potential in serial re-plating. Moreover, molecular analyses demonstrated EPCR+CD34- HSCs are uniquely enriched for expression of LT-HSC genes including Hoxb5, Fgd5 and Cpt1a . Nonetheless, EPCR+CD34- cells express Il1r1 and underwent accelerated myeloid differentiation in the presence of IL-1, suggesting they are directly responsive to this cytokine.To determine the impact of sustained IL-1 exposure on the blood-forming capacity of EPCR+CD34- HSCs in vivo, we assessed their growth and potential ex vivo following treatment of mice with PBS or IL-1β for 20 days. Notably, prior exposure to IL-1 did not significantly alter the growth or short-term potential of EPCR+CD34- HSCs based on in vitro assays. Strikingly, ongoing transplantation experiments of IL-1-exposed EPCR+CD34- HSCs versus total SLAM HSCs into irradiated recipient mice have revealed that while the total SLAM HSC compartment exhibits immediate impairment of reconstitution capacity, EPCR+CD34- HSCs exhibit no functional impairment, suggesting IL-1 signaling may not directly impact their self-renewal capacity. We are currently examining the molecular and epigenetic features of these cells to understand how they retain their self-renewal capacity in the face of inflammation, and assessing whether this compartment retains its functionality in a range of inflammatory disease contexts.Overall, our findings have uncovered a distinct fraction within the phenotypic SLAM HSC that retains essentially normal function despite exposure to chronic inflammation. This supports the notion that HSCs respond to inflammation in a heterogeneous manner and that a functional LT-HSC compartment is present even during chronic inflammatory contexts. These investigations stand to provide critical insights that will increase our understanding of the impact of inflammation on long-term blood system function in the context of chronic inflammatory disease, bone marrow transplant and hematological malignancy. DisclosuresNo relevant conflicts of interest to declare.

MiRNome profiling of LSC-enriched CD34+CD38−CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38−CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38−CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1 +) vs. CD26− (BCR-ABL1 −) CD34+CD38− fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38− fractions that distinguishes between CD26+ (BCR-ABL1 +) and their CD26− (BCR-ABL1 -) counterparts, providing valuable data for future studies.

Employing the CRISPR-Cas System for Clonal Hematopoiesis Research.

Clonal hematopoiesis is a state in which substantial fraction of hematopoietic stem cells acquire mutations in specific driver genes and expand in the absence of an overt hematological malignancy. Recent clinical studies have shown that clonal hematopoiesis increases likelihood of hematological malignancy and cardiovascular disease. While clinical studies have identified countless candidate driver genes associated with clonal hematopoiesis, experimental studies are required to evaluate causal and mechanistic relationships with disease processes. This task is technically difficult and expensive to achieve with traditional genetically engineered mice. The versatility and programmability of CRISPR-Cas system enables investigators to evaluate the pathogenesis of each mutation in experimental systems. Technical refinements have enabled gene editing in a cell type specific manner and at a single base pair resolution. Here, we summarize strategies to apply CRISPR-Cas system to experimental studies of clonal hematopoiesis and concerns that should be addressed.