Hematopoietic Stem Cells Division Research Articles

Asymmetric cell division of hematopoietic stem cells: recent advances, emerging concepts, and future perspectives

Hematopoietic stem cells (HSCs) can self-renew and differentiate for the entire life of an organism to produce new blood cells when needed. This process is regulated by asymmetric cell division (ACD), an evolutionarily conserved mechanism whereby cell fate determinants are unequally segregated into the daughter cells during division to instruct different cell fates. After many years of controversy, recent technical advances in microscopy, imaging, and bioinformatics make it now possible to visualize and quantify how factors segregate asymmetrically in dividing HSCs and lead to predictable changes in daughter cell fates many days later. While the molecular processes behind ACD in HSCs are still poorly understood, accumulating evidence suggests that lysosomes and other organelles, including mitochondria, autophagosomes, mitophagosomes, and recycling endosomes can segregate asymmetrically and act as cell fate determinants during divisions. Asymmetric segregation of lysosomes and mitochondria has been shown to predict mitochondrial activity, translation, and differentiation of HSC daughter cells and their offspring. This discovery and recent seminal findings show that lysosomes, once considered to be merely the trash bin of the cell, regulate many aspects of HSC biology and are crucial for the maintenance of quiescence and stem cell function. Here we provide a historical perspective and discuss the recent advances in our understanding of ACD and the role of lysosomes in HSC function. We discuss the limitations of past studies, talk about emerging concepts, and suggest critical next steps required to move the field forward.

Age-related changes in the hematopoietic stem cell pool revealed via quantifying the balance of symmetric and asymmetric divisions.

Hematopoietic stem cells (HSCs) are somatic stem cells that continuously generate lifelong supply of blood cells through a balance of symmetric and asymmetric divisions. It is well established that the HSC pool increases with age. However, not much is known about the underlying cause for these observed changes. Here, using a novel method combining single-cell ex vivo HSC expansion with mathematical modeling, we quantify HSC division types (stem cell-stem cell (S-S) division, stem cell-progenitor cell (S-P) division, and progenitor cell-progenitor cell (P-P) division) as a function of the aging process. Our time-series experiments reveal how changes in these three modes of division can explain the increase in HSC numbers with age. Contrary to the popular notion that HSCs divide predominantly through S-P divisions, we show that S-S divisions are predominant throughout the lifespan of the animal, thereby expanding the HSC pool. We, therefore, provide a novel mathematical model-based experimental validation for reflecting HSC dynamics in vivo.

M 6a-Son-CCL5 Axis Controls HSC Fate and Inflammation

Blood stem cells intricately regulate their fate, balancing self-renewal and differentiation through symmetric and asymmetric divisions. When exposed to various stresses they can rapidly undergo symmetric commitment divisions to generate differentiated progenitors for immune regeneration or driving inflammation. In previous studies, we and others found that m 6A RNA methylation controls symmetric commitment and inflammation in hematopoietic stem cells (HSCs) (Cheng and Luo et al., Cell Reports 2019). However, the precise molecular mechanisms underlying the role of m 6A and its influence on the inflammatory program in stem cells remain elusive. To uncover the dynamic RNA methylation during HSC commitment, we employed a recently developed tool called DART-seq, that fuses the YTH domain of the m 6A reader protein YTHDF2 with the RNA editing protein APOBEC1. We identified 300 m 6A sites and approximately 250-300 gene targets within HSCs and MPPs (padj beta binomial < 0.05). We found m 6A sites in HSCs were enriched for: innate immune response pathways including NFκB and Toll-like receptor cascades, cellular signaling processes encompassing RhoGTPase and ERK/MAPK signaling targets, and metabolic pathways associated with amino acid transport and fatty acid oxidation. In contrast, the m 6A targets enriched in MPPs were associated with lineage differentiation and myeloid programs such as MLL, GATA1, PU.1, and CEBPD. By integrating our findings with two additional m 6A mapping datasets, Son was a shared m 6A target with increased m 6A modification during the transition from HSCs to MPPs. SON, an RNA binding protein residing in nuclear speckles, has been implicated in a myriad of cellular processes including splicing control and transcriptional repression. Notably, de novo heterozygous loss-of-function variants in SON (ZTTK Syndrome) results in hematological symptoms, neurological impairments, and developmental delays. The genetic deletion of METTL3 ( Mettl3 conditional knockout mice; Mettl3 cKO) or METTL3 inhibitor STM2457 treatment, resulted in a twofold increase in Son transcripts in both HSCs and MPPs, but reduced SON protein abundance by 50%. Notably, SON exhibited asymmetric segregation during HSC division, with its abundance displaying a strong correlation with the commitment marker NUMB. Collectively, these findings suggest that m 6A modification regulates the abundance of SON protein and implicates SON in HSC commitment fate. Reintroduction of SON in m 6A-deficient HSCs effectively rescued commitment defects (cKO EV 9.74% vs. cKO+SON 21.43%). Furthermore, SON overexpression partially restored in vivo engraftment defects in Mettl3 cKO LSKs (cKO EV 1.5% vs. cKO+SON 4.5%). Intriguingly, the functional rescue was accomplished using a fragment containing the RNA-binding domain of SON (cKO EV 1.2% vs. cKO+SON-RB 12.3%), highlighting the significance of SON's RNA-binding ability. Additionally, deletion of SON in RosaCas9 WT LSKs using sgRNAs resulted in a substantial reduction in engraftment (WT EV 35.4% vs. WT+sgSON 11%), whereas SON overexpression in WT LSKs enhanced engraftment (WT EV 6% vs. WT+SON 39%). These results demonstrate that SON is a positive regulator of stem cell engraftment and function. Moreover, SON overexpression effectively rescued specific clusters identified through scRNA-seq analysis in Mettl3 KO (KOsp1-1 and CCL5+) by 50%. In bulk RNA-seq analysis, we identified 257 upregulated genes and 227 downregulated genes between the Mettl3 cKO EV and Mettl3 cKO SON groups (padj<0.05). Remarkably, SON overexpression significantly downregulated inflammatory pathways that were aberrantly upregulated upon m 6A loss. Furthermore, SON overexpression indirectly rescued dsRNA formation in Mettl3 cKO LSKs, aligning with the role of m 6A in suppressing dsRNA formation in HSPCs. SON overexpression reduced Ccl5 through binding to its transcript and decreasing nascent transcription. Strikingly, depletion of CCL5 significantly rescued Mettl3 cKO LSK engraftment (cKO 1.6% vs. cKO+shCCL5 5.3%), indicating that CCL5 is sufficient to mediate the effects downstream of SON. Moreover, co-culturing WT HSCs with CCL5 resulted in a symmetric commitment defect similar to that observed with m 6A loss. In summary, our study delineates the critical m 6A-SON-CCL5 axis governing HSC symmetric commitment fate and inflammation control.

Single Cell Analysis of Genes Involved in Asymmetric Hematopoietic Stem Cell (HSC) Division in Chronic Myeloid Leukemia (CML) : Evidence of the Regulation of Tetraspanin CD63 during Leukemia Progression

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history and survival of CML patients. However, the majority of the patients remain on long-lasting therapies and switch one TKI to another, either because of intolerance or resistance. All TKI cessation trials showed approximately 50% of leukemia recurrence rates even after several years of TKI therapies, suggesting the persistence of residual leukemic stem cells in patients in deep molecular response (DMR). The resistance and persistence of CML HSC are related to intrinsic (BCR-ABL independent survival) or extrinsic (leukemic niche ) mechanisms some of which have been extensively studied. However, there has been no data with regard to the role of asymmetric cell division pathways in the persistence and progression of CML stem cell clone. Indeed, During asymmetric division process of hematopoietic stem cells, some proteins are differently segregated between daughter HSCs such as endosome associated tetraspanin proteins (CD53, CD63) and mitochondria-associated fission regulator Dynamin related protein-1(DRP1). Similarly, it has been shown that asymmetric organelle inheritance can influence HSC decisions towards differentiation versus self-renewal. Interestingly, the tetraspanin CD63 is known to be involved in the quiescence of human and murine HSC via activation of TGF-beta pathway, CD63 hi cells exhibiting increased self-renewal potential after the asymmetric division with reduced differentiation potential. Materials and Methods We have analyzed the transcriptome performed in a cohort of CML patients in chronic phase (CP) and blast phase (BP) (GSE4170) as well as in a validation cohort of RNA-sequencing (GSE100026). Similarly, single cell transcriptome analysis of Lin- CD34+ CD38- CML progenitors (GSE76312) were included in the study evaluating the expression of genes involved in asymmetric cell division. We have then generated an asymmetric hematopoietic stem cell risk score using a panel of genes involved in the asymmetric division of HSCs and correlated these results with the transcriptome of the CML cohorts above. A pseudotime analysis was performed to evaluate single cell trajectories of the genes identified. Results To determine the potential role of asymmetric cell division markers in the progression of CML, we have analyzed the expression of several of these genes during CML progression in two independent transcriptome cohorts. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules which were found to be down-regulated during blast crisis as compared to chronic phase. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules down regulated during blast crisis as compared to chronic phase suggesting less of asymmetric properties during advanced phase of the disease. The down regulation in patients in blast crisis was confirmed at single cell level for the expression of SELL, CD63, NUMB, HK2 and LAMP2. CD63 expression has also been found to be of interest in CML chronic phase. Indeed, single cell neural network and deep learning in CP samples showed that the CD63 expression was the best asymmetric gene marker predicting a poor prognosis and this appeared to be induced by opposite regulation of HK2 and NUMB. In patients in CP-CML, the single cell trajectory reconstitution revealed the major regulatory role of CD63 highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, PDIA3, all genes known to be involved also in hematopoietic malignancies such as myelodysplasia, AML and ALL. Conclusion We show here for the first time the identification of the expression of genes involved in the asymmetric division of CML HSC. Among these genes we identified the tetraspanin CD63 as being down-regulated during CML progression. The expression of the genes involved in the asymmetric HSC division should require further studies in CML, either to predict persistence of CML or progressive disease. Figure 1 : Single cell transcriptome of Lin- CD34+ CD38- CML progenitors in CP links CD63 expression to progressive disease. A/ Piechart of counts for chronic phase cells analyzed according to prognosis; B/ pseudotime transformation of single cell transcriptome according patient prognosis; C/ Pseudotime expression heatmap; D/ Pseudotime expression of CD63 in CP.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

Predicting the number of lifetime divisions for hematopoietic stem cells from telomere length measurements

How many times does a typical hematopoietic stem cell (HSC) divide to maintain a daily production of over blood cells over a human lifetime? It has been predicted that relatively few, slowly dividing HSCs occupy the top of the hematopoietic hierarchy. However, tracking HSCs directly is extremely challenging due to their rarity. Here, we utilize previously published data documenting the loss of telomeric DNA repeats in granulocytes, to draw inferences about HSC division rates, the timing of major changes in those rates, as well as lifetime division totals. Our method uses segmented regression to identify the best candidate representations of the telomere length data. Our method predicts that, on average, an HSC divides 56 times over an 85-year lifespan (with lower and upper bounds of 36 and 120, respectively), with half of these divisions during the first 24 years of life.

Single cell analysis of the localization of the hematopoietic stem cells within the bone marrow architecture identifies niche-specific proliferation dynamics.

Hematopoietic stem cells (HSC) reside in the bone marrow (BM) in specialized niches which provide support for their self-replication and differentiation into the blood cells. Recently, numerous studies using sophisticated molecular and microscopic technology have provided snap-shots information on the identity of the BM niches in mice. In adults, HSC are localized around arterioles and sinusoids/venules whereas in juvenile mice they are in close to the osteoblasts. However, although it is well recognized that in mice the nature of the hematopoietic niche change with age or after exposure to inflammatory insults, much work remains to be done to identify changes occurring under these conditions. The dynamic changes occurring in niche/HSC interactions as HSC enter into cycle are also poorly defined. We exploit mice harboring the hCD34tTA/Tet-O-H2BGFP transgene to establish the feasibility to assess interactions of the HSC with their niche as they cycle. In this model, H2BGFP expression is driven by the TET trans-activator under the control of the human CD34 promoter which in mice is active only in the HSC. Since Doxycycline inhibits TET, HSC exposed to this drug no longer express H2BGFP and loose half of their label every division allowing establishing the dynamics of their first 1-3 divisions. To this aim, we first validated user-friendly confocal microscopy methods to determine HSC divisions by hemi-decrement changes in levels of GFP expression. We then tracked the interaction occurring in old mice between the HSC and their niche during the first HSC divisions. We determined that in old mice, most of the HSC are located around vessels, both arterioles which sustain quiescence and self-replication, and venules/sinusoids, which sustain differentiation. After just 1 week of exposure to Doxycycline, great numbers of the HSC around the venules lost most of their GFP label, indicating that they had cycled. By contrast, the few HSC surrounding the arterioles retained maximal levels of GFP expression, indicating that they are either dormant or cycle at very low rates. These results reveal that in old mice, HSC cycle very dynamically and are biased toward interactions with the niche that instructs them to differentiate.

Reconstruction of bone marrow microenvironment for expansion of hematopoietic stem cells by a histone deacetylase inhibitor.

Ex vivo expansion of hematopoietic stem cells (HSCs) is an approach for overcoming cell insufficiency for umbilical cord blood transplantation. It was suggested that in common ex vivo cultures, the stemness specificity of HSCs is rapidly reducing due to DNA hypermethylation. Here, Nicotinamide (NAM), a DNA methyltransferase and histone deacetylase inhibitor, is used with a bioengineered Bone Marrow-like niche (BLN) for HSC ex vivo expansion. The CFSE cell proliferation assay was used for tracking HSCs division. qRT-PCR was conducted to assay the HOXB4 mRNA expression levels. The morphology of BLN-cultured cells was analyzed using scanning electron microscopy (SEM). NAM boosted the induction of HSC proliferation in the BLN group compared to the control group. In addition, the ability of HSCs to colonize was more significant in the BLN group than in the control group. Our data suggest that the presence of NAM in bioengineered niches promotes HSC proliferation. The presented approach showed that small molecules could be used in the clinical setting to overcome the limited number of CD34+ cells in cord blood units.

The structure of the hematopoietic system can explain chronic myeloid leukemia progression

Almost all cancer types share the hallmarks of cancer and a similar tumor formation: fueled by stochastic mutations in somatic cells. In case of chronic myeloid leukemia (CML), this evolutionary process can be tracked from an asymptomatic long-lasting chronic phase to a final rapidly evolving blast phase. Somatic evolution in CML occurs in the context of healthy blood production, a hierarchical process of cell division; initiated by stem cells that self-renew and differentiate to produce mature blood cells. Here we introduce a general model of hierarchical cell division explaining the particular progression of CML as resulting from the structure of the hematopoietic system. Driver mutations confer a growth advantage to the cells carrying them, for instance, the BCR::ABL1 gene, which also acts as a marker for CML. We investigated the relation of the BCR::ABL1 mutation strength to the hematopoietic stem cell division rate by employing computer simulations and fitting the model parameters to the reported median duration for the chronic and accelerated phases. Our results demonstrate that driver mutations (additional to the BCR::ABL1 mutation) are necessary to explain CML progression if stem cells divide sufficiently slowly. We observed that the number of mutations accumulated by cells at the more differentiated levels of the hierarchy is not affected by driver mutations present in the stem cells. Our results shed light on somatic evolution in a hierarchical tissue and show that the clinical hallmarks of CML progression result from the structural characteristics of blood production.

M6A-BERT-Stacking: A Tissue-Specific Predictor for Identifying RNA N6-Methyladenosine Sites Based on BERT and Stacking Strategy

As the most abundant RNA methylation modification, N6-methyladenosine (m6A) could regulate asymmetric and symmetric division of hematopoietic stem cells and play an important role in various diseases. Therefore, the precise identification of m6A sites around the genomes of different species is a critical step to further revealing their biological functions and influence on these diseases. However, the traditional wet-lab experimental methods for identifying m6A sites are often laborious and expensive. In this study, we proposed an ensemble deep learning model called m6A-BERT-Stacking, a powerful predictor for the detection of m6A sites in various tissues of three species. First, we utilized two encoding methods, i.e., di ribonucleotide index of RNA (DiNUCindex_RNA) and k-mer word segmentation, to extract RNA sequence features. Second, two encoding matrices together with the original sequences were respectively input into three different deep learning models in parallel to train three sub-models, namely residual networks with convolutional block attention module (Resnet-CBAM), bidirectional long short-term memory with attention (BiLSTM-Attention), and pre-trained bidirectional encoder representations from transformers model for DNA-language (DNABERT). Finally, the outputs of all sub-models were ensembled based on the stacking strategy to obtain the final prediction of m6A sites through the fully connected layer. The experimental results demonstrated that m6A-BERT-Stacking outperformed most of the existing methods based on the same independent datasets.

Bmi1 suppresses protein synthesis and promotes proteostasis in hematopoietic stem cells.

The polycomb complex component Bmi1 promotes the maintenance of stem cells in multiple postnatal tissues, partly by negatively regulating the expression of p16Ink4a and p19Arf, tumor suppressors associated with cellular senescence. However, deficiency for p16Ink4a and p19Arf only partially rescues the function of Bmi1-deficient stem cells. We conditionally deleted Bmi1 from adult hematopoietic cells and found that this slowly depleted hematopoietic stem cells (HSCs). Rather than inducing senescence, Bmi1 deficiency increased HSC division. The increased cell division was caused partly by increased Aristaless-related homeobox (ARX) transcription factor expression, which also increased ribosomal RNA expression. However, ARX deficiency did not rescue HSC depletion. Bmi1 deficiency also increased protein synthesis, protein aggregation, and protein ubiquitylation independent of its effects on cell division and p16Ink4a, p19Arf, and ARX expression. Bmi1 thus promotes HSC quiescence by negatively regulating ARX expression and promotes proteostasis by suppressing protein synthesis. This highlights a new connection between the regulation of stem cell maintenance and proteostasis.

Three-Dimension Co-culture of Hematopoietic Stem Cells and Differentiated Osteoblasts on Gallic Acid Grafted-Chitosan Scaffold as a Model of Hematopoietic Stem Cells Niche.

The existing approaches of hematopoietic stem cells (HSCs) expansion in vitro were difficult to meet the needs of clinical application. While in vivo, HSCs efficiently self-renew in niche where they interact with three dimension extracellular matrix and stromal cells. Osteoblasts (OBs) are one of most significant stromal cells of HSCs niche. Here, we proposed a three-dimensional environment based on gallic acid grafted-chitosan (2c) scaffold and OBs differentiated from human umbilical cord mesenchymal stem cells (HUMSCs) to recapitulate the main components of HSCs niche. The results of alkaline phosphatase staining and alizarin red staining demonstrated that HUMSCs were successfully induced into OBs. The results showed that the expansions of CD34+cells, CD34+CD38- cells and CD34+CD38-CD45RA-CD49f+CD90+ cells (primitive hematopoietic stem cells, pHSCs) harvested from the biomimetic HSCs niche based on 2c scaffold and OBs (IV) group were larger than those harvested from other three culture groups. Importantly, it was found that the CD34+ cells harvested from IV group had better secondary expansion capability and colony forming potential, indicating better self-renewal ability. In addition, the biomimetic HSCs niche based on 2c scaffold and OBs protected HSCs apoptosis and promoted HSCs division. Taken together, the biomimetic HSCs niche based on 2c scaffold and OBs was an effective strategy for ex vivo expansion of HSCs in clinical scale.

Heterozygous lipoprotein lipase knockout mice exhibit impaired hematopoietic stem/progenitor cell compartment.

BackgroundHematopoietic stem cells (HSC) maintain the hematopoietic system homeostasis through self‐renewal and multilineage differentiation potential. HSC are regulated by the microenvironment, cytokine signaling, and transcription factors. Recent results have shown that lipid pathways play a key role in the regulation of HSC quiescence, proliferation, and division. However, the mechanism by which lipid metabolism regulates HSC proliferation and differentiation remains to be clarified. Lipoprotein lipase (LPL) is an essential enzyme in the anabolism and catabolism of very low‐density lipoprotein, chylomicrons, and triglyceride‐rich lipoproteins.MethodsThe percentage of hematopoietic stem/progenitor cells and immune cells were determined by fluorescence‐activated cell sorting (FACS). The function and the mechanism of HSCs were analyzed by cell colony forming assay and qPCR analysis. The changes in LPL+/− HSC microenvironment were detected by transplantation assays using red fluorescent protein (RFP) transgenic mice.ResultsTo explore the function of LPL in HSC regulation, heterozygous LPL‐knockout mice (LPL+/−) were established and analyzed by FACS. LPL+/− mice displayed decreased hematopoietic stem/progenitor cell compartments. In vitro single‐cell clonogenic assays and cell‐cycle assays using FACS promoted the cell cycle and increased proliferation ability. qPCR analysis showed the expression of p57KIP2 and p21WAF1/CIP1 in LPL+/− mice was upregulated.ConclusionsLPL+/− mice exhibited HSC compartment impairment due to promotion of HSC proliferation, without any effects on the bone marrow (BM) microenvironment.

Tracking Hematopoietic Precursor Division Ex Vivo in Real Time

Deciphering molecular mechanisms underlying division of hematopoietic stem cells (HSCs) and malignant precursors would improve our understanding of basis of stem cell-fate decisions and oncogenic transformation. Using a novel reporter of hematopoietic precursor Evi1-GFP, we track the division of hematopoietic precursors in culture in real time. First, we confirmed that Evi1-GFP is a faithful reporter of HSC activity and identified three dividing patterns of HSCs: symmetric renewal, symmetric differentiation, and asymmetric division. Moreover, we found that the cytokines and growth factors combination (STIF) promotes symmetric renewal, whereas OP9 stromal cells balance symmetric renewal and differentiation of HSCs ex vivo .Interestingly, we found that Tet2 knockout HSCs underwent more symmetric differentiation in culture compared to wild type control. Intriguingly, OP9 stromal cells reverse the phenotype of Tet2 knockout HSCs ex vivo . Furthermore, we demonstrated that Tet2-/- ; Flt3 ITD acute myeloid leukemia (AML) precursors primarily underwent symmetric renewal divisions in culture. Our study establishes a new system to explore the molecular mechanisms of the regulation of benign and malignant hematopoietic precursor division ex vivo . Theknowledge learned from these studies will provide new insights into the molecular mechanisms of HSC fate decision and leukemogenesis. Disclosures No relevant conflicts of interest to declare.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

Symmetric and asymmetric activation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are in an inactive quiescent state for most of their life. To replenish the blood system in homeostasis and after injury, they activate and divide. HSC daughter cells must then decide whether to return to quiescence and metabolic inactivity or to activate further to proliferate and differentiate and replenish lost blood cells. Although the regulation of HSC activation is not well understood, recent discoveries shed new light on involved mechanisms including asymmetric cell division (ACD). HSC metabolism has emerged as a regulator of cell fates. Recent evidence suggests that cellular organelles mediating anabolic and catabolic processes can be asymmetrically inherited during HSC divisions. These include autophagosomes, mitophagosomes, and lysosomes, which regulate HSC quiescence. Their asymmetric inheritance has been linked to future metabolic and translational activity in HSC daughters, showing that ACD can regulate the balance between HSC (in)activity. We discuss recent insights and remaining questions in how HSCs balance activation and quiescence, with a focus on ACD.

Increased stem cell proliferation in atherosclerosis accelerates clonal hematopoiesis

Clonal hematopoiesis, a condition in which individual hematopoietic stem cell clones generate a disproportionate fraction of blood leukocytes, correlates with higher risk for cardiovascular disease. The mechanisms behind this association are incompletely understood. Here, we show that hematopoietic stem cell division rates are increased in mice and humans with atherosclerosis. Mathematical analysis demonstrates that increased stem cell proliferation expedites somatic evolution and expansion of clones with driver mutations. The experimentally determined division rate elevation in atherosclerosis patients is sufficient to produce a 3.5-fold increased risk of clonal hematopoiesis by age 70. We confirm the accuracy of our theoretical framework in mouse models of atherosclerosis and sleep fragmentation by showing that expansion of competitively transplanted Tet2-/- cells is accelerated under conditions of chronically elevated hematopoietic activity. Hence, increased hematopoietic stem cell proliferation is an important factor contributing to the association between cardiovascular disease and clonal hematopoiesis.

Hematopoietic stem cell (HSC) divisional memory: The journey of mitochondrial metabolism through HSC division

Hematopoietic stem cells (HSCs) are characterized by their ability to produce all cells of the blood and immune system and have been used for transplantation for decades. Although the regenerative potential of HSCs is high, their self-renewal potential is limited. HSC functions are inversely correlated with their divisional history. Recent advances in our understanding of the regulation of HSCs through cell division suggest that HSCs may never replicate into identical self, but rather replicate into progeny that gradually lose functionality at each round of division. HSC division is accompanied by major transcriptional and metabolic changes. In this perspective, the possibility that mitochondrial metabolism confers HSC division memory and programs HSCs for extinction is discussed.

Effect of Lipids and Lipoproteins on Hematopoietic Cell Metabolism and Commitment in Atherosclerosis.

Hematopoiesis is the process that leads to multiple leukocyte lineage generation within the bone marrow. This process is maintained throughout life thanks to a nonstochastic division of hematopoietic stem cells (HSCs), where during each division, one daughter cell retains pluripotency while the other differentiates into a restricted multipotent progenitor (MPP) that converts into mature, committed circulating cell. This process is tightly regulated at the level of cellular metabolism and the shift from anaerobic glycolysis, typical of quiescent HSC, to oxidative metabolism fosters HSCs proliferation and commitment. Systemic and local factors influencing metabolism alter HSCs balance under pathological conditions, with chronic metabolic and inflammatory diseases driving HSCs commitment toward activated blood immune cell subsets. This is the case of atherosclerosis, where impaired systemic lipid metabolism affects HSCs epigenetics that reflects into increased differentiation toward activated circulating subsets.Aim of this review is to discuss the impact of lipids and lipoproteins on HSCs pathophysiology, with a focus on the molecular mechanisms influencing cellular metabolism. A better understanding of these aspects will shed light on innovative strategies to target atherosclerosis-associated inflammation.

Hematopoietic Stem Cells Self-Renew Symmetrically or Gradually Proceed to Differentiation

It is not known whether hematopoietic stem cells (HSCs) undergo symmetric or asymmetric cell divisions in the unperturbed bone marrow. Here, we integrate data from HSC fate mapping and cell-cycle-dependent labeling through mathematical inference and thus gain insight into how HSCs coordinate self-renewal with differentiation. We find that most HSC divisions in adult mice are symmetric self-renewing, replacing HSCs lost by direct differentiation and death, and slowly expanding the HSC population. This expansion maintains constant HSC output to multipotent progenitors (MPPs), despite declining HSC differentiation rate with age. We identify a linear hierarchy of differentiation states between tip HSCs and MPPs, where Tie2-driven HSC fate mapping fully covers the progression of the differentiating cells. A turning point from self-renewal to accelerated cell differentiation occurs between early-stage and late-stage MPPs, just before lineage differentiation becomes manifest in single-cell transcriptomes. This stem cell hierarchy precedes lineage differentiation and may limit mutation accumulation in the hematopoietic system.