Hematopoietic Stem Cells Fate Decisions Research Articles

How CBX proteins regulate normal and leukemic blood cells.

Hematopoietic stem cell (HSC) fate decisions are dictated by epigenetic landscapes. The Polycomb Repressive Complex 1 (PRC1) represses genes that induce differentiation, thereby maintaining HSC self-renewal. Depending on which chromobox (CBX) protein (CBX2, CBX4, CBX6, CBX7, or CBX8) is part of the PRC1 complex, HSC fate decisions differ. Here, we review how this occurs. We describe how CBX proteins dictate age-related changes in HSCs and stimulate oncogenic HSC fate decisions, either as canonical PRC1 members or by alternative interactions, including non-epigenetic regulation. CBX2, CBX7, and CBX8 enhance leukemia progression. To target, reprogram, and kill leukemic cells, we suggest and describe multiple therapeutic strategies to interfere with the epigenetic functions of oncogenic CBX proteins. Future studies should clarify to what extent the non-epigenetic function of cytoplasmic CBX proteins is important for normal, aged, and leukemic blood cells.

Divergent Roles of MLL3 and MLL4 in Hematopoietic Stem Cell Fate Decision

Complex gene regulatory mechanisms, orchestrated by transcription factors (TFs) and epigenetic regulators, govern cell fate decisions in normal and malignant hematopoiesis. While prior works have extensively characterized the TF-networks in blood development, we currently have limited understanding of epigenetic regulation, in particular molecular rules underlying cofactor usage, in hematopoietic cell fate decisions. Kmt2c and Kmt2d are homologous genes that encode transcriptional coactivators, MLL3 and MLL4, respectively. They have been shown to coordinate embryonic stem cell fate transitions and are recurrently mutated in leukemias, offering an ideal paradigm to study epigenetic regulation of hematopoiesis. While MLL3 and MLL4 are often believed to play redundant roles in stem cell fate decisions, few has characterized their molecular distinctions. MLL3 and MLL4 each nucleate a chromatin-bound complex called the Complex of Proteins Associated with Set1 (COMPASS) and both deposit H3K4me1 at enhancers. Despite similar biochemical functions, Kmt2c and Kmt2d loss-of-function mutations have been reported with contrasting phenotypes in hematopoiesis. Kmt2d loss impairs HSC self-renewal and enhances myeloid differentiation at the expense of lymphopoiesis, whereas Kmt2c deletions enhance HSC self-renewal and impair differentiation. We hypothesize that MLL3 and MLL4 are recruited by different transcription factors and bind chromatin at both overlapping and distinct enhancers to convey specific hematopoietic fates. To test whether MLL3 and MLL4 are redundant in licensing myeloid differentiation, we transplanted wildtype, Kmt2c/d single knockout, and Kmt2c/d double knockout (DKO) donor cells into lethally irradiated recipients. Surprisingly, DKO recipients showed significant expansion of phenotypic HSCs, MPPs, and pre-granulocyte-monocyte progenitors (pGMs), typically associated with a myeloid differentiation block. DKO mice progressively developed bone marrow failure 9-months after transplantation, indicating that MLL3/4-COMPASS is necessary to sustain hematopoiesis. To dissect how Kmt2c/d deletions shape lineage specification and whether DKO imposes a differentiation block, we performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) on donor Lin -Kit +Sca1 + (LSK) cells from the transplantation described above. While Kmt2c deletion does not markedly reprogram HSC/MPP identity, Kmt2d deletion results in precocious myeloid differentiation. Most notably, we observed that compound Kmt2c/d deletions fundamentally reprogramed HSCs/MPPs toward a B-lymphoid-like state, instead of imposing a myeloid differentiation block. The data establish a model wherein MLL3 and MLL4 have redundant roles in licensing HSC identity and myeloid potential. However, within the HSC to myeloid differentiation trajectory, MLL3 promotes differentiation while MLL4 promotes self-renewal. To understand how MLL3- and MLL4-COMPASS differentially regulate enhancer accessibility, we performed single-cell transposase-accessible chromatin sequencing (scATAC-seq) on the same donor LSK cells as in the CITE-seq. Compound Kmt2c/d deletions impose a unique epigenomic state on HSCs/MPPs (Figure 1A). Motif enrichment analyses revealed that DKO cells (i) were enriched for PAX5 and EBF1 motifs, consistent with B-lymphoid priming. HSCs (ii) were enriched for AP-1 and NFE2L2 motifs, suggesting that MLL4 may preferentially bind AP-1 and antioxidant response elements associated with HSC identity. Clusters of myeloid progenitors (iii and iv) were populated primarily by Kmt2d-deficient cells and enriched for CEBP and SPI1 motifs, suggesting that MLL3 is preferentially recruited to enhancers bound by myeloid TFs. This study provides several novel insights into epigenetic regulation of hematopoiesis. We have shown that HSC identity and myeloid fate specification are licensed by redundant MLL3/4-COMPASS-dependent TF-cofactor interactions, but once HSCs are established, MLL3- and MLL4-COMPASS engage distinct, antagonistic programs. Furthermore, early B-lymphoid priming is mediated via MLL3/4-COMPASS-independent mechanisms (Figure 1B). Ongoing work will link precise transcriptional codes to divergent cofactor utilization patterns and provide critical insights into how epigenetic regulators are deployed to effect distinct cell fates.

TAF1, the Largest Subunit of Tfiid, Is Dispensable for Adult Hematopoiesis

Temporal and spatial control of gene expression is central to normal hematopoietic stem cell (HSC) biology. Many sequence-specific transcription factors have been identified as key regulators of HSC fate decision, while the function of general transcription factors in HSC behavior is poorly understood. We previously reported that TAF1, the largest subunit of the TFIID complex, plays a critical role in AML1-ETO driven acute myeloid leukemogenesis. To evaluate the function of TAF1 in normal fetal and adult hematopoiesis, we generated TAF1 conditional knockout (CKO) mice and identified an essential role of TAF1 in fetal erythropoiesis. Surprisingly, deletion of TAF1 in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of hematopoietic stem and progenitor cell (HSPC) populations, with increased self-renewal and impaired differentiation capacity in these TAF1- null cells. Consistently, we found that TAF1-null HSPCs failed to up-regulate key differentiation-associated genes when induced to differentiate in vitro. Using a variety of genome-wide assays and biochemical approaches, we found that TAF1 loss not only disrupted TFIID complex formation and chromatin recruitment, but also reduced promoter accessibility, impaired RNA Polymerase II promoter recruitment and activity, as well as its promoter-proximal pausing mechanism. Thus, the effect of TAF1 loss on normal adult hematopoiesis primarily relates to the inability to upregulate genes involved in differentiation, while HSC self-renewal and the expression of self-renewal genes proceeds relatively normally.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Linking cell cycle to hematopoietic stem cell fate decisions.

Hematopoietic stem cells (HSCs) have the properties to self-renew and/or differentiate into any blood cell lineages. In order to balance the maintenance of the stem cell pool with supporting mature blood cell production, the fate decisions to self-renew or to commit to differentiation must be tightly controlled, as dysregulation of this process can lead to bone marrow failure or leukemogenesis. The contribution of the cell cycle to cell fate decisions has been well established in numerous types of stem cells, including pluripotent stem cells. Cell cycle length is an integral component of hematopoietic stem cell fate. Hematopoietic stem cells must remain quiescent to prevent premature replicative exhaustion. Yet, hematopoietic stem cells must be activated into cycle in order to produce daughter cells that will either retain stem cell properties or commit to differentiation. How the cell cycle contributes to hematopoietic stem cell fate decisions is emerging from recent studies. Hematopoietic stem cell functions can be stratified based on cell cycle kinetics and divisional history, suggesting a link between Hematopoietic stem cells activity and cell cycle length. Hematopoietic stem cell fate decisions are also regulated by asymmetric cell divisions and recent studies have implicated metabolic and organelle activity in regulating hematopoietic stem cell fate. In this review, we discuss the current understanding of the mechanisms underlying hematopoietic stem cell fate decisions and how they are linked to the cell cycle.

Cholesterol confers ferroptosis resistance onto myeloid-biased hematopoietic stem cells and prevents irradiation-induced myelosuppression

There is growing appreciation that hematopoietic alterations underpin the ubiquitous detrimental effects of metabolic disorders. The susceptibility of bone marrow (BM) hematopoiesis to perturbations of cholesterol metabolism is well documented, while the underlying cellular and molecular mechanisms remain poorly understood. Here we reveal a distinct and heterogeneous cholesterol metabolic signature within BM hematopoietic stem cells (HSCs). We further show that cholesterol directly regulates maintenance and lineage differentiation of long-term HSCs (LT-HSCs), with high levels of intracellular cholesterol favoring maintenance and myeloid bias of LT-HSCs. During irradiation-induced myelosuppression, cholesterol also safeguards LT-HSC maintenance and myeloid regeneration. Mechanistically, we unravel that cholesterol directly and distinctively enhances ferroptosis resistance and boosts myeloid but dampens lymphoid lineage differentiation of LT-HSCs. Molecularly, we identify that SLC38A9–mTOR axis mediates cholesterol sensing and signal transduction to instruct lineage differentiation of LT-HSCs as well as to dictate ferroptosis sensitivity of LT-HSCs through orchestrating SLC7A11/GPX4 expression and ferritinophagy. Consequently, myeloid-biased HSCs are endowed with a survival advantage under both hypercholesterolemia and irradiation conditions. Importantly, a mTOR inhibitor rapamycin and a ferroptosis inducer imidazole ketone erastin prevent excess cholesterol-induced HSC expansion and myeloid bias. These findings unveil an unrecognized fundamental role of cholesterol metabolism in HSC survival and fate decisions with valuable clinical implications.

Identification of the Differentiation Stages of Living Cells from the Six Most Immature Murine Hematopoietic Cell Populations by Multivariate Analysis of Single-Cell Raman Spectra.

Efforts to expand hematopoietic stem and progenitor cells (HSPCs) in vitro are motivated by their use in the treatment of leukemias and other blood and immune system diseases. The combinations of extrinsic cues within the hematopoietic stem cell (HSC) niche that lead to HSC fate decisions remain unknown. New noninvasive and location-specific techniques are needed to enable identification of the differentiation stages of individual hematopoietic cells on biomaterial microarray screening platforms that minimize the usage of rare HSCs. Here, we show that a combination of Raman microspectroscopy and partial least-squares discriminant analysis (PLS-DA) enables the location-specific identification of individual living cells from the six most immature hematopoietic cell populations, HSC, multipotent progenitor (MPP)-1, MPP-2, MPP-3, common myeloid progenitor, and common lymphoid progenitor. Better than 90% accuracy was achieved. We show that the accuracy of this differentiation stage identification was based on spectral features associated with cell biochemistries. This work establishes that PLS-DA can capture the subtle spectral variations between as many as six closely related cell populations in the presence of potentially significant within-population spectral variation. This noninvasive approach can be used to screen HSC fate decisions elicited by extrinsic cues within biomaterial microarray screening platforms.

The microbiota regulates hematopoietic stem cell fate decisions by controlling iron availability in bone marrow.

Host microbiota crosstalk is essential for the production and functional modulation of blood-cell lineages. Whether, and if so how, the microbiota influences hematopoietic stem cells (HSCs) is unclear. Here, we show that the microbiota regulates HSC self-renewal and differentiation under stress conditions by modulating local iron availability in the bone marrow (BM). In microbiota-depleted mice, HSC self-renewal was enhanced during regeneration, while the commitment toward differentiation was dramatically compromised. Mechanistically, microbiota depletion selectively impaired the recycling of red blood cells (RBCs) by BM macrophages, resulting in reduced local iron levels without affecting systemic iron homeostasis. Limiting iron availability in food (invivo) or in culture (exvivo), or by CD169+ macrophage depletion, enhanced HSC self-renewal and expansion. These results reveal an intricate interplay between the microbiota, macrophages, and iron, and their essential roles in regulating critical HSC fate decisions under stress.

CXCR4 Drives Lympho-Myeloid Fate of Hematopoietic Progenitors Via mTOR and Mitochondrial Metabolic Pathways

Blood production is a tightly regulated process that starts with hematopoietic stem cells (HSCs). In adults, HSCs are unique in their capacity to self-renew and replenish the entire blood system through production of a series of increasingly committed progenitor cells within the bone marrow (BM) microenvironment. HSCs form a rare, quiescent population that displays a metabolism skewed towards anaerobic glycolysis at the expense of mitochondrial oxidative phosphorylation (OXPHOS) to preserve its quiescent state and long-term reconstitution capacity. However, when HSCs differentiate, they undergo a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS, a process that is in part mediated by the metabolic sensor mTOR. It is well-established that HSCs in the BM adapt the production of myeloid and lymphoid cells depending on the needs of the body and that metabolic plasticity is a critical driver of HSC fate decisions. This has never been assessed for multipotent progenitors (MPPs) which constitute the stage at which the major divergence of lymphoid and myeloid lineages occurs. In mice, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) are generated from phenotypically and functionally distinct subpopulations of lineage-biased MPPs, i.e. MPP2 and MPP3 are reported as distinct myeloid-biased MPP subsets that operate together with lymphoid-primed MPP4 to control blood leukocyte production. This question is thus of paramount importance to understand how the lympho-myeloid specification process is regulated.Signaling by the G protein-coupled receptor CXCR4 on MPPs in response to stimulation by its natural ligand, the chemokine CXCL12, produced by BM perivascular stromal cells constitutes a key pathway through which the niches and MPPs communicate. However, the mechanisms whereby CXCR4 signaling regulates MPP specification are still unknown. We addressed this point using BM samples of patients with WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-CXCR4-function mutations affecting desensitization of CXCR4 and characterized by chronic lympho-neutropenia, as well as a unique WS mouse model which phenocopies severe pan-leukopenia.We unraveled myeloid skewing of the hematopoietic stem and progenitor cell (HSPC) compartment in BM of patients with WS and of WS mice. This relied on CXCR4 signaling strength that controls the output of the lymphoid and myeloid lineages by coordinating the composition and molecular identity of the MPP compartment. The fate of the lymphoid-biased MPP4 subset was central in such a process. Indeed, CXCR4 signaling termination was required for efficient generation and maintenance of the MPP4 pool, while regulating intrinsically their cell cycle status and lymphoid-myeloid gene landscape. In fact, we demonstrated for the first time that enhanced mTOR signaling, accumulation of damaged mitochondria and overactive OXPHOS-driven metabolism promoted cell-autonomous molecular changes that reprogram mutant MPP4 away from lymphoid differentiation. Consistent with this, in vivo chronic treatment with the CXCR4 antagonist AMD3100/Plerixafor or the mTOR inhibitor Rapamycin normalized mitochondrial metabolism and MPP4 differentiation.Thus, our study shows that CXCR4 signaling acts through the mTOR pathway as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4. DisclosuresNo relevant conflicts of interest to declare.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

The Spliceosomal Component Sf3b1is Essential for Hematopoietic Stem Cell Formation through the Regulation of the Jak/Stat Signaling Pathway

Hematopoietic stem cells (HSCs) maintain multi-lineage hematopoiesis for the lifetime of an organism. During embryogenesis, HSCs first emerge from specialized hemogenic endothelial cells within the dorsal aorta through a process termed the endothelial-to-hematopoietic transition. While much is known about the role of transcription factors and transcriptional control in HSC specification, it is ill defined how post-transcriptional RNA processing influences this first HSC fate decision. Using zebrafish loss-of-function mutants for the pre-mRNA spliceosomal component sf3b1 (splicing factor 3b, subunit 1), we demonstrated that impairing splicing hindered HSC production, specifically at the endothelial-to-hematopoietic transition. Loss-of-function sf3b1hi3394 mutants have diminished expression of thehemogenic endothelial and HSC markers runx1 and gata2b, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf (hours post fertilization). These data indicate that although sf3b1 is ubiquitously expressed, hemogenic endothelium and the downstream HSCs are more sensitive to its loss than other endothelial cells. To uncover the mechanism underlying the defect in hemogenic endothelium and HSCs in sf3b1 mutants, we performed RNA-sequencing on purified kdrl:gfp- positiveendothelial cells from sf3b1 mutants and wild-type siblings at 24 hpf. We observed nearly 900 genes were mis-spliced and of those only 144 were differentially expressed. This group was significantly enriched for genes involved in mRNA processing and Jak (Janus kinase)/Stat (Signaling transducer and activator of transcription) signaling. In particular, target genes of the Jak/Stat pathway were significantly enriched in the downregulated gene list and many components of the signaling pathway were mis-spliced including il6st (interleukin 6 signal transducer), the gene encoding the IL6-family common receptor Gp130 (Glycoprotein 130).To determine if mis-splicing of il6st was sufficient to decrease HSC production, we injected antisense morpholinos into wild-type zebrafish to mimic the sf3b1 mutant - specific exon-skipping event in il6st and found significantly diminished HSC levels at 28 hpf. Stat3 is the major transcription factor downstream of Gp130, thus we ubiquitously overexpressed a constitutively-active form of Stat3 and found it could significantly suppress the HSC defects in sf3b1 mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Mutations in SF3B1 are prevalent in myelodysplastic syndrome (MDS), a disorder originating in HSC dysfunction. Altered STAT3 signaling is also common in MDS, thus our data suggest that SF3B1 disruptions in HSCs could be contributing to MDS via mis-regulation of JAK/STAT signaling. DisclosuresNo relevant conflicts of interest to declare.

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.

Transcriptional and epigenetic control of hematopoietic stem cell fate decisions in vertebrates

Hematopoietic stem cells (HSCs) are the foundation of adult hematopoiesis that produce all types of mature blood lineages. In vertebrates, HSC development is a stepwise process, coordinately regulated by chromatin architectures and a group of transcriptional and epigenetic regulators. A deeper understanding of the molecular mechanisms governing the generation, expansion, and function of HSCs holds great promise in the generation and expansion of engraftable HSCs in vitro for clinical applications. This study reviewed recent advances in transcriptional and epigenetic control of hematopoietic stem cell fate decisions in vertebrates.

3D Scaffolds to Model the Hematopoietic Stem Cell Niche: Applications and Perspectives.

Hematopoietic stem cells (HSC) are responsible for the production of blood and immune cells during life. HSC fate decisions are dependent on signals from specialized microenvironments in the bone marrow, termed niches. The HSC niche is a tridimensional environment that comprises cellular, chemical, and physical elements. Introductorily, we will revise the current knowledge of some relevant elements of the niche. Despite the importance of the niche in HSC function, most experimental approaches to study human HSCs use bidimensional models. Probably, this contributes to the failure in translating many in vitro findings into a clinical setting. Recreating the complexity of the bone marrow microenvironment in vitro would provide a powerful tool to achieve in vitro production of HSCs for transplantation, develop more effective therapies for hematologic malignancies and provide deeper insight into the HSC niche. We previously demonstrated that an optimized decellularization method can preserve with striking detail the ECM architecture of the bone marrow niche and support HSC culture. We will discuss the potential of this decellularized scaffold as HSC niche model. Besides decellularized scaffolds, several other methods have been reported to mimic some characteristics of the HSC niche. In this review, we will examine these models and their applications, advantages, and limitations.

Immunometabolic control of hematopoiesis

Hematopoietic stem cells (HSC) lie at the center of the hematopoiesis process, as they bear capacity to self-renew and generate all hematopoietic lineages, hence, all mature blood cells. The ability of HSCs to recognize systemic infection or inflammation or other forms of peripheral stress, such as blood loss, is essential for demand-adapted hematopoiesis. Also of critical importance for HSC function, specific metabolic cues (e.g., associated with changes in energy or O2 levels) can regulate HSC function and fate decisions. In this regard, the metabolic adaptation of HSCs facilitates their switching between different states, namely quiescence, self-renewal, proliferation and differentiation. Specific metabolic alterations in hematopoietic stem and progenitor cells (HSPCs) have been linked with the induction of trained myelopoiesis in the bone marrow as well as with HSPC dysfunction in aging and clonal hematopoiesis of indeterminate potential (CHIP). Thus, HSPC function is regulated by both immunologic/inflammatory and metabolic cues. The immunometabolic control of HSPCs and of hematopoiesis is discussed in this review along with the translational implications thereof, that is, how metabolic pathways can be therapeutically manipulated to prevent or reverse HSPC dysfunction or to enhance or attenuate trained myelopoiesis according to the needs of the host.

Mitochondrial Fate of Regenerative Hematopoietic Stem Cells Is Sequentially Controlled By Two Specific Conformations of Connexin-43

Restricted mitochondrial metabolism with low mitochondrial reactive oxygen species (ROS) and membrane potential are essential properties of repopulating hematopoietic stem cells (HSC). Upon regenerative stress, as found after chemotherapy and/or radiotherapy, HSC exit quiescence, proliferate and differentiate into mature blood cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity on HSC. Recent evidence demonstrates that the coordinated regulation of mitochondrial dynamics and the clearance of damaged mitochondria are the critical determinants of HSC fate decisions. Upon myeloablative stress, hematopoietic connexin 43 (H-Cx43), a major component of the gap junctions (GJ) present in the cell, lysosome and mitochondrial membranes, preserves the survival and efficient blood formation of regenerating HSC and progenitors (HSPC) by the transfer of damaging excess ROS, preventing HSPC apoptosis and lethal hematology failure. The protective role of H-Cx43 depends on the regulation of cell-contact dependent mitochondrial transfer to BM mesenchymal stromal cells. Mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission, fusion and lysosome dependent mitophagy. We hypothesized that hematopoietic Cx43 may exert a mitochondrial autonomous activity affecting the ability of HSC to regenerate. We created HSC mitochondrial reporter mice with hematopoietic deficiency of Cx43(H-Cx43D/D) and analyzed mitochondrial dynamics and fate in quiescent and dividing HSC. While quiescent Cx43D/D HSC function normally, Cx43 deficiency results in increased mitochondrial ROS and membrane depolarization in cycling HSC. Time lapsed imaging of photo-converted mitochondria indicate that mitochondria of Cx43D/D cycling HSC split into highly-motile, smaller fragments. Interestingly, the activating phosphorylation (Ser616) of the mitochondrial fission protein, Drp1 and its accumulation within mitochondria is higher in Cx43D/D dividing HSC. The recruitment of Drp1 to mitochondria is regulated by mitochondrial membrane adaptors Mff and Fis1. Expression of Fis1, but not Mff, is significantly increased in Cx43D/D cycling HSC. In contrast, the components of mitochondrial fusion machinery Mfn2 and active Drp1 (phospho-Drp1-Ser637) are significantly attenuated in dividing Cx43D/D HSC, suggesting that HSC Cx43 promotes mitochondrial fusion and stability, and inhibits mitochondrial fragmentation. Increased mitochondrial fission in dividing Cx43D/D HSC facilitates mitophagy as indicated by increased co-localization of mitochondria with the ubiquitin kinase Pink1 which simultaneously recruits the E3 ubiquitin ligase Parkin, autophagosome p62 and Lc3, and the lysosomal membrane protein Lamp2 on the surface of dysfunctional mitochondria. Additionally, increased phosphorylation of Ampk (Tyr172) and Ulk1 (Ser555) in mitochondria of cycling Cx43D/D HSC demonstrate that H-Cx43 is a negative regulator of Ampk dependent mitophagy in diving HSC. Inhibition of the Drp1 GTPase activity by expression of the dominant negative Drp1-K38A mutant prevents mitochondrial fragmentation, motility and mitophagy of dividing Cx43D/D HSC, confirming that the inhibitory effect on mitophagy of Cx43 depends on its role on mitochondrial fission. Expression of Cx43 structure-function mutants (cys-less mutant with impaired head-to-head hemichannel docking, but not hemichannel function; and C-terminus truncated D257 mutant with impaired signaling and intramolecular interactions needed for channel gating) in H-Cx43D/D HSC demonstrated that the negative regulatory role of Cx43 on mitochondrial fission requires functional Cx43 hemichannels while the constitutive inhibitory effect of H-Cx43 on mitophagy depends on the formation of complete functional GJ channels. Our results identify for first time the sequential role of two distinct conformations of mitochondrial H-Cx43 dependent channels on the control of mitochondrial fate: fission and mitophagy, in cycling HSC. This data provides novel targets for ex-vivo intervention to preserve HSC activity by transfer of genetically manipulated mitochondria. Figure Disclosures Cancelas: TerumoBCT: Consultancy, Research Funding; Cerus Corp: Research Funding; Hemanext Inc.: Consultancy, Research Funding; Velico LLC: Consultancy, Research Funding; Cytosorbents: Research Funding; Westat Inc: Consultancy, Research Funding; US DoD: Research Funding; NIH: Consultancy, Research Funding.

Physiological Cues Involved in the Regulation of Adhesion Mechanisms in Hematopoietic Stem Cell Fate Decision.

Hematopoietic stem cells (HSC) could have several fates in the body; viz. self-renewal, differentiation, migration, quiescence, and apoptosis. These fate decisions play a crucial role in maintaining homeostasis and critically depend on the interaction of the HSCs with their micro-environmental constituents. However, the physiological cues promoting these interactions in vivo have not been identified to a great extent. Intense research using various in vitro and in vivo models is going on in various laboratories to understand the mechanisms involved in these interactions, as understanding of these mechanistic would greatly help in improving clinical transplantations. However, though these elegant studies have identified the molecular interactions involved in the process, harnessing these interactions to the recipients’ benefit would ultimately depend on manipulation of environmental cues initiating them in vivo: hence, these need to be identified at the earliest. HSCs reside in the bone marrow, which is a very complex tissue comprising of various types of stromal cells along with their secreted cytokines, extra-cellular matrix (ECM) molecules and extra-cellular vesicles (EVs). These components control the HSC fate decision through direct cell–cell interactions – mediated via various types of adhesion molecules –, cell-ECM interactions – mediated mostly via integrins –, or through soluble mediators like cytokines and EVs. This could be a very dynamic process involving multiple transient interactions acting concurrently or sequentially, and the adhesion molecules involved in various fate determining situations could be different. If the switch mechanisms governing these dynamic states in vivo are identified, they could be harnessed for the development of novel therapeutics. Here, in addition to reviewing the adhesion molecules involved in the regulation of HSCs, we also touch upon recent advances in our understanding of the physiological cues known to initiate specific adhesive interactions of HSCs with the marrow stromal cells or ECM molecules and EVs secreted by them.

A Unique Crosstalk between Tumor Cells and Hematopoietic Stem Cells Reveals a Myeloid Differentiation Pattern Signature Contributing to Metastasis

Metastasis is the major cause of death in cancer patients. Recent studies have demonstrated that the crosstalk between different host and tumor cells in the tumor microenvironment regulates tumor progression and metastasis. Specifically, immune cell myeloid skewing is a prominent promoter of metastasis. While previous studies have demonstrated that the recruitment of myeloid cells to tumors is a critical step in dictating tumor fate, the reservoir of these cells in the bone marrow (BM) compartment and their differentiation pattern has not been explored. Here we utilized a unique model system consisting of tumor cell clones with low and high metastatic potential (met-low and met-high, respectively) derived from melanoma and breast carcinoma cell lines. Hematopoietic stem cells (HSCs) and their early progenitor subset were defined as Lin-/Sca1+/CD117+, representing LSK cells. BM transplantation experiments using GFP-positive LSK cells derived from met-low and met-high tumor bearing mice were carried out to study lineage differentiation. The genetic signatures of LSK cells were analyzed by single cell RNA-sequencing (scRNA-seq). This analysis included unbiased automated annotation of individual cell types by correlating single-cell gene expression with reference transcriptomic data sets (SingleR algorithm) in order to evaluate the proportions of cell types in BM and reveal cell type-specific differentially expressed genes. Expression patterns of proteins originated from tumor cells were analyzed using a range of multi-omics techniques including nanostring, protein array, and mass spectrometry analysis. Tumor proteomic data was integrated with differential receptor expression patterns in BM cell types to reveal novel crosstalk between tumor cells and HSCs in the BM compartment. Mice bearing met-high tumors exhibited a significant increase in the percentage of LSK cells in the BM in comparison to tumor-free mice or mice bearing met-low tumors. These results were confirmed by functional CFU assays of peripheral blood of met-high compared to met-low tumor bearing mice. In addition, mice that underwent BM transplantation with GFP-positive LSK cells obtained from met-high inoculated donors exhibited an increased percentage of circulating GFP-positive myeloid cells in comparison to counterpart mice transplanted with LSK cells from met-low inoculated donors. Moreover, scRNA-seq analysis of LSK cells obtained from the BM of met-low and met-high tumor bearing mice revealed that met-high tumors induce the enrichment of monocyte-dendritic progenitor population (MDP), confirmed also by flow cytometry. To uncover the possible factors involved in myeloid programming of LSK cells, we performed a proteomic screen of tumor conditioned medium and integrated the results with the scRNA-seq data analysis. This analysis revealed that the IL-6-IL-6R axis is highly active in LSK-derived MDP cells from mice bearing met-high tumors. An adoptive transfer experiment using MDP-GFP+ cells obtained from BM of met-high tumor bearing mice demonstrated that met-high tumors directly dictate HSC fate decision towards myeloid bias, resulting in increased metastasis. Evidently, blocking IL-6 in mice bearing met-high tumors reduced the number of MDP cells, and consequently decreased metastasis. Our study reveals a unique crosstalk between tumor cells and HSCs. It provides new insight into the mechanism by which tumors contribute to the presence of supporting stroma. Specifically, tumors secreting IL-6 dictate a specific genetic signature in HSCs that programs them towards myeloid differentiation, thereby inducing a metastatic switch. Disclosures No relevant conflicts of interest to declare.

Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs

AbstractA major limitation preventing in vivo modulation of hematopoietic stem cells (HSCs) is the incomplete understanding of the cellular and molecular support of the microenvironment in regulating HSC fate decisions. Consequently, murine HSCs cannot be generated, maintained, or expanded in culture over extended periods of time. A significantly improved understanding of the bone marrow niche environment and its molecular interactions with HSCs is pivotal to overcoming this challenge. We here prospectively isolated all major nonhematopoietic cellular niche components and cross-correlate them in detail with niche cells defined by lineage marking or tracing. Compiling an extensive database of soluble and membrane-bound ligand-receptor interactions, we developed a computational method to infer potential cell-to-cell interactions based on transcriptome data of sorter-purified niche cells and hematopoietic stem and progenitor cell subpopulations. Thus, we establish a compendium of the molecular communication between defined niche components and HSCs. Our analysis suggests an important role for cytokine antagonists in the regulation of HSC functions.

Inflamm-aging of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are responsible for generating all blood cells throughout life. Apart from the role of HSCs in maintaining the homeostasis of blood cell production process, they must respond quickly to hematopoietic challenges, such as infection or blood loss. HSCs can be directly/indirectly activated and engage in blood formation for the acute needs in response to inflammation. Recent findings highlight the emerging role of inflammation signaling on HSC fate decision and shaping the hematopoietic system during aging. Here, we summarize recent studies identifying the changes in inflammation and their role in modulation of HSC function and discuss the interaction between inflammation and HSC biology in the contexts of aging and hematological malignancy.