Differentiation Of HSCs Research Articles

Ex vivo expansion of hematopoietic stem cells in two/ three-dimensional co-cultures with various source of stromal cells

The ex vivo expansion of hematopoietic stem cells, with both high quantities and quality, is considered a paramount issue in cell and gene therapy for hematological diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells reveal the importance of using 2D and 3D coculture as a physiological system simulator in the proliferation, differentiation, and homeostasis of HSCs. Herein, the capacity of mesenchymal stem cells derived from different sources to support the expansion and maintenance of HSPC was compared with each other. We evaluated the fold increase of HSPC, CD34 marker expression, cytokine secretion profile of different MSCs, and the frequency of hematopoietic colony-forming unit parameters. Our results show that there was no significant difference between adipose tissue-MSC, Wharton jelly-MSC, and Endometrial-MSCs in HSPC expansion (fold increase: 34.74±4.38 in Wj-MSC, 32.22±5.07 in AD-MSC, 25.9±1.27 in En-MSCs); However, there were significantly more than the expansion media alone (4.4±0.69). The results obtained from the cytokine secretion analysis also confirm these results. Also, there were significant differences in the clonogenicity of Wj-MSC, En-MSCs, and expansion media (CFU-GEMM: 7±1.73, 2.3±1.15, and 2.3±1.52), which indicated that Wj-MSC could significantly maintain the primitive state. As a result, using Wj-mesenchymal stem cells on a 3D coculture system effectively increases the HSPC expansion and maintains the colonization potential of hematopoietic stem cells.

Oncostatin M (OSM) Regulates an Inflammatory Cytokine Network in Dnmt3a-Mutant Hematopoietic Stem Cells

During aging, somatic mutations are known to accumulate in the hematopoietic stem and progenitor cell (HSPC) compartment. While most of these somatic mutations are considered ‘neutral’, a subset can confer a selective growth advantage to HSPCs, leading to a condition termed age-associated clonal hematopoiesis (CH). While CH is not a disease per se, it is associated with increased risk of age-associated pathologies such as blood cancers and ischemic stroke. Understanding the cellular and molecular mechanisms underlying the selective advantage of HSPCs harboring somatic mutations, and how and in whom this is a risk factor for age-associated disease, will further inform our understanding of CH-associated conditions. Using an inducible Dnmt3a-mutant mouse model of CH ( Dnmt3a R878H/+), we previously reported that transplant of Dnmt3a-mutant HSCs into 14-month-old recipient mice promotes their selective growth advantage compared to transplant into 2-month-old recipient mice (SanMiguel et al., Cancer Discovery 2022). This led us to hypothesize that differences in soluble factors in the middle-aged versus young bone marrow context contribute to Dnmt3a-mutant HSC expansion. We began with re-analysis of our HSC RNA-seq data from that study, identifying a hallmark inflammatory response signature in Dnmt3a-mutant versus control HSCs in middle-aged but not in young recipient mice. Examining this gene signature, we found that the top gene encoding a soluble factor and increased in expression in Dnmt3a-mutant HSCs was Oncostatin M ( Osm), anIL-6 family cytokine that activates JAK/STAT signaling and is involved in the immunopathogenesis of myeloma. Given that activation of OSM signaling is associated with expanded Dnmt3a-mutant hematopoiesis in the middle-aged BM microenvironment, we hypothesized that OSM as a single stimulus would be sufficient to promote the selective advantage of young Dnmt3a-mutant HSPCs. We found that recombinant OSM did not result in altered proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation of young Dnmt3a-mutant HSCs. Despite this, we found that young Dnmt3a-mutant HSCs do transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation as evidenced by significant upregulation of the genes encoding IL-6, IL-1β and TNFα. OSM-stimulated Dnmt3a-mutant HSCs also demonstrate upregulation of the anti-inflammatory genes Socs3, Atf3 and Nr4a1 which creates a negative feedback loop that limits sustained activation of the inflammatory network. In the context of an aged bone marrow context with chronically elevated levels of OSM, Dnmt3a-mutant HSCs upregulate pro-inflammatory genes but not the anti-inflammatory genes Socs3, Atf3 and Nr4a1. The results from our studies suggest that chronic inflammation with aging exhausts the regulatory mechanisms that HSCs in young mice use to resolve inflammatory states and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH.

Abstract 28 A Novel Chemically Defined Culturing System to Support Hematopoietic Stem Cell Expansion

Abstract Introduction Hematopoietic stem cells (HSCs) are known to be multipotent and can reconstitute the entire blood and immune system following HSC transplantation (HSCT). However, the current HSCTs have many unresolved issues including difficulties in finding donors for bone marrow transplantation, preparing a sufficient HSC dose in cord blood transplantation, and the risk of graft-versus-host disease (GvHD) for all allogeneic HSCTs. In recent years, there have been substantial efforts to research and understand the biology of HSCs to develop cell therapy products that meet these challenges and provide an alternative to traditional HSCTs. Several culturing methods have been developed to stimulate HSC expansion ex vivo, but the use of biological materials, such as albumin, has not been ideal for pharmaceutical manufacturing, and it has been difficult inhibiting differentiation of HSCs into lineage-committed cells while culturing in a cytokine-filled medium. We have developed a novel albumin-free culturing system that supports proliferation of long-term HSCs ex vivo by replacing cytokines and albumin with chemical agonists and a caprolactam-based polymer. Objectives The objective of this study is to characterize our expanded cell population as part of an analysis to determine whether the population reconstitutes bone marrow and is therefore suitable for development of a cell therapy product. Methods Here we report the in vitro and in vivo characterization of proliferated HSCs using our novel culturing system compared with a nicotinamide system which is known to support HSC proliferation ex vivo. A colony forming assay, cell surface marker analysis using a flow cytometer, and in vivo repopulating analysis using the NOD/Shi-scid IL-2Rγnull (NOG) mouse were conducted. Results Surface marker analysis of the proliferated population shows more than 70% remaining as undifferentiated CD34+. Of which, a higher number of CD201+CD90+CD45RA- exists compared with the nicotinamide-cultured population. Colony forming unit assays indicate that our proliferated cell population contains a higher number of multipotent progenitors. The repopulating cell assay also shows that the proliferated cells have bone marrow reconstitution capability in bone marrow-ablated NOG mice. Discussion We believe that our chemically defined culturing system can selectively proliferate HSCs while restraining their differentiation and will contribute to the development of novel HSC therapy products.

Fanca deficiency is associated with alterations in osteoclastogenesis that are rescued by TNFα

BackgroundHematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, which includes bone-forming and bone-resorbing cells, i.e., osteoblasts (OBs) and osteoclasts (OCs). OBs originate from mesenchymal progenitors, while OCs are derived from HSCs. Self-renewal, proliferation and differentiation of HSCs are under the control of regulatory signals generated by OBs and OCs within the BM niche. Consequently, OBs and OCs control both bone physiology and hematopoiesis. Since the human developmental and bone marrow failure genetic syndrome fanconi anemia (FA) presents with skeletal abnormalities, osteoporosis and HSC impairment, we wanted to test the hypothesis that the main pathological abnormalities of FA could be related to a defect in OC physiology and/or in bone homeostasis.ResultsWe revealed here that the intrinsic differentiation of OCs from a Fanca−/− mouse is impaired in vitro due to overactivation of the p53–p21 axis and defects in NF-kB signaling. The OC differentiation abnormalities observed in vitro were rescued by treating Fanca−/− cells with the p53 inhibitor pifithrin-α, by treatment with the proinflammatory cytokine TNFα or by coculturing them with Fanca-proficient or Fanca-deficient osteoblastic cells.ConclusionsOverall, our results highlight an unappreciated role of Fanca in OC differentiation that is potentially circumvented in vivo by the presence of OBs and TNFα in the BM niche.

BATF2 promotes HSC myeloid differentiation by amplifying IFN response mediators during chronic infection

Basic leucine zipper ATF-like transcription factor 2 (BATF2), an interferon-activated immune response regulator, is a key factor responsible for myeloid differentiation and depletion of HSC during chronic infection. To delineate the mechanism of BATF2 function in HSCs, we assessed Batf2 KO mice during chronic infection and found that they produced less pro-inflammatory cytokines, less immune cell recruitment to the spleen, and impaired myeloid differentiation with better preservation of HSC capacity compared to WT. Co-IP analysis revealed that BATF2 forms a complex with JUN to amplify pro-inflammatory signaling pathways including CCL5 during infection. Blockade of CCL5 receptors phenocopied Batf2 KO differentiation defects, whereas treatment with recombinant CCL5 was sufficient to rescue IFNγ-induced myeloid differentiation and recruit more immune cells to the spleen in Batf2 KO mice. By revealing the mechanism of BATF2-induced myeloid differentiation of HSCs, these studies elucidate potential therapeutic strategies to boost immunity while preserving HSC function during chronic infection.

Fecal microbiota transplantation from young mice rejuvenates aged hematopoietic stem cells by suppressing inflammation

AbstractHematopoietic stem cell (HSC) aging is accompanied by hematopoietic reconstitution dysfunction, including loss of regenerative and engraftment ability, myeloid differentiation bias, and elevated risks of hematopoietic malignancies. Gut microbiota, a key regulator of host health and immunity, has recently been reported to affect hematopoiesis. However, there is currently limited empirical evidence explaining the direct impact of gut microbiome on aging hematopoiesis. In this study, we performed fecal microbiota transplantation (FMT) from young mice to aged mice and observed a significant increment in lymphoid differentiation and decrease in myeloid differentiation in aged recipient mice. Furthermore, FMT from young mice rejuvenated aged HSCs with enhanced short-term and long-term hematopoietic repopulation capacity. Mechanistically, single-cell RNA sequencing deciphered that FMT from young mice mitigated inflammatory signals, upregulated the FoxO signaling pathway, and promoted lymphoid differentiation of HSCs during aging. Finally, integrated microbiome and metabolome analyses uncovered that FMT reshaped gut microbiota composition and metabolite landscape, and Lachnospiraceae and tryptophan-associated metabolites promoted the recovery of hematopoiesis and rejuvenated aged HSCs. Together, our study highlights the paramount importance of the gut microbiota in HSC aging and provides insights into therapeutic strategies for aging-related hematologic disorders.

CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage

Recently, Toll-like receptors (TLRs) have been extensively studied in radiation damage, but the inherent defects of high toxicity and low efficacy of most TLR ligands limit their further clinical transformation. CRX-527, as a TLR4 ligand, has rarely been reported to protect against radiation. We demonstrated that CRX-527 was safer than LPS at the same dose in vivo and had almost no toxic effect in vitro. Administration of CRX-527 improved the survival rate of total body irradiation (TBI) to 100% in wild-type mice but not in TLR4-/- mice. After TBI, hematopoietic system damage was significantly alleviated, and the recovery period was accelerated in CRX-527-treated mice. Moreover, CRX-527 induced differentiation of HSCs and the stimulation of CRX-527 significantly increased the proportion and number of LSK cells and promoted their differentiation into macrophages, activating immune defense. Furthermore, we proposed an immune defense role for hematopoietic differentiation in the protection against intestinal radiation damage, and confirmed that macrophages invaded the intestines through peripheral blood to protect them from radiation damage. Meanwhile, CRX-527 maintained intestinal function and homeostasis, promoted the regeneration of intestinal stem cells, and protected intestinal injury from lethal dose irradiation. Furthermore, After the use of mice, we found that CRX-527 had no significant protective effect on the hematopoietic and intestinal systems of irradiated TLR4-/- mice. in conclusion, CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage.

CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant.

Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2-/- and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2-/- cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk-/- mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.

Loss of De Novo DNA Methyltransferase DNMT3A Impacts Alternative Splicing in Hematopoietic Stem Cells

DNMT3A is a de novo DNA methyltransferase that is crucial to the maintenance of normal hematopoiesis. DNMT3A is frequently mutated in hematopoietic diseases, with mutations reported in over 20% of adult acute myeloid leukemia (AML), and 10-15% of myelodysplastic syndrome (MDS) patients. Our lab has shown that loss of Dnmt3a in mice leads to enhanced self-renewal of the HSC compartment and a block in differentiation, contributing to its role in clonal hematopoiesis (Challen et al. 2011). We have also shown that loss of DNMT3A leads to a spectrum of hematologic malignancies; however, changes in DNA methylation alone are insufficient to explain the leukemic transformation, suggesting that DNMT3A could also regulate stem cells independent of methylation. The mechanisms through which DNMT3A exerts its specific effects are unclear, in part due to our relatively poor understanding of its interactome.To address this gap in knowledge, we generated a doxycycline-inducible biotinylated-DNMT3A transgene in mouse ES cells. Using co-immunoprecipitation and mass spectrometry analysis of biotinylated-DNMT3A in murine ESCs, we identified several candidate interacting proteins. Along with known partners such as DNMT3L and DNMTB, factors associated with chromatin regulation (HDACs, NuRD complex), splicing factors (SF proteins, Hnrnps, and DDXs), and ribosomes (RPSs and RPLs) were pulled down. We validated these interactions by over-expressing myc-tagged DNMT3A in HEK293T cells. Since epigenetic regulators and splicing factors are recurrently co-mutated in hematologic malignancies, we validated its interaction with splicing factors such as DDX39B, SF3B3, and SF3B1 (among others) through co-IP of DNMT3A in CD34+ HSPCs. Furthermore, we found that these interactions are RNA and DNA-independent. N-terminal deletions of DNMT3A domains revealed that the catalytic domain is crucial for its interaction with splicing factors, and AML mutant, Arg882His, disrupts this interaction. These data suggest that through its interaction with splicing factors, DNMT3A may have a crucial role in RNA splicing efficacy.To determine how the putative interaction between DNMT3A and splicing factors affects hematopoiesis, we examined whether loss of Dnmt3a impacted RNA splicing in purified HSCs after competitive transplant using our Mx1-cre-inducible Dnmt3a knockout mouse model. Using intron retention as a measure of splicing efficacy, we performed deep RNA sequencing of poly-A-enriched mRNA from HSCs (LSK CD150+ CD48-). When comparing exon-intron junctions in Dnmt3a KO versus WT HSCs, Dnmt3a KO HSCs exhibited a greater degree of intron retention. Aberrant alternative splicing was observed in genes involved in HSC differentiation and self-renewal, splicing, ribosomal biogenesis, and cell cycle regulation, suggesting that its role in RNA splicing may be an important mechanism through which DNMT3A influences hematopoiesis. In addition, we delineate a key relationship between DNMT3A, intron retention, and HSC differentiation. Inducing differentiation of WT and Dnmt3a KO HSCs with 5-Fluorouracil (5-FU) revealed key differences in alternative splicing. As WT HSCs differentiate, intron retention decreases; however, Dnmt3a KO HSCs are unable to decrease intron retention in key hematopoietic differentiation genes, leading to a block in differentiation. This was also demonstrated after inducing differentiation in WT and Dnmt3a KO mouse ESCs with Retinoic Acid (RA).To determine if loss of DNMT3A also impacts splicing in human HSPCs, we used CRISPR/Cas9 to knock out DNMT3A in human HSPCs and transplanted CD34+ cells into NSG mice. RNA sequencing of CD34+ cells 3 months after transplantation revealed aberrant alternative splicing, specifically, greater intron retention in DNMT3A KO than control cells. Together, these data suggest that DNMT3A plays a crucial role in maintaining proper splicing in HSCs.In summary, this study reveals a novel role for DNMT3A in hematopoiesis. We show that DNMT3A interacts with splicing factors and impacts global alternative splicing patterns in HSCs. In addition, we demonstrate that decreasing intron retention has a specific role in HSC differentiation. The aberrant splicing patterns seen in Dnmt3a KO HSCs may be instrumental in decrypting the crosstalk between DNA methylation and alternative splicing, and their role in HSC differentiation. DisclosuresNo relevant conflicts of interest to declare.

Novel Roles of Separase in Hematopoiesis

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. In recent years, cancer genome sequencing has revealed several loss of function cohesin mutations in early stages of myeloid, hematologic and epithelial malignancies. These mutations affect the self-renewal and differentiation capacity of HSCs indicating cohesin as a tumor suppressor. Our prior studies indicated that Separase haploinsufficiency acts synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas. In contrast to cohesin proteins, the function of the cohesin protease, Separase in hematopoiesis and hematological malignancies has not been well defined.Here we examined the effect of Espl1 heterozygosity using our hypomorphic mouse model (mEspl1 +/hyp ) that has reduced germline Separase activity. The impact of heterozygous loss of mEspl1 allele on total number of HSCs was analyzed by staining with hematopoietic stem and progenitor cells (HSPCs) markers and FACS analysis of cells isolated from bone marrow. Our analysis showed that Separase mutant mice have significantly increased (~2 fold) immunophenotypic HSPCs compared to wild type littermates. To examine whether reduced Separase level elevates self-renewal capacity of HSCs in vivo , we performed competitive transplantation assays to compare differentiation and self-renewal capacity of HSCs in mEspl1+/hyp and WT littermate controls. The outcomes with significantly higher donor derived reconstitution suggested an expansion of HSPCs compartment in mutant mice. In addition, we also observed an altered hematopoietic differentiation with an increase in percentage of donor-derived T- and B-cells in Separase mutant mice. The increased number of HSCs could be a consequence of change in autonomous function of the cells due to reduced level of Separase. To test the cell autonomous function of mEspl1+/hyp , in vitro colony forming unit (CFU) assays were performed in methylocellulose medium. mEspl1+/hyp cells showed significantly higher serial re-plating capacity compared to WT littermate control cells, supporting the notion of cell autonomous function as a probable cause of the observed phenotype.Considering the role of cohesin on gene expression, the effect of heterozygous loss of Separase was analyzed with global transcriptional profiling. Several hematopoietic fingerprint genes like Tle1, Smarca1, Scl2a10, H19, Krt2-8 and Galnt3 were found upregulated. Other genes important in HSC function such as Runx1, Satb1, Hoxa9 and Pbx1 were also upregulated in mEspl1+/hyp mice. The transcriptional profiling identified genes involved in several biological processes such as leukocyte differentiation, cell apoptosis, and chromatin modifications. However, mechanism and direct gene targets of Separase in hematopoiesis or other biological pathways are yet to be identified. To study further on transcriptional regulatory roles and to characterize the direct targets of Separase, ChIP-Seq (cohesin and Separase) and RNA-Seq experiments are being performed in WT and mEspl1+/hyp HSCs.With these studies we hope to better understand the role of cohesin in association with Separase on hematopoiesis particularly the mechanisms that control the fate of HSCs, bone marrow engraftment and function; how cohesin mutations may give rise to malignant transformation, and if this pathway can be targeted for therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Liver type 1 innate lymphoid cells develop locally via an interferon-γ-dependent loop.

The pathways that lead to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. We show here that the adult mouse liver contains Lin-Sca-1+Mac-1+ hematopoietic stem cells derived from the fetal liver. This population includes Lin-CD122+CD49a+ progenitors that can generate liver ILC1s but not conventional natural killer cells. Interferon-γ (IFN-γ) production by the liver ILC1s themselves promotes the development of these cells in situ, through effects on their IFN-γR+ liver progenitors. Thus, an IFN-γ-dependent loop drives liver ILC1 development in situ, highlighting the contribution of extramedullary hematopoiesis to regional immune composition within the liver.

Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BackgroundHematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale.ResultsThrough the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state.ConclusionsOur results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

Single Cell Resolution Mapping of Hematopoietic Stem and Progenitor Cell States throughout Human Life

The hematopoietic system continuously generates and replenishes the supply of circulating blood cells from embryonic life throughout the entirety of human lifespan. Studies in mouse development have shown that the repertoire of mature blood cell types produced changes dramatically during development and aging, with hematopoietic stem and progenitor cells (HSPCs) adapting their output to meet age-specific physiologic needs. In humans, it is presumed that age-dependent changes in the production of mature blood lineages underlie the tendency of blood disorders to skew toward certain ages of onset. The observations that mature cell output changes throughout life but mechanisms of terminal hematopoietic differentiation within each lineage remain consistent suggest that age-specific hematopoietic states are programmed at the level of HSPCs. Although the developmental changes occurring in mouse hematopoiesis are well documented, the specific changes in human HSPC ontogeny occurring during prenatal development and postnatal aging from newborn, through childhood, and into adulthood are completely unknown. We hypothesized that temporal changes in human hematopoiesis are mediated by age-specific, occasionally transient, HSPC states and that mechanisms of HSPC lineage commitment change over time in order to meet the changing physiologic demands of the developing and aging human. To test this hypothesis, we comprehensively profiled human HSPC cell states from human fetal hematopoiesis through adulthood using single cell RNA sequencing (scRNAseq). We obtained CD34+ HSPCs from 14 different human donors covering a range of ages from first and second trimester fetal liver, umbilical cord blood, and pediatric and adult bone marrow. We obtained high quality sequencing data on a total of 38,873 individual HSPCs after filtering out apoptotic cells, hepatocytes, stromal cells, and endothelial cells. We then identified differentially expressed genes and performed dimensionality reduction and uniform manifold approximation and projection followed by Louvain clustering to identify 32 distinct cell types encompassing primitive stem and multipotent progenitor cells as well as committed progenitors in the myeloid, erythroid and lymphoid lineages. We found age-specific alterations in hematopoietic differentiation trajectories, particularly in the myeloid lineages. Additionally, we discovered an HSC, emerging in mid-gestation and diminishing at birth, with a characteristic immunophenotype and megakaryocyte (Meg) differentiation bias. Differential gene expression analysis of the Meg-biased fetal HSC identified increased expression of the MYB transcription factor relative to other HSCs, potentially illuminating a mechanistic role for MYB in driving megakaryocyte-erythroid progenitor (MEP) differentiation of fetal HSCs, that is distinct from the role of MYB in conferring erythroid differentiation bias at the MEP stage itself. Finally, we used this atlas of human developmental hematopoiesis to map lineage commitment and progenitor states in leukemia, highlighting the translational applicability of this resource. In summary, we have compiled the first comprehensive atlas of HSPCs across human development and aging. This resource allowed us to identify age-specific differentiation trajectories in human hematopoiesis and enabled identification of a Meg-biased fetal-specific HSC. Our research reveals novel mechanisms of maturation and aging of the human hematopoietic system, uncovers transient HSPC states and differentiation trajectories, and establishes a framework for interrogating the differentiation and maturation states of human leukemias that can likely be applied to other blood diseases. As a resource, we expect that this atlas will broadly impact the study of human hematopoietic development and aging, developmental immunology, and the pathophysiology of age-biased blood diseases. Disclosures No relevant conflicts of interest to declare.

Highly Efficient Gene Editing of Human Hematopoietic Stem Cells to Treat X-Linked Sideroblastic Anemia

X-linked sideroblastic anemia (XLSA) is an anemic disease caused by mutations in the gene encoding 5-aminolevulinate synthase 2 (ALAS2), which catalyzes the rate-limiting step of heme biosynthesis. With current interventions including pyridoxine treatment and allogeneic hematopoietic stem cell transplantation, severe unmet needs remain for patients with XLSA. Here, we used CRISPR/Cas9 technology to efficiently and functionally correct the pathogenic mutation in intron 1 of ALAS2 in CD34+ hematopoietic stem cell and progenitor cells (HSPCs) from two patients. Intriguingly, we found that the gene-editing efficiency of CD34+HSPCs from the elder patient was much lower than that from the younger patient, consistent with the poor hematopoiesis of older XLSA patients observed in clinical practice. Furthermore, we performed single cell RNA-sequencing (scRNA-seq) to investigate the causes of those phenomenon in-depth. Previously we and others identified the A>G mutation in the GATA1 binding region of intron 1 of ALAS2 in particular XLSA families and have demonstrated the key role of this site in regulating ALAS expression. Thus, we first designed a series of sgRNAs, along with single-stranded DNA oligonucleotide donors (ssODN). After co-electroporating with Cas9 mRNA into patient-derived hiPSCs, sgRNA-1 and ssODN were selected for further experiments based on optimal correction rate (43.93±3.43% via HDR). Next, using a well-established erythroid protocol, CD34+ HSPCs of control and gene-edited groups were differentiated into erythroid cells in vitro. Surprisingly, heme biosynthesis examined by benzidine staining showed that compared with the mock cells, the gene-corrected group significantly increased the frequency of benzidine-positive cells. To examine the multilineage differentiation potential of gene-corrected CD34+ HSCPs, we performed colony-forming unit (CFU) assays to quantify various types of colonies. Compared with mock cells, gene-edited group significantly enhanced the generation of total, CFU-GM and BFU-E colonies, suggesting higher clonogenic potential. Next, gene-corrected CD34+ HSPCs were transplanted into nonobese diabetic (NOD)/Prkdcscid/IL-2Rγnull (NPG) mice to evaluate the repopulating potential. All transplanted mice displayed engraftment in multiple organs at 10-16 weeks post transplantation, and the gene-corrected cells showed greater engraftment potential than mock group. In addition, hematopoietic reconstitution analysis indicated that the gene-corrected cells maintained normal lineage distribution, while the B cell development of mock group was impaired. Moreover, gene-editing efficiency analysis of bone marrow samples 16 weeks after transplantation exhibited high editing rate (34±7.18% via HDR), comparable to the in vitro efficiency. The specificity of the Cas9 mRNA-based gene editing system was examined using unbiased Digenome-Seq. In total, 32 potential off-target sites were identified and deeply interrogated via targeted PCR and NGS analysis of XLSA iPSCs electroporated with Cas9 mRNA and sgRNA. No off-target cleavage events were detected at these sites, suggesting a lack of detectable off-target events. Finally, scRNA-seq of CD34+ HSPCs from healthy donor and XLSA patients revealed more HSC/LMPP and erythroid progenitor cells in older XLSA patient. Further analysis showed that cell cycle and gene expression in older HSC/LMPP cells were significantly different from that from healthy donors and younger patients. Hence, we speculated that the compensatory differentiation of HSCs caused by long-term functional red blood cell deficiency caused the abnormal expansion of HSCs, which led to the poor hematopoiesis in elderly patients. Our study firstly uses CRISPR/Cas9 gene-editing technology to correct the disease mutation in patient's CD34+ HSPCs and rescues ALAS2 expression and heme biosynthesis, directly confirming that this mutation is the pathogenic factor for XLSA. In addition, we dissect the transcriptional profile of CD34+ HSCPs from XLSA patients at single cell resolution for the first time, shedding light on mechanistic insights into the XLSA pathogenesis. The robust gene-correction rates and significant function rescue in patient's CD34+ HSPCs further suggest a curable option of gene-edited HSC transplantation for the treatment of the patients with XLSA. Disclosures Fang: EdiGene Inc.: Current Employment. Yuan:EdiGene Inc.: Current Employment. Yang:EdiGene Inc.: Current Employment. Yu:EdiGene Inc.: Current Employment. Zhang:EdiGene Inc.: Current Employment. Shi:EdiGene Guangzhou Inc.: Current Employment. Qi:Novogene Co, Ltd: Current Employment. Wei:EdiGene Inc.: Current Employment.

Cell-Extrinsic Stressors from the Aging Bone Marrow (BM) Microenvironment Promote Dnmt3a-Mutant Clonal Hematopoiesis

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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Foxp3+ Regulatory T Cells in Bone and Hematopoietic Homeostasis.

The bone represents surprisingly dynamic structures that are subject to constant remodeling by the concerted action of bone-forming osteoblasts and bone-resorbing osteoclasts - two cell subsets of distinct developmental origin that are key in maintaining skeletal integrity throughout life. In general, abnormal bone remodeling due to dysregulated bone resorption and formation is an early event in the manifestation of various human bone diseases, such as osteopetrosis/osteoporosis and arthritis. But bone remodeling is also closely interrelated with lympho-hematopoietic homeostasis, as the bone marrow niche is formed by solid and trabecular bone structures that provide a framework for the long-term maintenance and differentiation of HSCs (>blood lineage cells and osteoclasts) and MSCs (>osteoblasts). Numerous studies in mice and humans have implicated innate and adaptive immune cells in the dynamic regulation of bone homeostasis, but despite considerable clinical relevance, the exact mechanisms of such immuno-bone interplay have remained incompletely understood. This holds particularly true for CD4+ regulatory T (Treg) cells expressing the lineage specification factor Foxp3: Foxp3+ Treg cells have been shown to play an indispensable role in maintaining immune homeostasis, but may also exert critical non-immune functions, which includes the control of metabolic and regenerative processes, as well as the differentiation of HSCs and function of osteoclasts. Here, we summarize our current knowledge on the T cell/bone interplay, with a particular emphasis on our own efforts to dissect the role of Foxp3+ Treg cells in bone and hematopoietic homeostasis, employing experimental settings of gain- and loss-of-Treg cell function. These data make a strong case that Foxp3+ Treg cells impinge on lympho-hematopoiesis through indirect mechanisms, i.e., by acting on osteoclast development and function, which translates into changes in niche size. Furthermore, we propose that, besides disorders that involve inflammatory bone loss, the modulation of Foxp3+ Treg cell function in vivo may represent a suitable approach to reinstate bone homeostasis in non-autoimmune settings of aberrant bone remodeling.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation invitro and myeloid lineage-biased reconstitution invivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

Hematopoietic stem/progenitor cell differentiation towards myeloid lineage is modulated by LIGHT/LIGHT receptor signaling.

The cytokine LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a member of the tumor necrosis factor (TNF) superfamily. It is expressed primarily on activated T lymphocytes, and detectable on monocytes, granulocytes, and immune dendritic cells. It mainly plays a role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role as an inducer in embryonic stem cell differentiation, but its role in regulation of adult stem cell has not been defined. In the present study, we examined the expression of LIGHT receptor in Lin- c-kit+ Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte-monocyte colony stimulating factor (GM-CSF). Further studies showed that LIGHT enhances both GM-CSF and GM-CSF receptor (GM-CSFR) expression in HSCs. LIGHT stimulation increases PU.1 expression in HSC/HPCs. In vivo administration of LIGHT increases the colony-forming unit-granulocyte/monocyte (CFU-GM) colony formation and plasma GM-CSF level. Altogether, the data suggest LIGHT promote myeloid differentiation of HSC/HPCs.

HDAC Inhibitors Lead to Significant Survival Benefit in a Novel Fusion Protein TBLR1-Rarα Induced Murine Promyelocytic Leukemia Model

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL.Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice.Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn’t survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions.Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn’t benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What’ more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. DisclosuresNo relevant conflicts of interest to declare.