Primitive Hematopoietic Stem Cells Research Articles

Crosstalk between DNA Damage Repair and Metabolic Regulation in Hematopoietic Stem Cells.

Self-renewal and differentiation are two characteristics of hematopoietic stem cells (HSCs). Under steady physiological conditions, most primitive HSCs remain quiescent in the bone marrow (BM). They respond to different stimuli to refresh the blood system. The transition from quiescence to activation is accompanied by major changes in metabolism, a fundamental cellular process in living organisms that produces or consumes energy. Cellular metabolism is now considered to be a key regulator of HSC maintenance. Interestingly, HSCs possess a distinct metabolic profile with a preference for glycolysis rather than oxidative phosphorylation (OXPHOS) for energy production. Byproducts from the cellular metabolism can also damage DNA. To counteract such insults, mammalian cells have evolved a complex and efficient DNA damage repair (DDR) system to eliminate various DNA lesions and guard genomic stability. Given the enormous regenerative potential coupled with the lifetime persistence of HSCs, tight control of HSC genome stability is essential. The intersection of DDR and the HSC metabolism has recently emerged as an area of intense research interest, unraveling the profound connections between genomic stability and cellular energetics. In this brief review, we delve into the interplay between DDR deficiency and the metabolic reprogramming of HSCs, shedding light on the dynamic relationship that governs the fate and functionality of these remarkable stem cells. Understanding the crosstalk between DDR and the cellular metabolism will open a new avenue of research designed to target these interacting pathways for improving HSC function and treating hematologic disorders.

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Single-Cell Whole-Genome Sequencing after Chemotherapeutic Treatment Reveals Cell Type-Specific Mutational Burdens

Introduction The hematopoietic system is especially vulnerable to chemotherapeutic treatment-related long-term consequences, including clonal and malignant hematopoiesis. These diseases are caused by the acquisition of driver mutations in hematopoietic stem and progenitor cells (HSPCs). An increased rate of mutation accumulation, as identified in HSPCs of patients who receive chemotherapeutic treatment, can therefore result in disrupted blood production. Accordingly, childhood cancer survivors have a five-fold increased risk of secondary cancers, amongst which therapy-related myeloid neoplasms (t-MN) are one of the most prevalent. Here, we aim to increase the understanding of the origin of t-MN through the characterization of the short- and long-term mutational consequences of chemotherapy in different hematopoietic stem and progenitor cell types. Methods We studied the genome-wide mutation burden in the HSPC compartment at the time of an initial cancer diagnosis, following chemotherapeutic treatment, and upon a second cancer diagnosis. We performed whole-genome sequencing (WGS) of single HSPCs, including hematopoietic stem cells (HSCs, CD34+CD38-CD45RA-CD90+), multipotent progenitor cells (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitor cells (CMPs, CD34+CD38+CD45RA-), and granulocyte-monocyte progenitor cells (GMPs, CD34+CD38+CD45RA+). In order to obtain enough DNA for WGS, we performed clonal expansion of single cells or primary template-directed amplification of the DNA of one cell. Next, we normalized the mutation burden in each cell to the expected number of mutations (Osorio et al. Cell Reports 2018). In addition, we performed both whole genome and transcriptome sequencing of individual HSCs/MPPs. Results First, we investigated pediatric ten t-MN patients with a latency between the initial and second cancer diagnosis spanning 1-12 years. We applied WGS to clonally expanded HSPCs (CD34+CD38-CD45RA-) and found that the relative increase in mutation burden in HSPCs is restricted to the first five years after initial treatment. To gain more insight into the heterogeneous HSPC compartment, we subsequently investigated cell-type specific mutational loads based on immunophenotypic markers. So far, we have included three pediatric cancer patients with comparable treatment regimens and diagnoses, namely medium risk acute lymphoblastic leukemia. Here, we found that the mutation burden is lower in HSCs when compared to the progenitor cell types (Figure 1A). When comparing the mutation load in the HSCs to the linear mutation accumulation observed in HSCs and MPPs of healthy donors, we identified a fraction of cells overlapping with the expected mutation load based on the age of the patient (Figure 1B). These data suggest that HSCs are more protected against chemotherapy-induced mutagenesis than progenitor cells, although this protective effect varies between individual cells. Therefore, we also performed both whole genome and transcriptome sequencing from a single cell, to characterize the transcriptional state of HSCs/MPPs showing a relatively high or low mutation load within their respective cell type. Conclusions Our results indicate that primitive hematopoietic stem cells are more protected against chemotherapy-induced mutations than the more proliferative progenitor cells. In addition, we find that in t-MN patients with a long latency (>5 years), the mutation load in HSPCs returns to the expected baseline. Based on our data, we hypothesize that the more damaged progenitor cells may be replaced over time, fueled by the undamaged primitive stem cell pool. Thus, our results may explain why the risk of t-MN drops 5-7 years after treatment end.

Circadian Rhythms Metabolically Regulate Bone Marrow Retained Primitive Hematopoietic Stem Cell Size and Function

Most primitive hematopoietic stem cells (pHSC; LSK SLAM CD34 - EPCR +) are bone marrow (BM) retained in a quiescent non motile state, and are the only chemotherapy resistant HSC which requires their rapid exit from homeostatic quiescence on demand to prevent lethal hematology failure. Circadian rhythms induce daily release of progenitor cells and low levels of rarely cycling pHSCs to replenish the blood and lymphatics with new maturing cells. Metabolically, pHSCs are characterized by their dependence on glycolysis, with low mitochondrial activity and membrane potential (MMP), associated with higher competitive repopulation potential. However, if their metabolism is influenced by circadian cues is currently unknown. We report here that pHSCs at night are more glycolytic with higher surface expression of the glucose transporter Glut1, and higher glucose uptake. Repeated administration of the glucose analog, 2-deoxyglucose (2-DG), during daylight mimicked the night phenotype and enhanced HSC engraftment potential. However, nocturnal administration of 2-DG did not further improve glucose uptake nor HSC engraftment potential, suggesting glycolytic saturation. To further understand these metabolic oscillations of glucose uptake, we investigated the circadian, transcriptional signatures by single pHSC RNA-seq analysis. We found higher expression of mitophagy-related genes at night. Genes regulating mitochondrial function, were also significantly down regulated at night leading to low MMP in pHSC. While mitochondrial ROS levels were higher, cytoplasmic ROS levels were significantly reduced in pHSC at night. Importantly, we further found at night, low-functioning pHSC mitochondria to also undergo higher levels of fission as assessed by increased expression of the active mitochondrial fission marker, phospho-Drp1 (S616). To better understand the molecular pathways mediating daily circadian metabolic reprogramming between glycolysis and mitochondrial activity, we monitored our gene set enrichment analysis. We found the Wnt/b-catennin and HIF-1a signaling pathways to be significantly increased at night in pHSC. Active Wnt signaling, leads to enhanced b-catenin localization to the nucleus which is enhanced by downstream effector of Wnt activation, FOXM1. We observed higher total protein expression of FOXM1 at night. While its nuclear and cytoplasmic localization were reduced, we observed significantly higher FOXM1 levels in the mitochondria. FOXM1 localization to the energy powerhouse has been shown to inhibit mitochondrial activity. Blocking mitochondrial recruitment of FOXM1 with a specific canonical Wnt inhibitor at night led to upregulation of MMP, reversing mitochondrial metabolic responses in pHSC. Finally, daily genetic changes between night Wnt, HIF-1a and glycolysis, versus daylight GSK3b, m-TOR signaling and enhanced mitochondrial activity in BM retained pHSC, also led to cell size changes. We documented reduced quiescent pHSC size at night associated with their increased BM maintenance and higher repopulation potential. While larger sized pHSCs in the morning during the time of blood replenishment, increased their migration and development potential, for their rapid exit from quiescence on demand. Our study reveals daily circadian cues dynamically determine Wnt-mediated FOXM1 bidirectional shuttling between the nucleus and the mitochondria, leading to metabolic switching of pHSCs to maintain their cellular fitness. Our data define key signaling pathways controlling pHSC quiescence and diverse circadian metabolic activities for accumulated ROS and old mitochondria clearance and turnover, keeping them ready for rapid activation in alarm situations.

Compound Loss of Dnmt3a and Kmt2c in Myeloid Malignancies

Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine 4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Individually, loss in DNMT3A activity in hematopoietic stem cells (HSCs) has shown bias towards self-renewal due to a failure to shut off stem-cell-related genes, whereas a loss of KMT2C leads to aberrant myelopoiesis. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that may accelerate development of MDS and progression to AML. To address this, we generated mice that lacked one copy of both Kmt2c and Dnmt3a in the bone marrow by conditional deletion with a hematopoietic-specific inducible Mx1-driven Cre recombinase. After inducing Cre-mediated recombination by poly(I:C) treatment creating Kmt2c(+/del):Dnmt3a(+/del) animals, the hematopoietic stem and progenitor compartment was subjected to comprehensive immunophenotypic and functional characterization. In serial competitive bone marrow repopulation assays, cells with combined heterozygous loss of both Dnmt3a and Kmt2c demonstrated increased competitive advantage in secondary transplants and retained ability to reconstitute tri-lineage hematopoiesis in tertiary transplants, unlike single gene deletion and wild-type controls. Consistently, immunophenotypic analysis of the bone marrow compartment 16 weeks after the third transplant demonstrated persistence of the primitive long-term hematopoietic stem cells (LT-HSCs) defined as Lineage -Sca1 +cKit + (LSK) CD150 +CD48 -, which were absent in other genotypes. Further, we found expansion of the LT-HSCs in aged 80-week old Kmt2c(+/del):Dnmt3a(+/del) animals that were also characterized by a gain in self-renewal as seen by their ability to serially replate in semisolid media (MethoCult GM3434). To test if combined loss of Kmt2c and Dnmt3a led to disease development, next we established a large cohort of animals non-competitively transplanted with Kmt2c(+/del):Dnmt3a(+/del), single gene deletion, and wild-type control bone marrow, and initiated a tumor watch by monthly monitoring the composition of their peripheral blood by CBCs and flow cytometry. Sixty weeks after transplantation, over a half of double knock-out animals developed myeloid disease characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly most consistent with myelodysplasia. Immunophenotypically, we found expanded LSK populations (LT-HSCs specifically) in the bone marrow and evidence of extramedullary hematopoiesis in the spleen. At the same time, the majority of mice with single gene deletion and all wild-type controls remained healthy. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in the development of myelodysplastic disease with long latency through endowing the HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles and gene expression patterns resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.

A Pro-Inflammatory Gene Signature Characterizes a Better Risk Aged AML Patient Group in ECOG-ACRIN Cancer Research Group's Clinical Trial E3999

Background: Acute myeloid leukemia in patients over the age of 60 (aAML) is associated with poor prognosis. aAML patients are the majority diagnosed but are underrepresented in large molecular studies. We have identified two clinical subgroups of aAML patients with distinct survival outcomes (low-risk and high-risk) in ECOG-ACRIN Cancer Research Group's clinical trial E3999 (NCT00046930). We hypothesize that these two groups are characterized by distinct biological mechanisms that contribute to leukemogenesis. We also hypothesize that the low-risk group harbor molecular and phenotypic signatures characteristic of known better-risk AMLs. Methods: Diagnostic specimens were obtained from patients enrolled in NCT00046930. Disease enriched cells were isolated by negative selection of lymphocytes (magnetic beads). Flow cytometry was used to characterize the blast population. Bulk RNA-sequencing was performed on 220 blast enriched samples and 10 age matched normal controls (CD34+ bone marrow cells). Transcriptional differences were determined using DESeq2. Differentially expressed genes (DEGs) were identified as those with an absolute log2 fold change greater than 1 and q less than 0.05. Gene set enrichment analysis (GSEA) was performed against the MSigDB databases. Upstream regulators associated with gene expression changes were identified using Ingenuity Pathway Analysis (IPA). DESeq2 was used to identify genes that were differentially associated with overall survival times between the risk groups. Enrichment for transcription factor gene targets was calculated using EnrichR based on the CHEA Transcription Factor Target Dataset. Cytokines were quantified in a subset of patient serum samples using a Luminex assay. Results: We identified 2314 DEGs between the low-risk and high-risk aAMLs with high-risk as the baseline. The upregulated genes in the low-risk aAMLs were positively enriched for gene signatures derived from: 1. Favorable risk AML ( NPM1 mutated), 2. M4 and M5 AML patients, 3. Differentiated hematopoietic cells and 4. Inflammatory response pathways. On the contrary, upregulated genes in the high-risk aAMLs were enriched for gene signatures identified in hematopoietic and leukemic stem cells and known poor-risk AMLs (for example: EVI1 fusion). Accordingly, immunophenotypic data from flow cytometry results gated on the blast populations validated differences in the surface expression of hematopoietic cell markers between the two groups. Next, we sought to determine upstream regulators associated with the DEGs using IPA. We identified 107 potential upstream regulators that associated with the changes in gene expression observed between the low- and high-risk groups (87 activated and 20 inhibited). The results suggested that several pro-inflammatory cytokines (ex. tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)) were predicted activators in the low-risk aAML group. Accordingly, we detected significantly higher levels of TNFα and IFNγ in the serum levels of a subset of low-risk aAML patients. Finally, we assessed for genes whose expression significantly associated with overall survival. Interestingly, in agreement with a previously proposed inflammation score (iScore), we identified higher iScore associated with the high-risk group. Of note, the upstream regulators identified do not overlap with those contributing to the calculation of iScore. Additionally, longer overall survival times were associated with higher levels of expression of genes associated with heme metabolism and erythroblast differentiation in the low-risk aAML patient group. Conclusions: Our results suggest that the low-risk aAML patients were characterized by a more mature hematopoietic cell gene expression signature and cell surface markers as well as a pro-inflammatory state. Our study supports the possibility that pro-inflammatory cytokines and/or signaling could mediate AML cell survival in the low-risk aAML patient group. The high-risk aAML patients were characterized by a more primitive hematopoietic and leukemic stem cell gene expression signature. Collectively, we propose that differences in the underlying molecular and/or immune-based mechanisms could contribute to leukemogenesis in the two risk groups. These findings suggest that distinct therapeutic approaches may need to be considered to improve clinical outcomes in the two aAML risk groups.

Mechanisms of Aging Vary with Developmental Stages in the Human Hematopoietic Trajectory

Background : Hematopoietic stem and progenitor cells (HSPCs) undergo functional changes with age. An unresolved issue is whether aging is induced by a shift in intrinsic state in which all HSPCs undergo coordinated changes in functional potential, or by clonal evolution of a senescent population.Identification of senescent cells in human hematopoiesis poses another challenge. In this study, we have identified lineage-specific, senescence-associated pathways at different developmental stages of HSPCs and have developed an “aging signature HSC” for non-primed human HSCs. Methods: HSPCs (CD34+ cells) were isolated from the bone marrow of healthy human subjects (old = >60 years; young = 21 to <35 years). Single-cell RNA-sequencing (scRNAseq) studies and downstream analysis were performed using different R packages for normalization, dimension reduction, clustering, classification, pseudotime ordering, and developmental trajectory analyses. Gene Set Enrichment Analysis, GSEA (version 4.3.2., Broad Institute, Inc) was applied to identify pathways and processes that were coordinately up- or down-regulated with aging. Results: HSPCs (CD34+ cells) were derived from 13 healthy human subjects (old subjects n = 6; young subjects n = 7). There were 26,076 genes identified. The HSPCs were ordered and classified along their developmental trajectory during differentiation processes from (1) hematopoietic stem cells (HSC) to (2) myeloid-lymphoid progenitors (MLP), (3) megakaryocyte-erythrocyte progenitors (MEP), (4) granulocytic-monocytic progenitors (GMP), (5) granulocyte- (G), and (6) monocyte- (Mono) progenitors, (7) dendritic cell (DC), (8) erythroid progenitors (E), as well as various subsets of lymphocytes (Ly1 to Ly4). The results are depicted in Figure 1A. Remarkable are the increases in HSCs (p < 0.05) and in Ly4 (p < 0.001) in old subjects. The Kolmogorov-Smirnov test showed a significant delay in the developmental “pseudotimes” of HSCs (p < 0.001) and of Ly4 (p < 0.05). We then compared the differences in GSEA between old and young subjects in each of the following clusters: (A) whole HSPCs; (B) HSPCs along myeloid pathway (mye-HSPC); (C) HSC-MLP-MEP; (D) HSC. The major findings relevant to aging are summarized in Fig. 1B. Pathways that are significantly increased in the old subjects and throughout all comparisons include: “Formation of senescence associated heterochromatin foci”, “Aging signature HSC”, “DNA damage telomere stress induced senescence”, and “TNFR1 induced NFKB signaling pathway”. “Hypoxia”, “P53 pathway”, “IL2 signaling”, “TNFA signaling via NFKB” were elevated upon enrichment of the primitive HSC population, while other pathways decreased: “Senescence associated secretory phenotype (SASP)”, “DNA methylation”. “Cell cycle” and “DNA repair” are consistently more abundant in young subjects. We analyzed the 512 cells in the HSC cluster with higher resolution and were able to identify a sub-cluster “MPP2A” that is significantly increased in old subjects (4.5% of all HSPCs and 28.5% of HSCs, versus 1.9% and 12.9% respectively in young subjects; p < 0.005). The results are depicted in the highlighted circles in Fig. 1A. This population is quiescent and is highly enriched in “aging signature HSC” in old subjects (p <0.0001). Prominent examples of these signature genes within this list are shown in Fig. 1C). Further along the trajectory, a unique population of B cells “Ly4” was found predominantly in old subjects. The transcriptome profile Ly4 is similar to immature and age-associated B cells (ABCs). This cluster of lymphoid cells may represent the precursors of ABCs described thus far only in peripheral blood, providing another evidence that the aging mechanisms at various differentiation stages are different. Conclusions: In this study, we have defined specific pathways that distinctly separate aging HSCPs from young ones, and shown that the definition of “aging signature” varies with developmental stages. The accumulation a small cluster in the non-primed HSC compartment, with “aging signature HSC” and cell cycle arrest in old subjects indicates that aging of human hematopoiesis is induced by clonal evolution. The abundance of a unique population among lymphoid precursors with expression profiles similar to ABCs demonstrates that lymphoid and myeloid progenitors age differently.

Synergistic effect and molecular mechanism of PVA and UM171 in ex vivo expansion of primitive hematopoietic stem cells.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) used for transplantation; the number of cells in a single UCB is too small to quickly establish bone marrow (BM) implantation, and ex vivo expansion of HSCs has the potential to overcome this limitation. The purpose of this study is to explore the culture conditions conducive to the maintenance and expansion of hematopoietic stem and progenitor cells(HSPCs) and long-term hematopoietic stem cells (LT-HSCs) derived from human umbilical cord blood, compare the different effects of albumin (HSA) and polyvinyl alcohol (PVA), optimize the culture system using UM171 and investigate the molecular mechanism of PVA and UM171 promoting the expansion of primitive hematopoietic stem cells. CD34+ cells were purified from UCB using MacsCD34 beads, and then cultured in serum-free medium supplemented with cytokines for 12 days, with PVA or UM171 added according to experimental requirements; the relative percentage of different HSCs subsets after culture were detected by flow cytometry; CFU Assay Setup for detecting the multilineage differentiation potential of HSCs; RT-PCR detection of gene expression levels; reactive oxygen detection assessment of intracellular ROS levels. (1) The conditions of 20 ng/mlSCF, 100 ng/mlTPO, and 5% oxygen concentration are conducive to the maintenance of LT-HSCs. (2) Compared with HSA, PVA significantly increased the proportion of HSPCs and LT-HSCs, as well as dramatically promoted the expression of antioxidant enzymes and reduced the production of reactive oxygen species (ROS). (3) After adding UM171 to PVA-based medium, the proportion of HSPCs and LT-HSCs further increased, and downstream genes of Notch and Wnt pathways were selectively activated. (1) PVA may inhibit ROS production by upregulating the expression of antioxidant enzymes, which is beneficial for maintaining stemness and inhibiting differentiation of HSCs. (2) The antioxidant properties of PVA can delay differentiation, while UM171 can promote self-renewal by regulating the stem cell pathway, and the combination of them is beneficial for the maintenance and expansion of HSCs in vitro.

Aplastic anaemia treated using eltrombopag in a dog

AbstractA 2‐year‐old, neutered, female beagle was presented to a referral hospital for investigation of pancytopenia. The patient had a history of recurrent infections and, on previous blood tests, anaemia, leukopenia and thrombocytopenia were detected. At the time of presentation, the patient had been treated for 2 weeks with omeprazole, amoxicillin‐clavulanic acid, marbofloxacin and gabapentin, and for 3 months with prednisolone, in decreasing doses. A bone marrow aspirate and core biopsy were performed. Hypoplastic bone marrow was observed. Once underlying infectious diseases and exposure to toxins were ruled out, a diagnosis of aplastic anaemia was made. The patient was treated with eltrombopag, a thrombopoietin receptor agonist that stimulates primitive haematopoietic stem cells, used for the treatment of aplastic anaemia in humans. One week after starting eltrombopag treatment, all the affected cellular lines were recovered. The patient continued in remission 7 months after treatment.

The CD133 and CD34 cell types in human umbilical cord blood have the capacity to produce infectious dengue virus particles

Although dengue virus (DENV) can establish infections in hematopoietic stem progenitor cells (HSPCs), there is little information on dengue virus persistent infection in CD34+ and CD133+ cell surface glycoproteins of hematopoietic stem cells (HSCs). CD34 and CD133 also function as cell–cell adhesion factors, which are present in umbilical cord blood (UCB). In this study, we sought to establish a persistent infection model of DENV infection in UCB using a prolonged period of infection lasting 30 days. Post-infection, the results exhibited a productive and non-productive phase of DENV production. Using a plaque assay, Western blot, and confocal microscopy, we demonstrated that CD133 and CD34 cells are target cells for DENV infection. Moreover, we showed that DENV particles can be recovered from the non-productive phase of DENV-infected CD34 and CD133 cells after co-incubation with Vero cells. We concluded that CD133 and CD34 retain their capacity to produce the infectious virus due to proliferation and their ability to repopulate, as deduced from a BrdU proliferation assay and flow cytometry analysis using t-distributed stochastic neighbor embedding. In summary, the platform to co-culture infected primitive HSCs from their non-productive phase onto Vero cells will give new insights into understanding the DENV dynamics in cell-to-cell transmission and reactivation of the virus.

Role of Nitric Oxide in Megakaryocyte Function.

Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally known for maintaining hemostasis. Megakaryocytes are characterized as large polyploid cells that reside in the bone marrow but may also circulate in the vasculature. They are generated directly or through a multi-lineage commitment step from the most primitive progenitor or Hematopoietic Stem Cells (HSCs) in a process called "megakaryopoiesis". Immature megakaryocytes enter a complicated development process defined as "thrombopoiesis" that ultimately results in the release of extended protrusions called proplatelets into bone marrow sinusoidal or lung microvessels. One of the main mediators that play an important modulatory role in hematopoiesis and hemostasis is nitric oxide (NO), a free radical gas produced by three isoforms of nitric oxide synthase within the mammalian cells. In this review, we summarize the effect of NO and its signaling on megakaryopoiesis and thrombopoiesis under both physiological and pathophysiological conditions.

Abstract LB363: Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia

Abstract The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human cancers. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, “Complete Capture of Mutational Burden” (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines representing all major genetic groups of acute myeloid leukemia (AML). Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSGS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Citation Format: Andriana Kotini, Saul Carcamo, Nataly Cruz-Rodriguez, Malgorzata Olszewska, Dan Hasson, Eirini Papapetrou. Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB363.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

Impact of the Aryl Hydrocarbon Receptor on Aurora A Kinase and the G2/M Phase Pathway in Hematopoietic Stem and Progenitor Cells

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.

Cytogenetic Analysis of Chronic Myeloid Leukemia in an Eastern Indian Population

Chronic myeloid leukaemia (CML) is a common translocation known as BCR-ABL1, also referred to as the Philadelphia chromosome, that affects the primitive hematopoietic stem cell. The GLOBOCAN 2020 report estimates that there will be roughly 19.3 million new cancer diagnoses and 10 million cancer-related deaths in 2020. A fusion oncoprotein called BCR-ABL1, a constitutively active tyrosine kinase that is essential for the development of CML, can be inhibited to slow the course of the illness. The bone marrow and peripheral blood pile up different forms of immature and mature granulocytes or blast cells. In the previous study of CML in India, the annual incidence was reported to be 0.8 to 2.2 per 100,000 population. In leukemia, the most, common type is CML in India. Therefore, chromosomal analysis of CML plays an essential aspect in diagnosing leukemia patients. In the current study, 130 CML cases from the Eastern Indian population ranging in age from 7 to 79 years were cytogenetically analysed (mean of 36). In the study population, the male to female ratio was 1.24:1, with 72 males (55.38%) and 58 women (44.61%) participating. 122 (93.7%) of the 130 cases could be successfully karyotyped, whereas 8 (6.3%) failed culture tests. Out of the 130 cases that were noted, 25 (19.25%) had karyotypes that were normal, while 97 (74.61%) had the Philadelphia (Ph’) chromosome, which has the distinctive translocation t(9;22); Ph’+ve. Furthermore, we conclude that while more advanced molecular techniques cannot entirely replace cytogenetics, which continues to be the focus of laboratory studies into the condition, they can be used in conjunction with it to help diagnose of chronic myeloid leukemia.

Role of Rap1 GTPases in Growth, Differentiation, and Migration of Myeloid Cells

Background:Rap1 is a Ras-like small-molecular-weight GTP-binding protein involved in signal transduction cascades. It cycles between a GDP-bound inactive and a GTP-bound active form. This switching is regulated by specific GEFs and GAPs. Rap1 exists in two isoforms - Rap1a and Rap1b. While Rap1 has been implicated in regulating several hematologic disorders, including chronic lymphocytic leukemia, its role in the development and function of hematopoietic stem cells and progenitors (HSC/Ps) has not been investigated. Macrophages play an essential role in the retention of hematopoietic cells in the mesenchymal niche. Resident macrophages in the spleen retain HSCs through VCAM1 adhesion. Previous studies have shown that loss of both isoforms of Rap1 in mice results in enhanced peripheral blood leukocyte counts and mobilization of primitive hematopoietic stem cells (Lin-KIT+Sca1+) into peripheral blood circulation. Given this loss of retention of primitive hematopoietic cells in the bone marrow, we hypothesized that perhaps Rap1 plays an essential role in adhesive interactions of HSC/Ps in the bone marrow.
 Methods:To test this hypothesis, we derived macrophages from the bone marrow of wild-type (WT) and Rap1a/b double knockout (DKO) mice and compared their growth, survival and differentiation using proliferation and adhesion assays, as well as flow cytometry to assess apoptosis, macrophage differentiation, MCSF receptor expression, and expression of integrins.
 Results:Our studies show that macrophages derived from Rap1 DKO mouse bone marrow show impaired growth, survival and differentiation along with impaired adhesion in response to extracellular matrix components including fibronectin.
 Future Directions:In the future, we hope to study macrophage adhesive interactions in response to SDF1, collagen, and other extracellular matrix proteins in myeloid-specific deletion of Rap1 in mice, and to study the role of Rap1 in different lineages of hematopoietic cells using different CRE drivers.

The scaffolding function of LSD1/KDM1A reinforces a negative feedback loop to repress stem cell gene expression during primitive hematopoiesis

Lsd1/Kdm1a functions both as a histone demethylase enzyme and as a scaffold for assembling chromatin modifier and transcription factor complexes to regulate gene expression. The relative contributions of Lsd1's demethylase and scaffolding functions during embryogenesis are not known. Here, we analyze two independent zebrafish lsd1/kdm1a mutant lines and show Lsd1 is required to repress primitive hematopoietic stem cell gene expression. Lsd1 rescue constructs containing point mutations that selectively abrogate its demethylase or scaffolding capacity demonstrate the scaffolding function of Lsd1, not its demethylase activity, is required for repression of gene expression invivo. Lsd1's SNAG-binding domain mediates its scaffolding function and reinforces a negative feedback loop to repress the expression of SNAG-domain-containing genes during embryogenesis, including gfi1 and snai1/2. Our findings reveal a model in which the SNAG-binding and scaffolding function of Lsd1, and its associated negative feedback loop, provide transient and reversible regulation of gene expression during hematopoietic development.

A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma β-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.

A Novel Role for PLK1 in Leukemia Stem Cell Function in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. While most patients achieve initial remission following standard chemotherapy, the majority will relapse and ultimately die. Poor outcomes in AML are tied to the properties of leukemia stem cells (LSC), which drive therapy resistance and relapse. For successful therapeutic targeting, it is critical to understand the pathways that operate in LSC function and survival and how they are impacted by drug activity. We carried out an innovative stemness-focused high-throughput drug screen employing a hierarchical AML cell model and a Nanostring-based LSC signature assay to probe for genes and pathways that impact LSC function. All 4 small molecule inhibitors of Polo-like Kinase 1 (PLK1) included in the compound library were among the top hits. PLK1 inhibitor treatment selectively targeted the CD34⁺CD38⁻ fraction of OCI-AML-8227 cells, which is enriched for quiescent LSCs. PLK1 is an important cell cycle regulator; although there are reports for cell cycle-independent roles, they have not been systematically characterized. Importantly, the role of PLK1 has not been previously studied in the context of cancer stem cells. To investigate the role of PLK1 in LSCs, we performed single-cell (sc) multi-omics incorporating both scRNAseq and scATACseq on the CD34⁺CD38⁻ compartment of OCI-AML-8227 cells. Enrichment analysis of previously-identified chromatin accessibility signatures of primitive long-term hematopoietic stem cells (LT-HSC) and progenitors was used to identify cell clusters with distinct stemness and differentiation properties. Interestingly, PLK1 mRNA expression in OCI-AML-8227 was restricted to cells enriched for the LT-HSC epigenetic signature (Fig 1A). In healthy human bone marrow, PLK1 expression was restricted to committed progenitors and not detected in HSCs (Fig 1B). These data strongly suggest a previously unexplored role of PLK1 in regulating stemness that may be specific LSCs and not normal HSCs. To determine the effects of PLK1 against LSCs in vivo, we carried out PLK1 inhibitor treatment using the specific PLK1 inhibitor volasertib in a cohort of 9 primary AML samples in PDX models. Volasertib treatment significantly reduced leukemic burden in 7 of 9 samples, all of which had a high LSC17 score (associated with poor prognosis and standard chemotherapy response). To quantify effects on LSCs, we serially transplanted 2 samples at limiting doses into untreated secondary recipients and demonstrated a 5- to 25-fold reduction in LSC frequency following volasertib treatment, indicating that functional LSCs were targeted by PLK1 inhibition. Together, these data strongly suggest that PLK1 impacts LSC function and stemness, and that PLK1 inhibitors are acting through an uncharacterized cell-cycle independent mechanism to target quiescent LSCs. To discover novel PLK1 signaling pathways, we carried out proximity-dependent biotinylation (PDB) to characterize the PLK1 interactome in serum-starved cells. Preliminary analysis has revealed a number of previously uncharacterized PLK1 interactors, including components of autophagy and apoptosis machinery, that are currently being validated in the hematopoietic cell context. Our study highlights PLK1 as a specific vulnerability of LSCs, and furthers the understanding of malignant PLK1 signaling in non-cell cycle related processes. Further insights into the underlying mechanisms will enable the development of effective therapies to target these pathways and improve patient outcomes in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia

The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human leukemia. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, "Complete Capture of Mutational Burden" (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines for the modeling of human acute myeloid leukemia (AML) representing all major genetic groups of AML. Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/ multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSG-3GS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells (with NPM1 mutations the most notable) and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal