Phenotypic Hematopoietic Stem Cells Research Articles

490 A CTS team approach to Gold Nanorod (GNR) Theranostics in Adoptive Cell Therapy (ACT)

OBJECTIVES/GOALS: The objective ofthis study is to use GNR technology to track immune cells infiltrating malignant brain tumors that are delivered as part of a novel immunotherapeutic strategy. We seek to implement this new platform to elucidate the underlying mechanisms of therapeutic benefit from ACT via correlation between biodistribution and efficacy. METHODS/STUDY POPULATION: Utilizing the inherent two-photon luminescent signal of GNRs, we will identify uptake and phenotype of lineage negative hematopoietic stem cells (HSCs) in vitro. HSCs will be isolated from the bone marrow of 6-week-old C57bl/6 female mice. Following isolation, HSCs will be co-cultured with varying concentrations of GNRs in DMEM w/o sodium pyruvate for 24 hours, tested for viability, and images to quantify uptake and identify phenotyping. CT contrast of our novel Iodine-capped PEGylated gold nanorods will be confirmed through microCT and biodistribution of HSCs at time points after injection will be identified via CT visualizationin vivo. RESULTS/ANTICIPATED RESULTS: We expect that increased GNR signaling 24 hours post-transplant in the tumors of glioma-bearing mice will be positively correlated with long term survival following ACT. Published data from our labs have revealed that CCR2+ lineage-negative HSCs significantly accumulate in tumor of glioma-bearing mice12. Importantly, CCR2+ lineage-negative HSCs promote differentiation to dendritic cells in the tumor, increase antitumor T cell responses mediated by cross-priming and cross-presentation, and improve efficacy of immune checkpoint inhibition12. Given that HSCs are important in mediating immunotherapy efficacy, we seek to correlate the accumulation of GNR signaling within the tumor as a marker of treatment response. DISCUSSION/SIGNIFICANCE: Adoptively transferred cells have been imaged using numerous published methods. While promising to the field of immunotherapy, these methods lack significant clinical validation. GNRs have not been used to study hematopoietic stem cells in the context of ACT and brain malignancies. Our research is poised to address this gap.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype.

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

Phosphatase, Mg2+/Mn2+ dependent 1B regulates the hematopoietic stem cells homeostasis via the Wnt/β-catenin signaling.

Hematopoietic stem cells (HSCs) are primarily dormant in a cell-cycle quiescence state to preserve their self-renewal capacity and long-term maintenance. How HSCs maintain the balance between activation and quiescence remains largely unknown. Herein, we found that Phosphatase, Mg2+/Mn2+ Dependent 1B (Ppm1b) is required for the expansion of phenotypic HSCs in vitro. By using a conditional knockout mouse model in which Ppm1b was specifically depleted in hematopoietic cells, we demonstrated that loss of Ppm1b impaired the HSC homeostasis and hematopoietic reconstitution. Ppm1b deficiency mice also exhibited B-cell leukocytopenia, which is due to the compromised commitment and proliferation of B-biased lymphoid progenitor cells from CLPs. With the aid of a small molecular inhibitor, we confirmed the roles of Ppm1b in adult hematopoiesis that phenocopied the effects with loss of Ppm1b. Furthermore, transcriptome profiling of Ppm1b-deficient HSCs revealed the disruptive quiescence of HSC. Mechanistically, Ppm1b interacted with β-catenin and mediated its dephosphorylation. Loss of Ppm1b led to the decrease of the active β- catenin (non-phosphorylated) that interrupted the Wnt/β-catenin signaling in HSC, which consequently suppressed HSC expansion. Together, our study identified an indispensable role of Ppm1b in regulating HSC homeostasis via Wnt/β-catenin pathway.

15-Pgdh Inhibition Alternatively Activates Macrophages to Promote Hematopoietic Function during Aging

Aging results in the progressive decline of organ function and integrity across species. In the hematopoietic system aging is associated with diminished stem cell regenerative capacity and contributes to compromised tissue homeostasis and heightened vulnerability to age-related diseases including anemia, myelodysplastic syndromes, and hematological malignancies. Macrophages, integral components of the innate immune system, demonstrate remarkable functional plasticity and play diverse roles in tissue homeostasis, inflammation, and immunity. The balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages significantly influences immune and inflammatory responses, and thus the progression of hematopoietic aging. We have previously demonstrated that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) regulates hematopoietic stem cell (HSC) activity, that 15-PGDH inhibition (PGDHi) following transplantation results in a markedly accelerated recovery of neutrophils, platelets, and HSCs, and that 15-PGDH knockout animals display enhanced HSC function with age. Here we expand on our previous work and demonstrate in aged mice that (1) 15-PGDH expression and activity remains conserved (2) that PGDHi induces a gene expression signature consistent with alternatively activated M2 macrophages, (3) that prolonged PGDHi results in a significant increase in phenotypic HSCs in both the bone marrow and spleen and induces a gene signature consistent with enhanced HSC function, and (4) that PGDHi treated bone marrow (BM) displays a advantage during primary competitive repopulation studies and prevents age-associated myeloid bias during secondary transplants. Taken together we propose PGDHi represents an in vivo switch to alternatively activate macrophages, which has significant implications to not only prevent age-associated hematopoietic decline, but also as a novel strategy to treat age-associated hematologic disease.

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

Reduction in Lymphoid-Supporting Factors By Mesenchymal Stromal Cells Drives Myeloid-Biased Hematopoiesis at Middle Age

The bone marrow “niche” for hematopoietic stem cells (HSCs) encompasses various cell types, extracellular matrices and secreted factors that maintain the specialized functions of HSCs such as quiescence, self-renewal, and production of multipotent progenitor cells. In old age, changes in this niche contribute to dysfunction of HSCs and the hematopoietic system including increased inflammation and myeloid-biased molecular programs. We have observed that HSC and hematopoietic aging phenotypes become apparent by mid-age (Young et al. Cell Stem Cell 2021, Young et al. J Exp Med 2016), leading to the hypothesis that evaluating alterations in the niche at this stage would reveal the earliest changes initiating HSC aging. To test this hypothesis, we examined both the hematopoietic and non-hematopoietic compartments in individual middle-aged (12-14 months of age, n = 9) and compared them to young C57BL/6 mice (2-4 months of age, n = 5). Immunostaining of femurs showed an increase in sinusoid volume, vessel dysmorphia, and an increase in megakaryocyte abundance in the bone marrow of middle-aged mice. No alterations were observed in arterioles nor adipocyte volume. This data provides supporting evidence that a subset of HSC niche remodeling phenotypes observed in old age are present by mid-age. We then performed single-cell RNA-seq to comprehensively assess changes in abundance and transcriptomes of hematopoietic and non-hematopoietic cells at mid-age. Analysis of this data revealed significant expansion of HSCs in middle-aged mice. In the expanded HSC population, a reduction in B lymphoid differentiation signatures was observed without alterations in inflammation or myeloid differentiation signatures. Computational predictions suggested that the reduced B lymphopoiesis program in middle-aged HSCs resulted from decreased interaction with soluble factors produced by subsets of mesenchymal stromal cells (MSCs), rather than endothelial cells or other hematopoietic cell compartments. Specifically, reduced MSC-to-HSC interactions were linked to a decline in signaling mediated by adiponectin (ADIPOQ; Adipoq), stem cell factor (SCF; Kitl), midkine (MDK; Mdk), and insulin-like growth factor 1 (IGF1; Igf1). Among these, reductions in Kitl and Igf1 in MSCs were correlated with the loss of B lymphopoiesis programs in HSCs comparing individual middle-aged mice. To test the functional impact of the decline in Igf1 in MSCs on lymphopoiesis, we created three MSC-selective Igf1 conditional knockout mouse models using distinct Cre recombinases (Lepr-Cre, nestin-CreER and Prx1-CreER). In all three of these models, MSC-selective knockout of Igf1 reduced B lymphopoiesis resulting in myeloid-biased hematopoiesis, replicating hematopoietic aging phenotypes observed at middle age. These findings indicate that the decline in B lymphopoiesis from HSCs during aging is initiated by a reduction in lymphoid-supporting factors produced by MSCs in the bone marrow microenvironment, and this occurs independently of inflammation or activation of myeloid-promoting transcriptional programs in HSCs.

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=<0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Efficient and Specific In Vivo Genetic Engineering of Human Hematopoietic Stem Progenitor Cells without Selective Conditioning

Achieving in vivo genetic engineering of hematopoietic stem progenitor cells (HSPC) has the potential to transform treatment for hematological disorders. An ideal in vivo delivery platform should provide access to resting HSPC at low doses, in different in vivo compartments and with high specificity. We developed a multi-model strategy to assess efficiency and specificity of access to human HSPC, without reliance on conditioning to selectively enrich for gene-modified cells, and we achieved efficient genetic engineering of Lin-CD34+ cells and individual HSPC subtypes using highly potent lentiviral vectors (LV) and gene editing virus-like particles (VLP). We first conducted a high-resolution characterization of access to resting HSPC and individual FACS-sorted HSPC subtypes in vitro with different LV envelopes, including a detailed analysis of insertion site distribution in resting vs cytokine activated CD34+ cells. With a BaEVTR LV we achieved transduction of ~100% resting HSPC at MOI 66 (vs ~40% with a VSV-G LV at MOI 50,750) and ~20% of FACS-sorted hematopoietic stem cells (HSC) at MOI 32. This translated into 2-4% transduction of bone marrow (BM) huCD45+ cells when the same BaEVTR LV was dosed in NBSGW long-term humanized mice. However, such models carry a highly lymphoid-skewed human hematopoiesis and are not well suited for measuring access to all HSPC subtypes in all compartments. We therefore established 2 additional in vivo experimental scenarios. To measure LV access to HSPC in the peripheral blood (PB), we followed long-term NBSGW mice where BaEVTR LV was dosed intravenously (IV) immediately after HSPC infusion. In this model we achieved targeting of 23% early engrafting BM HSPC which was mirrored by a ~20% transduction in PB myeloid cells during the first 6 weeks, later stabilizing at 5-7% from week 8 up to week 16. To then measure access to HSPC in the BM niche, we characterized early human HSPC dynamics in the BM of NBSGW mice along 5 timepoints over the first 19 days after CD34+ cell infusion, with or without AMD3100/G-CSF mobilization. We then dosed BaEVTR LV at D7 post-humanization, a timepoint when we established that engrafted human cells have a more physiological lineage distribution, are few and remained confined in the BM even upon mobilization. In this model we demonstrated that a single BaEVTR LV dose injected IV can reach 5-6.4% of BM-resident HSPC. Based on these data, we developed a BaEVTR VLP carrying a CRISPR/Cas9 editor targeting the B2M locus. This VLP displayed 93% editing efficiency in vitro in resting HSPC and 38 to 61% in FACS-sorted HSC at MOI 16 and MOI 83 respectively. When dosed in vivo in the D7 post-humanization model, our VLP achieved B2M KO in 16% and 31% BM-resident Lin-CD34+ cells with 1 and 3 consecutive IV injections respectively, including 14% of phenotypic HSC. Importantly, a comparable level of in vivo editing of BM Lin-CD34+ cells (12%) was achieved upon equivalent pilot single VLP dosing in long-term humanized mice despite them carrying substantially higher numbers of human HSPC in the BM and a complete and stable PB human blood cell engraftment. Secondary transplantation experiments to confirm editing of long-term HSC are ongoing. We proved delivery of genetic payloads to resting HSPC in vitro and access to HSPC in different compartments in vivo with high efficiency. Adding a specific tropism for HSPC has the potential to enhance on-target bioavailability impacting effectiveness and safety of our delivery systems. To this aim, we are now testing LV and VLP carrying HSPC-targeted envelopes engineered with viral fusion proteins (retargetable fusogens). We will present comprehensivedata showing that a LV pseudotyped with a CD133-targeted fusogen combines in vitro and in vivo potency on par with BaEVTR LV with high specificity for CD133+ cells, as seen upon titrations on 8 cell lines and 10 primary human cell types as well as in spike-in experiments aimed at measuring CD133+ HSPC transduction when diluted in a mixture of CD133- human blood cells in vitro and in NBSGW mice. Furthermore, we will show that a CD117-targeted LV can transduce, in vivo, a cell population exclusively located in the BM and comprising as little as 0.3% of total huCD45+ cells with a 175-fold increased specificity as compared to envelopes with broad specificity. These results set the basis for generating a potent candidate for in vivo delivery of genetic payloads to HSPC with high efficiency and specificity.

Single-Cell RNA-Seq Reveals Intermediate Cell States and Identifies Features Defining Cellular Heterogeneity in Inv(16) Acute Myeloid Leukemia (AML)

AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. One recurrent chromosomal translocation common in AML is chromosome 16 inversion, inv(16)(p13.1q22), which results in the leukemogenic fusion gene CBFB- MYH11. Using a conditional Cbfb-MYH11 (CM) knock-in mouse model to mimic the somatic expression of the CM fusion gene, we demonstrated that CM expression leads to impaired hematopoietic differentiation as well as accumulation of phenotypic hematopoietic stem cells (HSC) and pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitors, which are predisposed to leukemic transformation. Flow cytometry-based phenotypic analysis revealed that the transformed leukemic blasts are predominantly characterized as pre-granulocyte-macrophage progenitor (pre-GM) (Lin −cKit +Sca1 −CD16/32 -/loCD150 −CD105 −). However, cell type characterization via conventional surface markers is limited by known cell markers and is incapable of resolving pathologic heterogeneities. We previously reported that modeling AML in a continuum of differentiation states using single-cell RNA-seq (scRNA-seq) data predicts intermediate states of differentiation in AML. Herein, we utilized scRNA-seq to characterize CM leukemic blasts, and identified transcriptomic alternations and features that defined the leukemic cell states and heterogeneities. CM expression was induced by poly (I:C) and mice (n=3) were monitored for leukemia progression. At the leukemic end-stage (detection of >50% of circulating cKit + cells), we collected unfractionated and ckit + sorted peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells. Similarly poly(I:C)More than 105,000 cells harvested from the WT and CM leukemic mice were analyzed by scRNA-seq. We applied uniform manifold approximation and projection (UMAP) for dimensionality reduction and performed cell type annotation by SingleR. First, we mapped mature hematopoietic cell types in the WT PB and hematopoietic stem/progenitor cell populations (HSPC) in the WT BM, and in contrast, leukemic PB and BM were highly similar and showed distinct cell clusters compared to WT. To characterize HSPC subpopulations, we focused on cKit+ sorted WT and leukemic BM cells, visualized them via UMAP, and performed clustering and cell type annotation. The leukemic cKit+ cells were mostly identified as megakaryocytic-erythroid progenitor (MEP)-like HSC, revealing a discrepancy between flow cytometry-based phenotypic characterization and transcriptome-based cell type assignment. These leukemic MEP-like HSCs showed up-regulation of erythroid differentiation genes, including Hba-a1, Hba-a2, Hbb-bs, Hbb-bt and Sox6, together with decreased expression of granulocytic markers such as Elane, Prtn3, S100a8, S100a9 and Mpo. Comparison of leukemic MEP-like HSCs vs WT MEP clusters via gene set enrichment analysis (GSEA) with a threshold of nominal p-value ≤ 0.05 and FDR ≤ 0.05 in 50 hallmark gene sets (MSigDB) indicated dysregulated pathways, including up-regulation of “TNFα signaling pathway via NFκB”, “inflammatory response”, and “UV responsive downregulated genes”; and down-regulation of “MYC target V1”, “MYC target V2”, “E2F targets”, “G2M checkpoint”, and “oxidative phosphorylation”. Similarly, GSEA in Gene Ontology (GO) showed downregulation of pathways relevant to mitochondrial protein translation and ribosomal biogenesis/assembly in leukemic MEP-like HSCs, compared to WT MEP. In addition, cKit+ BM cells can be separated into 31 clusters (C0-C30), of which 9 leukemic clusters were identified. Through gene expression-based cell cycle scoring, single sample gene set enrichment (ssGSEA), and marker identification, these 9 leukemic clusters can be defined by several features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). Altogether, these data reveal intermediate differentiation cell states in inv(16) leukemic cells which are highly heterogeneous populations with distinct cell cycle stages, metabolic activities, and marker expression.

Hyperglycemia epigenetically propels senescence-associated secretory phenotype of CD34 hematopoietic stem cells and their myeloid expansion into inflammatory monocyte subpopulations

Abstract Background The "leitmotif "underlying the cardiovascular disease initiation and progression in diabetic patients is the chronic inflammation in which monocytes/macrophages are the key players [1]. Diabetes mellitus (DM) showed to alter hematopoiesis, by promoting myelopoiesis and the generation of monocyte subsets with inflammatory and atherogenic phenotype [2-4]. Epigenetic modifications have been described in the inflammation and in control of monocyte-derived macrophage polarization [5,6]. However, there is no notion of when these epigenetic modifications take place and whether can be transferred to more differentiated cells. Purpose The aims of the study were 1) to assess in vitro if high glucose (HG) exposure promotes epigenetic changes and alteration in myeloid differentiation of hematopoietic stem/progenitor cells HSPCs; 2) if such epigenetic modifications are transferred to cell progeny and 3) if similar mechanisms are at work in bone marrow (BM)-derived HSPCs of DM patients. Methods HSPCs were isolated form the cord blood (CB) and from BM of DM patients. CB-HSPCs were grown in normal-glucose (NG;30 mM mannitol) or HG (30 mM) conditions and counted after 10, 20 and 30 days. The expression of p27, p21, IL6, TNFα, RELA/p65, KAT2B/PCAF, SetD7 genes and telomere length was assessed by qPCR; cytokines by ELISA. Western Blot was used to evaluate NFkB-p65 expression and acetylation at lysine-310. H3K9me3, H3K4me1 modifications and SetD7, RNA polymerase II at RELA/p65 gene promoter were assessed by ChIP-qPCR assay. Flow cytometry was used to assess ROS, CD14/CD16 monocyte subpopulations and NFkB-p65 nuclear translocation. Results HG-exposure of CB-HSPCs induced a senescent-associated secretory phenotype characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27 genes, increased secretion of TNFα and IL6 cytokines, and upregulation of NFkB-p65 transcription factor (fig.1). NFkB-p65 gene promoter analysis showed H3K9me3 and H3K4me1 histone mark alterations associated with increased SetD7 and RNA polymerase II recruitment. Importantly, the upregulation of KAT2B gene linked with increased lysine-310 acetylation, nuclear translocation and binding activity of NFkB-p65 (fig.1). HG-HSPCs, once differentiated into myeloid lineage, generated higher level of intermediate (CD14++CD16+) proinflammatory monocyte subpopulations (Fig.2). Similarly, BM-HSPCs from DM patients displayed a senescent-associated secretory phenotype and abnormal intermediate (CD14++CD16+) monocyte output when differentiated into myeloid lineage. Importantly, epigenetic changes at the level of NFkB-p65 gene promoter persisted in both HG-HSPC- and DM-HSPC-derived monocytes populations (fig.2). Conclusions Overall, our data show that hyperglycemia elicits epigenetic changes in HSPCs that persist in monocyte progeny and are potentially involved in their inflammatory phenotype.fig.1fig.2

Aging and Age-Related Epigenetic Drift in the Pathogenesis of Leukemia and Lymphomas: New Therapeutic Targets.

One of the traits of cancer cells is abnormal DNA methylation patterns. The idea that age-related epigenetic changes may partially explain the increased risk of cancer in the elderly is based on the observation that aging is also accompanied by comparable changes in epigenetic patterns. Lineage bias and decreased stem cell function are signs of hematopoietic stem cell compartment aging. Additionally, aging in the hematopoietic system and the stem cell niche have a role in hematopoietic stem cell phenotypes linked with age, such as leukemia and lymphoma. Understanding these changes will open up promising pathways for therapies against age-related disorders because epigenetic mechanisms are reversible. Additionally, the development of high-throughput epigenome mapping technologies will make it possible to identify the "epigenomic identity card" of every hematological disease as well as every patient, opening up the possibility of finding novel molecular biomarkers that can be used for diagnosis, prediction, and prognosis.

Aging-disturbed FUS phase transition impairs hematopoietic stem cells by altering chromatin structure

AbstractAged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity. The molecular mechanisms behind this phenomenon are not fully understood. Here, we observed that the expression of FUS is increased in aged HSCs, and enforced FUS recapitulates the phenotype of aged HSCs through arginine-glycine-glycine–mediated aberrant FUS phase transition. By using Fus-gfp mice, we observed that FUShigh HSCs exhibit compromised FUS mobility and resemble aged HSCs both functionally and transcriptionally. The percentage of FUShigh HSCs is increased upon physiological aging and replication stress, and FUSlow HSCs of aged mice exhibit youthful function. Mechanistically, FUShigh HSCs exhibit a different global chromatin organization compared with FUSlow HSCs, which is observed in aged HSCs. Many topologically associating domains (TADs) are merged in aged HSCs because of the compromised binding of CCCTC-binding factor with chromatin, which is invoked by aberrant FUS condensates. It is notable that the transcriptional alteration between FUShigh and FUSlow HSCs originates from the merged TADs and is enriched in HSC aging-related genes. Collectively, this study reveals for the first time that aberrant FUS mobility promotes HSC aging by altering chromatin structure.

Combination of CD49b and CD229 Reveals a Subset of Multipotent Progenitors With Short-Term Activity Within the Hematopoietic Stem Cell Compartment.

Hematopoiesis is maintained by hematopoietic stem cells (HSCs) that replenish all blood lineages throughout life. It is well-established that the HSC pool is functionally heterogeneous consisting of cells differing in longevity, self-renewal ability, cell proliferation, and lineage differentiation. Although HSCs can be identified through the Lineage-Sca-1+c-Kit+CD48-CD34-CD150+ immunophenotype, the cell surface marker combination does not permit absolute purification of functional HSCs with long-term reconstituting ability. Therefore, prospective isolation of long-term HSCs is crucial for mechanistic understanding of the biological functions of HSCs and for resolving functional heterogeneity within the HSC population. Here, we show that the combination of CD229 and CD49b cell surface markers within the phenotypic HSC compartment identifies a subset of multipotent progenitor (MPP) cells with high proliferative activity and short-term reconstituting ability. Thus, the addition of CD229 and CD49b to conventional HSC markers permits prospective isolation of functional HSCs by distinguishing MPPs in the HSC compartment.

Foxm1 haploinsufficiency drives clonal hematopoiesis and promotes a stress-related transition to hematologic malignancy in mice.

Clonal hematopoiesis plays a critical role in the initiation and development of hematologic malignancies. In patients with del(5q) myelodysplastic syndrome (MDS), the transcription factor FOXM1 is frequently downregulated in CD34+ cells. In this study, we demonstrated that Foxm1 haploinsufficiency disturbed normal hematopoiesis and conferred a competitive repopulation advantage for a short period. However, it impaired the long-term self-renewal capacity of hematopoietic stem cells, recapitulating the phenotypes of abnormal hematopoietic stem cells observed in patients with MDS. Moreover, heterozygous inactivation of Foxm1 led to an increase in DNA damage in hematopoietic stem/progenitor cells (HSPCs). Foxm1 haploinsufficiency induced hematopoietic dysplasia in a mouse model with LPS-induced chronic inflammation and accelerated AML-ETO9a-mediated leukemogenesis. We have also identified Parp1, an important enzyme that responds to various types of DNA damage, as a target of Foxm1. Foxm1 haploinsufficiency decreased the ability of HSPCs to efficiently repair DNA damage by downregulating Parp1 expression. Our findings suggest that the downregulation of the Foxm1-Parp1 molecular axis may promote clonal hematopoiesis and reduce genome stability, contributing to del(5q) MDS pathogenesis.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Basal type I interferon signaling has only modest effects on neonatal and juvenile hematopoiesis.

Type I interferon (IFN-1) regulates gene expression and hematopoiesis both during development and in response to inflammatory stress. We previously showed that during development in mice, hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) induce IFN-1 target genes shortly before birth. This coincides with the onset of a transition to adult hematopoiesis, and it drives the expression of genes associated with antigen presentation. However, it is not clear whether perinatal IFN-1 modulates hematopoietic output, as has been observed in contexts of inflammation. We have characterized hematopoiesis at several different stages of blood formation, from HSCs to mature blood cells, and found that loss of the IFN-1 receptor (IFNAR1) leads to depletion of several phenotypic HSC and MPP subpopulations in neonatal and juvenile mice. Committed lymphoid and myeloid progenitor populations expand simultaneously. These changes had a surprisingly little effect on the production of more differentiated blood cells. Cellular indexing of transcriptomes and epitopes by sequencing resolved the discrepancy between the extensive changes in progenitor numbers and modest changes in hematopoiesis, revealing stability in most MPP populations in Ifnar1-deficient neonates when the populations were identified based on gene expression rather than surface marker phenotype. Thus, basal IFN-1 signaling has only modest effects on hematopoiesis. Discordance between transcriptionally and phenotypically defined MPP populations may affect interpretations of how IFN-1 shapes hematopoiesis in other contexts, such as aging or inflammation.

Aging-induced pseudouridine synthase 10 impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSC) exhibit compromised reconstitution capacity and differentiation-bias towards myeloid lineage, however, the molecular mechanism behind it remains not fully understood. In this study, we observed that the expression of pseudouridine (Ψ) synthase 10 is increased in aged hematopoietic stem and progenitor cells (HSPC) and enforced protein of Ψ synthase 10 (PUS10) recapitulates the phenotype of aged HSC, which is not achieved by its Ψ synthase activity. Consistently, we observed no difference of transcribed RNA pseudouridylation profile between young and aged HSPC. No significant alteration of hematopoietic homeostasis and HSC function is observed in young Pus10-/- mice, while aged Pus10-/- mice exhibit mild alteration of hematopoietic homeostasis and HSC function. Moreover, we observed that PUS10 is ubiquitinated by E3 ubiquitin ligase CRL4DCAF1 complex and the increase of PUS10 in aged HSPC is due to aging-declined CRL4DCAF1- mediated ubiquitination degradation signaling. Taken together, this study for the first time evaluated the role of PUS10 in HSC aging and function, and provided a novel insight into HSC rejuvenation and its clinical application.