Hematopoietic Stem And Progenitor Cells Subsets Research Articles

Molecular characterization of human HSPCs with different cell fates in vivo using single-cell transcriptome analysis and lentiviral barcoding technology.

Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single-Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent-lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation-bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa-generated tracking trajectory to improve the accuracy of pseudo-time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. KEY POINTS: SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function. The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness. The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent-lineage differentiation. The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo. The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset. SCALeBa improves the accuracy of differentiation trajectories in monocle2-based pseudo-time analysis.

Different inflammatory, fibrotic, and immunologic signatures between pre-fibrotic and overt primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloid proliferative neoplasm (MPN) characterized by bone marrow (BM) fibrosis. Pre-fibrotic PMF (pre-PMF) progresses to overt PMF. Megakaryocytes (MKs) play a primary role in PMF; however, the functions of MK subsets and those of other hematopoietic cells during PMF progression remain unclarified. Therefore, we analyzed BM aspirates in pre-PMFs, overt PMFs, and other MPNs using single-cell RNA sequencing (scRNA-seq). We identified 14 cell types with subsets, including hematopoietic stem and progenitor cells (HSPCs) and MKs. HSPCs in overt PMF were MK-biased and inflammation/fibrosis-enriched. Among MKs, the epithelial-mesenchymal transition (EMT)-enriched subset was abruptly increased in overt PMF. MKs in non-fibrotic/non-PMF MPN were MK differentiation-enriched, whereas those in fibrotic/non-PMF MPN were inflammation/fibrosis-enriched. Overall, the inflammation/fibrosis signatures of the HSPC, MK, and CD14+ monocyte subsets increased from pre-PMF to overt PMF. Cytotoxic and dysfunctional scores also increased in T and NK cells. Clinically, MK and HSPC subsets with high inflammation/fibrosis signatures were frequent in the patients with peripheral blood blasts ≥1%. scRNA-seq predicted higher cellular communications of MK differentiation, inflammation/fibrosis, immunologic effector/dysfunction, and tumor-associated signaling in overt PMF than pre-PMF. However, no decisive subset emerged during PMF progression. Our study demonstrated that HSPCs, monocytes, and lymphoid cells contribute to PMF progression, and subset specificity existed regarding inflammation/fibrosis and immunologic dysfunction. PMF progression may depend on multiple cell types' alterations, and EMTenriched MKs may be potential targets for the diagnosis and therapy of the progression.

Expansion effect of romiplostim on hematopoietic stem and progenitor cells versus thrombopoietin and eltrombopag

Romiplostim, a thrombopoietin (TPO) receptor agonist, is a clinically approved drug that is clearly effective in reconstituting hematopoiesis in refractory aplastic anemia and idiopathic thrombocytopenic purpura. However, the mechanism underlying its biological effect is unknown, and its differences from other TPO receptor agonists remain unclear. Therefore, we determined the in vitro expansion effect of romiplostim on human CD34 + hematopoietic stem and progenitor cells (HSPCs) versus recombinant human TPO (rhTPO) and another clinically available drug, eltrombopag. We also performed single-cell RNA-seq to determine effects of romiplostim on CD34 + HSPCs at the molecular level. The maximum expansion effect of romiplostim on total CD34 + cells, CD34 + CD38 + progenitor cells, and CD34 + CD38 − immature cells was comparable to that of rhTPO, but higher than that of eltrombopag, particularly on CD34 + CD38 − immature cells. Single-cell RNA-seq analysis revealed that both romiplostim and eltrombopag induced signatures driven by rhTPO, but romiplostim induced molecular changes related to RHOA signaling in the most primitive HSPC subsets that were partially driven or not driven by eltrombopag. Additionally, romiplostim did not induce TFRC expression as was observed with eltrombopag. In conclusion, romiplostim expands and affects human HSPCs similar to rhTPO, but partially different from eltrombopag in terms of induction of gene expression.

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

Modeling Cell Population Dynamics in Early Hematopoiesis Reveals Mechanisms of Myeloid Bias Mediated By NFκB-Dysregulation

Alterations in hematopoiesis are associated with immune dysregulation and perturbations in NFκB signaling. Aging, for example, is correlated with increased myeloid and decreased lymphoid cell output, as well as elevated levels of NFκB activity in hematopoietic stem and progenitor cells (HSPC) and inflammatory cytokines in the bone marrow. Further, patients with myelodysplastic syndrome (MDS) show increased expression of components of the NFκB signaling pathway in HSPC and mesenchymal cells, in addition to increased production of inflammatory cytokines. Despite being implicated in these contexts, it remains unknown how elevated NFκB signaling might affect the production and abundance of certain HSPC subsets. Here we interrogate how increased NFκB activity may alter the developmental dynamics within the HSPC compartment to give rise to a biased progenitor population. To investigate how myeloid cell overproduction arises in the bone marrow, we developed mathematical models to recapitulate HSPC population dynamics. To address the role of NFκB in the process, we leveraged data from the IκB - mouse model ( Nfkbia +/- Nfkbib -/- Nfkbie -/-), which has constitutively elevated NFκB activity. Cell counts of HSPC subpopulations demonstrated a myeloid biased alteration in multipotent progenitor (MPP) abundances in the IκB -mouse, reminiscent of what occurs with aging. Fitting the cell population dynamics model to these data suggested that accelerated differentiation of Short-Term HSC (STHSC) with skewing in their fate commitment is insufficient to account for the increase in MPP2 and MPP3 cells. To explore additional mechanisms contributing to myeloid bias, we utilized single-cell RNA sequencing (scRNAseq) data from IκB - versuscontrol HSPC. We first used pseudotime reconstruction to position cells along a continuous differentiation trajectory from HSCs to erythrocyte-megakaryocyte- (MPP2), myeloid- (MPP3), and lymphoid- (MPP4) primed progenitors and then fit these data to a partial differential equation (PDE) model of population dynamics. This analysis suggested not only a loss of lymphoid fate specification amongst HSC but also an increase in the expansion of early myeloid primed cells to produce more MPP3 cells. Transplantation of wildtype HSPC into a lethally irradiated IκB - mouse additionally showed that an inflamed microenvironment is sufficient to drive myeloid bias. Utilizing the same mathematical models to describe these data revealed similar perturbations in population dynamics of the transplanted HSPC. Finally, the scRNAseq pseudotime analysis identified differentially expressed genes along the differentiating lineages that indicated decreased HSC commitment towards the lymphoid fate and increased proliferation of early myeloid primed cells, supporting the model predictions. We next leveraged scRNAseq datasets from HSPC isolated from MDS patients and healthy aged-matched controls to explore if these features of NFκB-mediated immune dysregulation are clinically relevant. These data demonstrated myeloid bias in progenitors at the transcriptional level and suggested elevated NFκB signaling amongst the HSPC from MDS patients. Finally, applying the same mathematical modeling and gene expression analyses developed for the murine datasets revealed common perturbations in developmental dynamics of MDS HPSC as the IκB - mouse models. These analyses motivate further study of the downstream consequences of immune dysregulation on hematopoiesis and the identification of perturbations in NFκB signaling in pathological contexts.

Trafficking Kinetics of Steady State Versus Mobilized Hematopoietic Stem and Progenitor Cells in Non-Human Primates

The safety and efficacy of hematopoietic stem and progenitor cell (HSPC) allogeneic transplantation and autologous gene therapies are dependent on mobilization of HSPCs out of bone marrow (BM) niches into peripheral blood (PB) and homing of infused HSPCs into open niches. However, the disposition of HSPCs exiting the marrow in steady state and following mobilization are not well understood. We characterized short-term (≤ 48 hours (h)) HSPC dynamics during steady state and following mobilization (single dose AMD3100 (plerixafor), 1mg/kg) in rhesus macaques (RMs), a model with direct preclinical relevance. We used serial intravascular staining (SIVS) (Potter et al., 2021; Morlock et al., 2021), based on timed intravenous infusions of multiple fluorescently labeled anti-CD45 antibodies (Ab) (half-life ~8.5 min), to specifically and instantaneously label all blood leukocytes at the time of each infusion, providing a window into blood residence time and tissue entry and egress. SIVS Abs are stably retained on the surface of leukocytes for up to one week and persist with migration into tissues, allowing determination of the passage of cells in and out of circulation. SIVS Abs were administered at multiple time points over 48 h, and PB was collected 5 (minutes (min)) before and 5 min, 2 h, and 4 h after each infusion. PB and BM were collected 5 min after the final Ab infusion, which was used to designate cells as intravascular (-5 min +/IVas +) or tissue-homed (-5 min -/IVas -). HSPC subsets and SIVS Abs were analyzed via flow cytometry. Additionally, trafficking to extramedullary HSPC tissue reservoirs (spleen, thymus, lymph nodes) was studied in a RM euthanized immediately following the final Ab infusion. Under steady state (n=3 RMs), we documented rapid (≤ 2 h) homing of ~55-95% of blood HSPCs out of the circulation to alternate tissue reservoirs. Of the circulating HSPC subsets, no significant differences were observed between the trafficking of HSC-enriched (CD34 +CD45RA -CD90 +), lympho-myeloid progenitor-enriched (CD34 +CD45RA +CD90-), and multipotent and erythro-myeloid progenitor-enriched (CD34 +CD45RA -CD90 -) HSPCs (Radtke et al., 2020). Following 48 h, minimal (≤ 10%) circulating HSPCs from all earlier times were found in BM, suggesting that steady state circulating HSPCs rarely “rehome,” at least within 48 h (Figure 1). Of the extramedullary reservoirs studied, minimal HSPCs trafficked to the thymus or lymph nodes; however, the spleen had a population (< 14 %) of HSPCs moving in and out over a 24 h period. To investigate the impact of AMD3100 on HSPC trafficking (n=1 RM), four SIVS Abs were administered 5 min prior to mobilization (baseline), at peak of mobilization (4 h post-AMD3100), and 10 h and 12 h post-AMD3100, as the concentration of blood HSPCs decreased. PB was collected 5 min before and 5 min, 0.5 h, and 2 h post each SIVS Ab infusion to monitor HSPC kinetics; PB and BM were collected at 12 h post-AMD3100, and final PB was collected at 24 h post-AMD3100. Compared to steady state HSPC dynamics described above, mobilized HSPCs labeled at the peak of mobilization exhibited prolonged circulation (Figure 2), with a higher portion of the mobilized cells remaining in the blood at the 2 h time window compared to steady state. However, there was no increase in the fraction of the HSPCs rehoming to the BM (Figure 1), suggesting that mobilized HSPCs do not exhibit a greater propensity for rehoming compared to steady state and that BM niches are still open 12 h after mobilization. The extended availability of these niches holds potential relevance for the use of potent mobilizing regimens to facilitate engraftment of transplanted autologous gene-modified or allogeneic HSPCs. In summary, under both steady-state and mobilization conditions, HSPCs exhibit minimal rehoming to bone marrow niches, and mobilized HSPCs linger in circulation for up to 24 h post-mobilization. Further studies in progress aim to investigate longer term BM rehoming and better define extramedullary sites of HSPC egress at steady state or following mobilization. Harnessing knowledge of HSPC dynamics holds potential to improve mobilization approaches and provide key insights into developing efficient mobilization-based conditioning for safer HSPC transplantation.

Efficient and Stable Makassar Base Editing in Nonhuman Primates

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin chain. This nucleotide can be modified using adenine base editors (ABEs) to convert the defective SCD β-globin gene (HBB S) into the non-pathogenic Makassar β-globin (HBB M) variant. Proof-of-concept for this editing strategy in human and mouse hematopoietic stem cells (HSCs) has been previously demonstrated by transplantation after ex vivo modification as well as by in vivo adenoviral editing followed by selection. Here, we evaluated the feasibility, long-term efficiency, and safety of autologous ex vivo Makassar base editing in the preclinical nonhuman primate (NHP) large animal model to pave the way towards clinical applications in humans. As NHPs don't carry the sickle mutation (position A7 in the protospacer), bystander editing at position A9 was monitored as a surrogate. NHP CD34 + hematopoietic stem and progenitor cells (HSPCs) were enriched, CD34 +CD90 + HSCs FACS-purified as previously reported to increase the editing efficiency of short- and long-term engrafting cells, primed over-night, and ABE8e-NRCH base editor mRNA was delivered by electroporation. Base-edited HSCs were cultured overnight and then combined with unmodified CD34 +CD90 - cells and infused into animals after myeloablative total body irradiation. The infusion product was quality controlled by flow-cytometry, next generation sequencing (NGS), and assessing the mono-/bi-allelic base editing efficiency on a single cell level from colony-forming cell (CFC) assays. Animals were followed for up to 200 days analyzing the editing efficiency in peripheral blood (PB) white blood cells (WBCs), FACS-purified lineages from the PB, bone marrow (BM) WBCs, as well as FACS-purified BM HSPCs. Comprehensive bioinformatics analysis was performed on the generated NGS data to determine the frequency of editing over time as well as the on- and off-target editing within the protospacer at all four target locations. Finally, chromosomal integrity after base editing was confirmed performing Kromatid karyotyping assays on gene-edited BM-derived HSPCs. Ex vivo base editing of CD34 +CD90 + was highly reproducible at the Makassar locus reaching more than 60% efficiency within bulk HSCs. No impact of base editing on the myeloid and erythroid differentiation of HSCs was seen in CFC assays. Clonal analysis of colonies revealed that more than 90% of HSCs in the infusion product were edited with > 20% of cells showing biallelic editing. Gene-edited HSCs combined with unmodified progenitors rapidly engrafted in myeloablated animals without any noticeable delay in the recovery of neutrophils, red blood cells, as well as platelets and multilineage reconstitution was established within 3-6 months. Most importantly, stable editing of >20% was observed in PB WBCs and across all lineages throughout the entire follow-up. Similarly, the BM stem cell compartment fully recovered to baseline within 6-month post-transplant. Engrafted HSPCs demonstrated normal erythro-myeloid colony-formation potential. Assessment of editing in colonies showed persistence of HSCs with mono- and bi-allelic Makassar editing 6-month post-transplant. Closely matching the editing efficiency in the PB, all BM lineages, HSPC subsets, and colonies demonstrated >20% editing efficiency confirming successful editing of long-term persisting multipotent HSCs. Finally, comprehensive assessment of NGS data from all tissues in conjunction with the Kromatid assay revealed no unwanted off-target effects or chromosomal rearrangements. Editing was primarily observed at positions A9 and A12 confirming highly specific on-target editing. In summary, we demonstrate efficient, persistent, and safe base editing of NHP HSCs for the treatment of SCD. The editing efficiency of the bystander adenine in NHP HSCs is almost identical to the previously observed editing in human patient HSCs to generate the Makassar HBB. Base edited HSCs showed no impairment in homing, long-term engraftment, or multilineage differentiation in the NHP. These findings have major implications for the application of base editing in patients with hematological disease and disorders.

Mycobacterium tuberculosis infection drives differential responses in the bone marrow hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These HSPC responses can be influenced, directly and indirectly, by pathogens as well. Infection with Mycobacterium tuberculosis (Mtb) can drive lymphopoiesis through modulation of type I interferon (IFN) signaling. We have previously found that the presence of a drug resistance (DR)-conferring mutation in Mtb drives altered host-pathogen interactions and heightened type I IFN production in vitro. But the impacts of this DR mutation on in vivo host responses to Mtb infection, particularly the hematopoietic compartment, remain unexplored. Using a mouse model, we show that, while drug-sensitive Mtb infection induces expansion of HSPC subsets and a skew toward lymphopoiesis, DR Mtb infection fails to induce an expansion of these subsets and an accumulation of mature granulocytes in the bone marrow. Using single-cell RNA sequencing, we show that the HSCs from DR Mtb-infected mice fail to upregulate pathways related to cytokine signaling across all profiled HSC subsets. Collectively, our studies report a novel finding of a chronic infection that fails to induce a potent hematopoietic response that can be further investigated to understand pathogen-host interaction at the level of hematopoiesis.

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.

Identification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

The preclinical development of hematopoietic stem cell (HSC) gene therapy/editing and transplantation protocols is frequently performed in large animal models such as nonhuman primates (NHPs). Similarity in physiology, size, and life expectation as well as cross-reactivity of most reagents and medications allows for the development of treatment strategies with rapid translation to clinical applications. Especially after the adverse events of HSC gene therapy observed in the late 1990s, the ability to perform autologous transplants and follow the animals long-term make the NHP a very attractive model to test the efficiency, feasibility, and safety of new HSC-mediated gene-transfer/editing and transplantation approaches.This protocol describes a method to phenotypically characterize functionally distinct NHP HSPC subsets within specimens or stem cell products from three different NHP species. Procedures are based on the flow-cytometric assessment of cell surface markers that are cross-reactive in between human and NHP to allow for immediate clinical translation. This protocol has been successfully used for the quality control of enriched, cultured, and gene-modified NHP CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as sort-purified CD34 subsets for transplantation in the pig-tailed, cynomolgus, and rhesus macaque. It further allows the longitudinal assessment of primary specimens taken during the long-term follow-up post-transplantation in order to monitor homing, engraftment, and reconstitution of the bone marrow stem cell compartment.

Characterization of myelodysplastic syndromes hematopoietic stem and progenitor cells using mass cytometry.

Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. Bone marrow samples from healthy donors (n =10), low risk (n =12) and high risk (n =13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n =20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

3161 – CELLULAR AND MOLECULAR EFFECTS OF DMPGE₂ RADIOMITIGATION IN MOUSE AND HUMAN BONE MARROW CELLS IN VIVO USING C57BL/6 AND HUMANIZED NSG MOUSE MODELS OF ACUTE RADIATION SYNDROME

The hematopoietic system is highly sensitive to radiation exposure, and bone marrow (BM) failure from the hematopoietic acute radiation syndrome (H-ARS) is the primary threat to human life following a nuclear accident or attack. Dimethyl prostaglandin E2 (dmPGE₂) is a promising medical countermeasure (MCM) which significantly increases H-ARS survival in mice. Here mechanisms of dmPGE₂ radiomitigation were defined when given 24 h post-total body irradiation (TBI) in C57BL/6 mice and in humanized NOD scid gamma null (NSG) mice. In C57BL/6 mice exposed to the LD50/30 (8.53 Gy), dmPGE₂ increased cycling rates of hematopoietic stem and progenitor cells (HSPCs) within 6h of treatment, increased total BM cellularity and viability within 24h of treatment, and accelerated peripheral blood recovery. In CD34-humanized NSG mice exposed to 1 Gy, total BM counts were depressed by 48h post-TBI, but % huCD45+ BM chimerism was increased in dmPGE₂-treated animals, suggesting a slight human cell advantage from dmPGE₂. Within the huCD45+ population, significant effects of both TBI and dmPGE₂ on various human HSPC subsets in vivo regarding frequencies, cycling, DNA damage, and gene expression were delineated, including upregulation of transcriptional activators FOS, JUNB, EGR1, and NR4A1 within both mouse and human HSCs in response to dmPGE2 radiomitigation. These proof-of-concept studies support that dmPGE₂ effects related to radiomitigation can be detected in irradiated human HSPCs in vivo in NSG mice. Further work will investigate a more radioresistant humanized mouse system to better model the radiation-damaged BM environment after lethal exposure. Overall, this work describes an innovative translational platform for testing radiation MCMs, and represents a significant step toward human translation of dmPGE₂ for this purpose. The hematopoietic system is highly sensitive to radiation exposure, and bone marrow (BM) failure from the hematopoietic acute radiation syndrome (H-ARS) is the primary threat to human life following a nuclear accident or attack. Dimethyl prostaglandin E2 (dmPGE₂) is a promising medical countermeasure (MCM) which significantly increases H-ARS survival in mice. Here mechanisms of dmPGE₂ radiomitigation were defined when given 24 h post-total body irradiation (TBI) in C57BL/6 mice and in humanized NOD scid gamma null (NSG) mice. In C57BL/6 mice exposed to the LD50/30 (8.53 Gy), dmPGE₂ increased cycling rates of hematopoietic stem and progenitor cells (HSPCs) within 6h of treatment, increased total BM cellularity and viability within 24h of treatment, and accelerated peripheral blood recovery. In CD34-humanized NSG mice exposed to 1 Gy, total BM counts were depressed by 48h post-TBI, but % huCD45+ BM chimerism was increased in dmPGE₂-treated animals, suggesting a slight human cell advantage from dmPGE₂. Within the huCD45+ population, significant effects of both TBI and dmPGE₂ on various human HSPC subsets in vivo regarding frequencies, cycling, DNA damage, and gene expression were delineated, including upregulation of transcriptional activators FOS, JUNB, EGR1, and NR4A1 within both mouse and human HSCs in response to dmPGE2 radiomitigation. These proof-of-concept studies support that dmPGE₂ effects related to radiomitigation can be detected in irradiated human HSPCs in vivo in NSG mice. Further work will investigate a more radioresistant humanized mouse system to better model the radiation-damaged BM environment after lethal exposure. Overall, this work describes an innovative translational platform for testing radiation MCMs, and represents a significant step toward human translation of dmPGE₂ for this purpose.

Hematopoietic Cell Transplantation of Higher CD34+ Cell Doses and Specific CD34+ Subsets Mobilized with Motixafortide and/or G-CSF Is Associated with Rapid Engraftment - a Post-Hoc Analysis of the Genesis Trial

Hematopoietic Cell Transplantation of Higher CD34+ Cell Doses and Specific CD34+ Subsets Mobilized with Motixafortide and/or G-CSF Is Associated with Rapid Engraftment - a Post-Hoc Analysis of the Genesis Trial

Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis

BackgroundHematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry.ResultsWith fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling.ConclusionsThese comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

IL-6 contributes to metastatic switch via the differentiation of monocytic-dendritic progenitors into prometastatic immune cells

BackgroundMetastasis is the major cause of death in patients with cancer. Myeloid skewing of hematopoietic cells is a prominent promoter of metastasis. However, the reservoir of these cells in the...

Metabolic dysfunction induced by a high-fat diet modulates hematopoietic stem and myeloid progenitor cells in brown adipose tissue of mice.

Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high-fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPCs)-which give rise to DCs-in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the 'whitening' effect of eating a high-fat diet upon interscapular (i) BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPCs and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional DCs (cDCs) increased in both of these tissues. When compared with a low-fat diet, consumption of a high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed the high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin [blocked using the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)], but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction .

Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells.

Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production.

Heterogeneity of Hematopoietic Stem and Progenitor Cell (HSPC) Composition in Αβ T-Cell/CD19 B-Cell Depleted Peripheral Blood Cell Stem Cell (PBSC) Transplant Grafts and Correlation with Immune and Hematopoietic Recovery

INTRODUCTION. While total CD34 counts in PBSC graft products have correlated with overall likelihood of hematopoietic recovery after allogeneic hematopoietic stem cell transplantation (HSCT), analyses of the HSPC composition and its relationship to relevant post-transplant clinical outcomes are lacking. In fact, the biological basis for different dynamics of hematopoietic/immune recovery, the risk of infection, and graft-versus-host disease (GvHD) is not fully understood. We have performed the first analysis of HSPC graft composition in 6 αβ T-cell/CD19 B-cell depleted haploidentical (αβhaplo) HSCT. Additionally, we correlated the HSPC graft composition with the distribution of the same HSPC subsets in serial post-HSCT bone marrow aspirates performed at Days 30, 60, and 90, with the peripheral blood counts [neutrophils, monocytes, platelet (PLT)] and with the immune recovery (CD3+, CD3+CD4+, CD3+CD8+, αβT, γδT, NK cells) at the same time points. The patients were divided in two groups: 3 patients had a robust and sustained hematopoietic recovery (Group 1) while 3 patients experienced mild cytopenia after Day 60 (Group 2). All patients were transplanted for acute leukemia and received a myeloablative TBI-based conditioning regimen. See Table 1 for details about patients and graft composition. METHODS. All patients were enrolled in the Stanford IRB approved BMT Protocols 179/351/361 and had peripheral blood (PB) and bone marrow (BM) evaluated at Day 30, 60 and 90 post HSCT for the primitive CD34+ Lin- HSPC subsets: HSC (CD38-CD45RA-CD90+), MPP (CD38+CD45RA+), CMP (CD38+CD45RA-CD123+), GMP (CD38+CD45RA+CD123+), MEP (CD38+CD45RA-CD123+) and CLP (CD38+CD127+). Aliquots of αβhaplo-HSCT grafts were cryopreserved for later analyses. Mononuclear cells were isolated from PBSC, PB and BM by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. FACS analyses were performed on either fresh or frozen cells on Becton Dickinson (BD) Aria II flow cytometer. At least 5x105 events were acquired and analyzed using FlowJo software (BD). RESULTS. Despite consistent levels of αβT-cell depletion and CD34 enrichment, the frequency of the HSPC subsets varied between the grafts. Notably, while CMP and GMP were very consistent across the 6 grafts, the frequency of HSC, CLP, MEP and MPP showed a 2-fold range of variation (Fig1A). No significant correlation was observed between the HSC frequency in the graft and the hematopoietic/immune recovery. However, the frequency of HSC in the BM at Day 30 is statistically correlated (P=0.027) with the PLT at Day 90 (Fig1B). In these preliminary results, the different distribution of CMP, GMP, MEP and MPP did not impact on the hematopoietic/immune recovery. However, there was a significant correlation (P=0.02) between CLP and γδ T cells reconstitution at Day 90 in Group 1 patients (Fig1C). Additionally, the neutrophils, monocytes and phagocytic cells recovery at Day 90 is statistically correlated with the GMP frequency in the BM at Day 30 (P=0.017; P=0.018; P=0.0132, respectively) (Fig1D). Interestingly, the same strong correlation is observed between the CMP in the BM at Day 60 and the recovery of neutrophils and phagocytic cells (P=0.016, P=0.019) at Day 90 (Fig1E), but the CMP at day 30 are already predictive of a robust engraftment in the Group 1 patients (data not shown). CONCLUSIONS. In αβhaplo-HSCT, previously identified factors influencing hematopoietic recovery have been mainly limited to the enumeration of bulk CD34 counts and of mature effector cells, such as αβ/γδ T and NK cells. On the other hand, the presence of GvHD and thymic injury have been correlated to the kinetics of immune reconstitution. We hypothesized that the HSPC composition of the graft would impact lymphohematopoietic recovery in αβhaplo-HSCT recipients. Although preliminary, our data indicate that even with a consistent method of graft manipulation, the HSPC graft composition is heterogeneous. Variations in HSPC subsets frequency and number can contribute to significant differences in lymphohematopoietic recovery and, therefore, clinical outcome. The evaluation of a larger number of patients with longer follow up after HSCT are required. Comparative studies with unmanipulated T-cell replete and cord blood grafts are ongoing. Such analyses will be instrumental not only for prediction of clinical outcome, but also for optimization of novel graft engineering strategies. Disclosures No relevant conflicts of interest to declare.

A Role for TNF-α in Chronic Lymphocytic Leukemia Bone Marrow Hematopoietic Dysfunction

The current paradigms of known peripheral immune abnormalities in B-Chronic Lymphocytic Leukemia (CLL) are not able to consistently explain patient complications and it is difficult to correct a given CLL patient's immune status. Here, we expand on our initial report demonstrating bone marrow (BM) hematopoietic dysfunction in untreated CLL patients (Manso et al., Leukemia volume 33, pages 638-652, 2019). CLL patient BM had significantly reduced frequencies and short-term functional capacity of hematopoietic stem and progenitor cells (HSPCs). Additionally, the remaining progenitors exhibited increased protein levels of the key hematopoietic transcriptional regulators GATA-2 and PU.1. We further evaluated the frequency and function of myeloid stem cells from controls and untreated CLL patients by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays. Over the 5 week duration of the assay, we observed delayed and partial recovery of myelopoiesis from CLL-derived HSPCs (Figure 1A). These data suggest that removal of HSPCs from the CLL leukemic microenvironment partially recovers their ability to sustain myelopoiesis. A known inflammatory mediator and hematopoiesis-modulating cytokine that is constitutively produced by CLL cells, TNF-α, induced increased expression of GATA-2 and PU.1 in specific HSPC subsets and reduced formation of short-term colony forming units in vitro. Addition of TNF-α to LTC-IC assays resulted in a striking ablation of myelopoiesis in a dose-dependent manner, partially reproducing the ex vivo results (Figure 1B). To further assess the direct impact of CLL cells on HSPC biology, isolated HSPCs from controls were exposed in vitro to leukemic CLL cells. The co-culture induced overexpression of GATA-2 and PU.1 in distinct HSPC populations, recapitulating our ex vivo findings (Figure 1C-D). When cell-cell contact was inhibited by use of Transwell inserts, an intermediate increase in GATA-2 and PU.1 was observed, highlighting the contributions of both soluble mediators and cell-cell contact to HSPC alterations. In both direct and Transwell co-culture conditions, overexpression of GATA-2 and PU.1 was reversed when TNF-α was neutralized (Figure 1E-F). Taken together, these findings indicate a significant role for CLL-derived TNF-α in HSPC modulation and expand our previous observations of BM dysfunction in untreated CLL patients. This data offers new molecular insight into the contribution of the leukemic microenvironment to altered hematopoiesis, contributing to immunodeficiency in CLL, and identifies TNF-α as a potential therapeutic target for correction of hematopoiesis in CLL disease. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Parikh:AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Novak:Celgene Coorperation: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board.

VE-Cadherin and ACE Co-Expression Marks Highly Proliferative Hematopoietic Stem Cells in Human Embryonic Liver.

Despite advances to engineer transplantable hematopoietic stem and progenitor cells (HSPCs) for research and therapy, an in-depth characterization of the developing human hematopoietic system is still lacking. The human embryonic liver is at the crossroad of several hematopoietic sites and harbors a complex hematopoietic hierarchy, including the first actively dividing HSPCs that will further seed the definitive hematopoietic organs. However, few are known about the phenotypic and functional HSPC organization operating at these stages of development. In this study, using a combination of four endothelial and hematopoietic surface markers, that is, the endothelial-specific marker vascular endothelial-cadherin (Cdh5, CD144), the pan-leukocyte antigen CD45, the hemato-endothelial marker CD34, and the angiotensin-converting enzyme (ACE, CD143), we identified distinct HSPC subsets, and among them, a population co-expressing the four markers that uniquely harbored an outstanding proliferation potential both ex vivo and in vivo. Moreover, we traced back this population to the yolk sac (YS) and aorta-gonad-mesonephros (AGM) sites of hematopoietic emergence. Taken together, our data will help to identify human HSPC self-renewal and amplification mechanisms for future cell therapies.