Hematopoietic Stem And Progenitor Cells Populations Research Articles

DNA fork remodeling proteins, Zranb3 and Smarcal1, are uniquely essential for aging hematopoiesis.

Over a lifetime, hematopoietic stem and progenitor cells (HSPCs) are forced to repeatedly proliferate to maintain hematopoiesis, increasing their susceptibility to DNA damaging replication stress. However, the proteins that mitigate this stress, protect HSPC replication, and prevent aging-driven dysregulation are unknown. We report two evolutionarily conserved, ubiquitously expressed chromatin remodeling enzymes with similar DNA replication fork reversal biochemical functions, Zranb3 and Smarcal1, have surprisingly specialized roles in distinct HSPC populations. While both proteins actively mitigate replication stress and prevent DNA damage and breaks during lifelong hematopoiesis, the loss of either resulted in distinct biochemical and biological consequences. Notably, defective long-term HSC function, revealed with bone marrow transplantation, caused hematopoiesis abnormalities in young mice lacking Zranb3. Aging significantly worsened these hematopoiesis defects in Zranb3-deficient mice, including accelerating the onset of myeloid-biased hematopoietic dysregulation to early in life. Such Zranb3-deficient HSPC abnormalities with age were driven by accumulated DNA damage and replication stress. Conversely, Smarcal1 loss primarily negatively affected progenitor cell functions that were exacerbated with aging, resulting in a lymphoid bias. Simultaneous loss of both Zranb3 and Smarcal1 compounded HSPC defects. Additionally, HSPC DNA replication fork dynamics had unanticipated HSPC type and age plasticity that depended on the stress and Zranb3 and/or Smarcal1. Our data reveal both Zranb3 and Smarcal1 have essential HSPC cell intrinsic functions in lifelong hematopoiesis that protect HSPCs from replication stress and DNA damage in unexpected, unique ways.

Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

BackgroundTransplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34+ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34+ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34+ isolated from cord blood (CB-CD34+) or fetal liver (FL-CD34+ and FL-CD34+CD14−) and their engraftment into immunocompromised NOD/Shi-scid Il2rgnull (NOG) mice.MethodsNOG mice were transplanted with either CB-CD34+, FL-CD34+ or FL-CD34+CD14− to generate CB-NOG, FL-NOG and FL-CD14−-NOG, respectively. After 15–20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles.ResultsWe show that CB-CD34+ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34+ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34+ isolates contain non-hematopoietic CD14+ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14+CD34+ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14+CD34+ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection.ConclusionOur results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

Cellular and Molecular Analysis of Hematopoietic Stem and Progenitor Cells in Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an inherited multi-organ disorder with bone marrow failure (BMF), which may include neutropenia, anemia, and thrombocytopenia, as well as a high risk of myelodysplasia and leukemia. Hematological complications are common causes of morbidity and mortality in SDS. Around 90% of SDS patients have biallelic mutations in the Shwachman-Bodian-Diamond Syndrome ( SBDS) gene, which plays an important role in ribosome biogenesis and hematopoietic cell survival. In a collaborative study led by Dr. John Dick (Science 2016) novel hematopoietic progenitor subsets were identified (noted as F1, F2 and F3), of which the abundance, clonal capacity, and function in disease states remain unknown. We hypothesized that cellular and molecular analysis of hematopoietic stem and progenitor cells (HSPC) including these novel progenitors would provide valuable insights into the pathogenesis of BMF in SDS. Using multiparametric flow cytometry we quantified the following HSPC populations: hematopoietic stem cells (HSC), multipotent progenitors (MPP F1-F3), common myeloid progenitors (CMP F1-F3), megakaryocyte-erythroid progenitors (MEP F1-F3) and granulocyte-monocyte progenitor (GMP) in 13 SDS patients with SBDS mutations and 10 normal control donors. We found a significant reduction in CD34+/CD38- and CD34+/CD38+ cells in SDS patients. It is particularly noteworthy that the sub-populations of CMPs (F1 and F2), and MEPs (F2 and F3) were prominently decreased. These cells contribute to the production of myeloid, erythroid, and megakaryocytic cells with different proportions. Using single-cell colony-forming assays, we assessed the cloning efficiency of each HSPC, revealing that HSCs, MPPs and GMPs from SDS patients exhibited a significantly lower cloning efficiency compared to those from control subjects. Notably, the typical erythroid-committing progenitor cells (CMP F2, CMP F3, MEP F2, and MEP F3) in SDS collectively produced approximately 46% fewer erythroid cells per colony compared to control progenitors, while myeloid-committing progenitor cells (CMP F1, MEP F1) from these patients produced approximately 86% fewer myeloid cells, including neutrophils, per colony compared to the corresponding control cells. Further, HSCs and MPPs-F1 cells from SDS patients had a reduced cloning efficiency of approximately 22% and 27%, respectively, compared to control HSCs and MPP-F1 progenitors. These data suggest that multiple HSPC populations from SDS patients are characterized by reduced cell numbers, functional impairments, and dysregulation compared to normal subjects. Next, we performed transcriptome analysis of each HSPC by RNA sequencing. We revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS. Using KEGG pathway analysis, we found that downregulation of the ribosome pathway reached statistical significance in HSC, CMP F1 and MEP F3. Downregulation of the hematopoietic cell lineage pathway reached statistical significance in MEP F1-2 and MPP F1-2. Additionally, the TGF-beta signaling pathway was suppressed in CMP F1-3, MPP F1, and GMP, while the NF-kappa B signaling pathway, crucial for regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells, was also found to be decreased in GMP. Interrogation of specific dysregulate genes, we found that the hematopoietic transcription factor, ZFP36 (ZFP36 ring finger protein) which involved in cellular response to cytokines and growth factors, was significantly downregulated in five subpopulations of SDS HSPCs, particularly noticeable in the four progenitors (CMP F1-3, MEP F2, and GMP) which produced substantially fewer colonies in SDS patients compared to controls. Additionally, we observed downregulation of TGFB2 and upregulation of NFKBIA and NFKBIB may inhibit the NF-kappa B signaling pathway in SDS patients. These alterations have the potential to influence the hematopoietic cell lineage pathway, leading to BMF. Functional assays are ongoing and aim to assess the impact of these dysregulated genes on formation and behavior of HSPCs. In summary, our data suggest that multiple SDS HSPC populations are characterized by reduced cell numbers and functional impairments. As well, we revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Maternal Obesity Causes Sex Specific Long-Term Hematopoietic and Metabolic Defects

Maternal obesity is increasingly common and can negatively impact offspring health. Children born to obese mothers are at higher risk of developing diseases involved with metabolic health (e.g., type 2 diabetes). Many of these diseases are associated with abnormalities within the hematopoietic system such as atypical immune profiles or hematopoiesis. Interestingly, disease incidence and/or severity is often dependent on the offspring's gender. This suggests that there may be sex-specific reprogramming of hematopoietic stem and progenitor cells (HSPCs) during immune cell development. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. To test this, C57BL/6 (CD45.2+) dams were fed standard chow (2018SX, Envigo, IN) or western-style diet (TD.88137, Envigo, IN) for four weeks prior to mating, through pregnancy, and the lactating period. At postnatal day 21, HSPCs (Lineage negative, Sca1 positive, cKit positive cells) were flow-sorted from bone marrow (BM) of offspring from dams fed chow (Con) or western-style diet (MatOb) and used for competitive transplant (Boy/J BM competitor cells; CD45.1+) into same-sex lethally irradiated C57BL/6 x Boy/J F1 (CD45.1+CD45.2+) recipient mice (8-10 weeks of age) or for RNA-sequencing analysis. Secondary transplants were performed to look at long-term engraftment potential of HSPCs. Body weight, body adiposity, fasting insulin, glucose tolerance test (GTT), and pancreatic β and α-cell area were assessed on male and female recipients of both primary and secondary transplants. Compared to mice that received male Con HSPCs, recipient mice that received male MatOb HSPCs had significantly lower donor chimerism in the blood and BM post-secondary transplantation. This was associated with a significant reduction in donor hematopoietic stem cell (HSC) numbers in the BM 16 weeks post-secondary transplant, indicating male MatOb HSPCs had decreased long-term engraftment capability due to an exhaustion of the HSC pool. This reduction in HSPC engraftment seen between male MatOb versus Con HSPCs was not seen when using HSPCs from female offspring. Next, metabolic parameters of our recipient mice was examined. Sixteen weeks after primary transplant, there were no differences detected in body weight and adiposity in the Con and MatOb HSPC recipient mice. Compared to sex-matched controls, we found that recipients transplanted with male MatOb HSPCs developed glucose intolerance, increased fasting serum insulin concentrations, and decreased β-cell area, supporting the presence of impaired glucose metabolism due to a combination of insulin resistance and lower β-cell mass. There was no difference in recipients of HSPCs from female Con and female MatOb pups. RNA-seq of BM HSPCs at time of transplant (p21) from Con or MatOb revealed 100 differentially expressed genes (DEG) in male MatOb HSPCs and 1788 DEG in female MatOb HSPCs. There were more and higher magnitude of differential gene expression in female mice than male, despite the long-term phenotypic differences being evident in males but not females, and the majority of genes changed in males were also altered in females. This suggests that the gene programs responsible for the observed phenotypes may be altered by maternal obesity in both male and female offspring, but that female offspring have a transcriptomic profile that confers protection on the HSPC population. One of the most striking findings from the transcriptomic analysis was a marked enrichment in gene programs associated with immune response, interferon signaling, and cytokine production in female MatOb offspring compared to sex matched controls that is not seen in male MatOb offspring. Pathway analysis identified upregulation of programs involving both IFNγ and IL-10 in the female MatOb HSPCs when compared to sex-mated Con HSPCs which correlated with an increase in IL-10 concentrations in the serum of p21 female MatOb mice. These pathways were downregulated or unaltered in males with no change in systemic IL-10 concentration.Our results demonstrate that maternal obesity results in sex-specific changes in offspring HSPC function and a novel role HSPCs may play in regulating glucose metabolism in the offspring of obese dams.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

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.

Cooperation between SF3B1 and JAK2V617F Mutations Accelerates Fibrotic Progression in Myeloproliferative Neoplasms By Enhancing STAT1 Signaling

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1 mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1 K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1 mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies. To address this, we applied two complimentary single-cell multiomic methods to study 15 individuals (healthy donor [HD, n=5], JAK2V617F-single mutated [J+, n=5] and JAK2V617F- SF3B1K666double mutated MPN cases [JS+, n=5]). First, we characterised the cellular landscape of JS+ MPN using CITE-seq/10X genomics platform. Then, we analyzed SF3B1-mutant ( SF3B1mut) disease-driving hematopoietic stem and progenitor cells (HSPCs) and their associated aberrant splicing signatures using TARGET-seq, which permitted single-cell genotyping with allelic resolution and intra-patient comparison of mutant versus wild-type (wt) cells. In an analysis of 109,498 cells, we observed abnormally expanded erythroid (ERP), megakaryocyte (MK) and unexpectedly, eosinophil-basophil-mast cell (EBM) progenitors in JS+ as compared with J+ or HD HSPCs (Fig 1A). Two HSC clusters were present, one of which (HSC_mpp2) principally comprised of JS+ cells and had a distinct molecular program (Fig 1A); upregulation in MK (eg. PF4, VWF) and EBM lineage signature genes (eg. TPSB2), interferon signaling genes but downregulation in apoptosis genes and canonical stemness markers (eg. CD133). These signatures suggest the JS+ driven HSC cluster may be MK/EBM-transcriptionally primed and may therefore, promote expansion of these abnormal progenitors which are implicated in driving fibrosis development. Through genotyping of >5000 HSPCs, we resolved the clonal architecture of JS+ MPN, observing that in most cases double mutant cells were present and clonally dominant, with mutant SF3B1 the initiating event. Mutations arose from the HSC level and genotypes were equally distributed in the HSPC compartment. Highlighting the importance of single-cell analysis, and not apparent at the bulk level, JAK2V617F and SF3B1K666mutationsresided in separate clones for one case with a WHO fibrosis score 0 in contrast to the other 4 cases with double mutant cells and fibrosis scores 1-3. This reinforces the cooperative effect of thesemutationsto drive fibrosis. We performed full-length transcriptomics of select HSPCs to study SF3B1mut-specific aberrant splicing events (ASE). In a global splicing analysis, more events were detected in JAK2V617F- SF3B1K666 double mutant than single mutant HSPCs. Retained intron (RI) events were the most prevalent ASE, followed by skipped exon and alternative 3' splice site events. We identified previously described SF3B1mut-specific ASE ( MAP3K7, ERGIC3 and SEPTIN6) confirming the validity of our dataset to study new ASE. Inter- and intra-patient comparison of SF3B1mut versus SF3B1wt cells uncovered a number of novel biologically relevant ASE, including two RI events in STAT1. These RI events were reduced in SF3B1mut HSPCs with the predicted consequence of increased STAT1 expression and activation, of direct relevance to MPN pathobiology since STAT1 is known to play a key role in enhancing megakaryopoiesis in MPN (Fig 1B). We validated these events using long-read Nanopore sequencing of single-cell cDNA libraries and have developed a cell model system with transient expression of SF3B1K666 mutation versus SF3B1wt in HEK293T to corroborate STAT1 ASE occurring as a direct consequence of mutant SF3B1. In further support of upregulated STAT1 activation, we observed upregulation of STAT1 downstream targets in abnormally expanded JS+ HSPCs. In conclusion, this analysis resolved the clonal architecture and hematopoietic cellular composition of JS+ MPN, identifying abnormally expanded and transcriptionally primed HSPC populations with abnormal splicing of STAT1 enhancing JAK-STAT signaling, uncovering the mechanism by which these mutations promote an accelerated fibrotic phenotype.

Multiparametric Flow Cytometry Based Quantification of the Marrow Hematopoietic Stem and Progenitor Compartment in Post Allogeneic Stem Cell Transplant Recipients with Poor Graft Function to Evaluate Hematopoietic Stem Cell Quality and Predict Clinical Outcomes: A Pilot Study

Poor graft function (PGF) is a poorly defined, commonly occurring (estimated 5 to 27%) 1 and difficult to treat complication of allogeneic stem cell transplantation (Allo-HSCT). Thrombopoietin agonists such as eltrombopag (EPAG) or CD34-selected cell infusions are treatments offered for persistent cytopenias. However, with limited knowledge of pathophysiology, there is a lack of reliable prognostic biomarkers. We aimed to investigate if assessment of the BM hematopoietic stem and progenitor cell (HSPC) landscape by multiparametric flow cytometry (MP-FCM) could help evaluate stem cell quality and predict clinical outcomes in PGF patients. We screened the UHN biobank repository to identify banked BM samples of patients who underwent diagnostic BM aspirate for PGF post allo-HSCT. Patients who had progressive cytopenia in one or more lineages after achieving engraftment while having full donor chimerism were included. Relapse as a cause of cytopenia was excluded. Controls in 1:1 ratio were Allo-SCT recipients with stable counts with banked day 60 BM aspirate. The blinded BM samples were analyzed with a 14-parameter FCM adapted from panels previously utilized to assess HSPC and mature lineages in cord blood xenograft studies (2). We selected 12 each of PGF and controls. The mean age of all recipients was 50 years and donors was 30.5 years. 23/24 cases had AlloSCT for hematological malignancies with 15 having AML or MDS. There were 14 unrelated (9 matched, 5 mismatched) and 10 related (5 matched sibling, 5 haploidentical) donors. Mean cell dose of infused graft was 6.66(± 1.98)x 10E6/kg CD34 cells. The significant difference between PGF and controls were recipient age (median 57y vs 28y), allo-SCT number (3 vs 0 with 2 nd transplant), intensity of conditioning regimen (myeloablative in 16% PGF vs 83% controls), and incidence of ≥ grade 3 acute graft vs host disease (GVHD) (n=3 vs 0). Most common causes of PGF were GVHD (n=6), infections (n=4) and drug effects (n=5). 11 PGF patients were treated with EPAG with 5 patients (A3, B2, B3, B5, C4) showing complete response, 3 (A6, B6, C5) showing partial response, and 3 (A2, A4, C9) showing no response. Patient C1 showed complete recovery without the use of EPAG, while C9, who initially did not respond to EPAG, showed a slow spontaneous recovery almost a year later. Amongst non-responders A2 and A4 received CD34 boost; with A4 showing a good response to the second boost. Analyzing the HSPC population by MP-FCM, we found that the PGF vs control groups show significant differences in the percentage of CD34+CD19+ B-Lymphoid precursors (B-LP) and CD19+ B lymphocytes. The mean ± SD percentage of B Lymphocytes and B-LPs in the PGF cohort were 6.76% ± 0.096% and 0.39% ± 0.005% respectively, while in the control group they were 20.09% ± 0.136% and 1.17% ± 0.012% respectively. When the 50 th centile value of the B-LPs (0.375%) or CD19+ B cells (10.89%) was investigated as a cut off to predict the clinical syndrome, this strategy achieved a 75% success rate with 3 PGF (blue bars) or control (black bars) samples misclassified (Fig1). Next, we investigated if these HSPC fractions could help predict the clinical outcome of PGF cases. The cases A2 and A4 who demonstrated no count recovery and needed CD34 boost lie in the lower quartile of both B Lymphocytes as well as B Lymphoid precursors content (Fig 1 red arrows). Conversely the three PGF cases in the upper 50 centile of these cell populations (patient B3, C4 and C9) all showed either a spontaneous or EPAG induced recovery of counts. We further investigated if the proportion of immunophenotypic CD34+CD38-CD45RA-CD90+CD49f+ long-term HSC (LT-HSC) could provide additional prognostic information (Fig2). Again, patients A2 and A4 had LT-HSC in the lower 50 th centile. Patient C1 who showed a spontaneous recovery of counts without the need of EPAG had the highest proportion of LT-HSCs despite lying in the second quartile of the cohort for both B cells as well as B-LPs. In conclusion, MP-FCM based analysis of the HSPC landscape in PGF marrow could be a robust prognostic biomarker in Allo-SCT patients.

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

Clinical and Hematopoietic Profiles Associated with Sustained Hydroxyurea Response for Patients with Sickle Cell Disease

Background: Hydroxyurea improves the lives of patients with sickle cell disease (SCD) by inducing expression of fetal hemoglobin (HbF). Higher HbF levels are associated with improved clinical outcomes. However, there is significant heterogeneity in long-term response to the drug. The impact of quantitative changes in hematopoietic stem and progenitor cells (HSPCs) during hydroxyurea treatment remains poorly characterized. Objectives: (1) Determine the prevalence and clinical impact of declining hydroxyurea response in pediatric patients with SCD; and (2) Characterize HSPC profiles associated with hydroxyurea response. Methods: We conducted a retrospective review of longitudinal clinical data from 760 pediatric patients with SCD seen at Texas Children's Hematology Center between 2010-2021. We selected patients consistently on hydroxyurea for >5 years for this study. We classified each subject according to their individual HbF response to hydroxyurea into: 1) Sustained-Response if the patient maintained an absolute HbF level >20% or 2) Decreased-Response if a patient's HbF levels decreased to <10% over time despite medication compliance. We obtained clinical and laboratory hydroxyurea treatment information for each individual. We isolated peripheral blood mononuclear cells (PBMCs) from whole blood collected from these patients during outpatient appointments. Patients were on a stable hydroxyurea dose without recent blood transfusions prior to collection. Flow cytometry was used to quantify the frequencies of ten HSPC populations in the PBMC samples. Results: In total, 208 children with HbSS or HbSβ 0-Thalassemia were included in our analysis: 148 individuals (71%) had a Sustained Response and 60 (29%) experienced a Decreased Response to hydroxyurea. Subjects with a Decreased Response had a median initial hydroxyurea induced HbF of 23.9% that decreased to 9.8% over time compared to stable values of HbF >20% in the HU-Sustained Response group (p<0.001). These differences were consistent after adjusting for age ( Figure 1). We found that patients who developed a Decreased Response to hydroxyurea experienced a significantly higher incidence of clinical complications, including number of visits to the emergency department (153.3 vs. 120.3 events per 100 patient-years), number of transfusions (37.4 vs. 11.9), and SCD-related hospitalizations (57.7 vs. 42.0). The causes of SCD-related hospitalizations included vaso-occlusive events requiring admission (27.7 vs 21.1 events per 100 patient-years), acute chest syndrome (13 vs 9.9), splenic sequestration (6.4 vs 3.5), and surgical procedures (7.47 vs 3.02). We also profiled HSPCs by flow cytometry using PBMCs collected from a subset of 40 patients (20 Sustained Response individuals vs. 20 Decreased Response). Individuals with a Sustained Response had increased numbers of hematopoietic multipotent cells (51.75% vs. 43%), hematopoietic stem cells positive for CD49F ++ (20.5% vs. 13.55%), and CD235a +/CD71 + cells (11.55% vs. 4.54%). In contrast, individuals with a Decreased Response had an increased percentage of unipotent hematopoietic progenitor cells (38.55% vs. 52.9%) and committed megakaryocyte erythroid progenitor cells (33.2% vs 46.2%; Figure 2). Conclusions: Nearly 30% of children with SCD taking hydroxyurea had a significant decline in their HbF levels after years of treatment and consequently suffered an increased number of clinical complications. While higher dosages of hydroxyurea may restore some HbF induction in these patients, we have quantified broad changes in specific HSPC levels that may be responsible for this decline in HbF. Patients with a Decreased Response had a lower percentage of multipotent progenitor cells that maintain self-regeneration capacity and increase numbers of committed erythroid progenitor cells. Although many stressors can impact quantitative and functional changes seen in HSPCs, these findings suggest that premature aging of the hematopoietic system plays an important role in long-term hydroxyurea response. Better understanding of why certain individuals are more susceptible to this phenomenon would enable the development of personalized strategies to enhance HbF induction and facilitate early selection of alternative treatments for these individuals with SCD.

Uncovering perturbations in human hematopoiesis associated with healthy aging and myeloid malignancies at single-cell resolution.

Early hematopoiesis is a continuous process in which hematopoietic stem and progenitor cells (HSPCs) gradually differentiate toward specific lineages. Aging and myeloid malignant transformation are characterized by changes in the composition and regulation of HSPCs. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize an enriched population of human HSPCs obtained from young and elderly healthy individuals.Based on their transcriptional profile, we identified changes in the proportions of progenitor compartments during aging, and differences in their functionality, as evidenced by gene set enrichment analysis. Trajectory inference revealed that altered gene expression dynamics accompanied cell differentiation, which could explain aging-associated changes in hematopoiesis. Next, we focused on key regulators of transcription by constructing gene regulatory networks (GRNs) and detected regulons that were specifically active in elderly individuals. Using previous findings in healthy cells as a reference, we analyzed scRNA-seq data obtained from patients with myelodysplastic syndrome (MDS) and detected specific alterations of the expression dynamics of genes involved in erythroid differentiation in all patients with MDS such as TRIB2. In addition, the comparison between transcriptional programs and GRNs regulating normal HSPCs and MDS HSPCs allowed identification of regulons that were specifically active in MDS cases such as SMAD1, HOXA6, POU2F2, and RUNX1 suggesting a role of these transcription factors (TFs) in the pathogenesis of the disease.In summary, we demonstrate that the combination of single-cell technologies with computational analysis tools enable the study of a variety of cellular mechanisms involved in complex biological systems such as early hematopoiesis and can be used to dissect perturbed differentiation trajectories associated with perturbations such as aging and malignant transformation. Furthermore, the identification of abnormal regulatory mechanisms associated with myeloid malignancies could be exploited for personalized therapeutic approaches in individual patients.

Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following Chimeric Antigen Receptor T Cell Therapy

Chimeric antigen receptor (CAR) T cell (CAR-T) therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from the release of proinflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias, and those with the most severe CRS also have the longest delays in full marrow recovery. Although an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown. This study was conducted to test our hypothesis that delayed bone marrow recovery following CAR-T therapy is caused by elevation of proinflammatory cytokines, leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs). SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR T cells. Bone marrow was then harvested for analysis by flow cytometry, and HSPCs were isolated for whole-transcriptome analysis by RNA sequencing. Complete blood counts and serum cytokine levels were measured as well. A second model was developed in which SCID-beige mice were treated with murine IFN-γ (mIFN-γ), murine IL-6 (mIL-6), or both. Bone marrow was harvested, and flow cytometry assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T therapy developed CRS, with elevations of several proinflammatory cytokines, including profound elevation of human IFN-γ. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Endothelial protein C receptor (EPCR)-negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFN-γ and mIL-6, whereas short-term HSCs and multipotent progenitors exhibited increases in proliferation with mIFN-γ treatment alone. The results from this study provide evidence that the elevation of proinflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFN-γ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFN-γ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias. © 2023 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Bnip3lb Regulated Mitophagy Maintains the Embryonic Pool of Hematopoietic Stem Cells By Protecting Them from ROS Induced Apoptosis

We have previously shown that Reactive Oxygen Species (ROS) promote Hematopoietic Stem and Progenitor Cell (HSPC) specification in the dorsal aorta, however, in the adult marrow it is established that HSPCs must maintain low levels of ROS to protect the stem cell pool against excessive cycling, differentiation, loss of stemness and apoptosis. We aim to elucidate the developmental timing and the mechanism by which HSCs transition from their initial dependence on ROS, to their precise regulation of it in the adult marrow. Since adult HSPCs regulate ROS by maintaining high levels of mitophagy, a process in which damaged mitochondria, which disproportionately generate ROS, are removed from the cell and recycled, we sought to define the initiation, regulation and impact of mitophagy in embryonic HSPC development. Our recent data shows that oxidative stress limits HSPC numbers as early as their colonization of the secondary hematopoietic niche, the Caudal Hematopoietic Tissue (CHT). This is the zebrafish analog of the mammalian fetal liver in that it is the tissue to which the HSPCs migrate after emerging from the hemogenic endothelium of the dorsal aorta. Once in the CHT they mature and proliferate before migrating again to their terminal niche in the marrow. In contrast to published data showing that ROS promotes HSPC specification in the dorsal aorta, when ROS levels were increased in the embryo using a morpholino against PRDX1 we saw a decrease in runx1/c-myb expression in the CHT. Conversely, when ROS was decreased using the antioxidant N-acetyl cystine (NAC), there was an increase in HSPC marker expression. By live imaging of zebrafish embryos expressing a recently developed fluorescent transgenic mitophagy-reporter, Tg(ubi:mitoGR), we observed that mitophagy is low in the hemogenic endothelial tissue which gives rise to HSPCs, but is upregulated as HSPCs emerge and begin to migrate to the CHT, and is occurring at high levels at the developmental stage when ROS begins to become damaging to the HSPC population. Our scRNAseq analysis and in situ-hybridization experiments allowed us to examine which genes and pathways were regulating the induction of mitophagy in these cells. These data show that the increase in mitophagy induction in the HSPC population at this timepoint coincides precisely with the initialization of expression of the bnip3lb mitophagy receptor gene. Fluorescent whole-mount in situ hybridization experiments demonstrated that bnip3lb is co-expressed with runx1 (a HSPC marker), in the CHT (figure 1), and, by examining our Tg(ubi:mitoGR) mitophagy reporter line after morpholino knock down of bnip3lb we confirmed that it is this gene which is driving mitophagy in HSPCs at this developmental stage. NIX, the murine homolog of bnip3lb, is responsible for developmentally regulated mitophagy in reticulocytes, lens fibers and macrophages, but has, thus far, had no reported role in maintaining HSPCs. Functionally, mitophagy appears to be essential for the survival of the HSPC population in the CHT. Reducing mitophagy via morpholino directed knockdown of bnip3lb has no effect on the initial specification of HSPCs in the dorsal aorta, but significantly decreases HSPC marker expression in the CHT, as measured by runx1/c-myb in-situ hybridization, and quantified by CD41:GFP HSPC reporter expression. In contrast upregulating mitophagy, which can be done via a novel heat-shock driven ΔOTC transgenic line or via small molecule mitophagy induction, elevates HSPC numbers in the CHT. Mechanistically, we found that bnip3lb knockdown increases detection of CellROX, a fluorescent dye which reports ROS levels, and increases the number of acridine orange+ apoptotic runx1+ HSPCs. Further, reducing ROS with antioxidant NAC reduces apoptosis and rescues the HSPC population (figure 2). We therefore propose that developmentally programmed mitophagy directed by bnip3lb is responsible for protecting proliferative embryonic HSPCs from the harmful effects of oxidative stress while the HSPC pool is expanding to seed lifelong hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigation of Impact Induced By AML1-ETO on Self-Renewal and Differentiation of Hematopoietic Stem and Progenitor Cells in Pre-Leukemic Stage

AML1-ETO fusion gene is the most common cytogenetic subtype of AML, which occurs in approximately 15% of acute myeloid leukemia (AML) patients. More than 85% of patients could achieve complete remission after induction chemotherapy. However, it was less effective on the patients over the age of 60 who cannot tolerate standard chemotherapy. And nearly 30% of patients relapsed eventually with a dismal outcome. Therefore, identification of the initiating abnormal cells to inhibit the early occurrence induced by AML1-ETO is an urgent problem. In this study, an inducible conditional AML1-ETO knock-in murine model (Aml1Eto/+; Mx1-Cre) was successfully established, hereafter called AEKI. The stage of malignant transformation in hematopoietic stem and progenitor cells (HSPC) was identified. We found that the absolute number of LT-HSCs (1.43×106 vs 4.85×103, p<0.0001), ST-HSCs (7.90×106 vs 1.88×104, p<0.0001) and MPPs (6.07×105 vs 1.82×104, p=0.0358) in bone marrow (BM) increased significantly in AEKI group compared with that of control group, while CMPs (8.13×103 vs 1.83×105, p=0.0029), GMPs (8.48×103 vs 3.12×105, p=0.0026) and MEPs (1.25×104 vs 2.03×105, p=0.001) decreased. It suggested that the activation of AML1-ETO in vivo resulted in excessive accumulation of HSCs and differentiation blockage from HSCs to HPCs. However, AML1-ETO alone was insufficient to induce the onset of overt leukemia after 300-day observation of survival. Furthermore, the characteristics of AEKI HSPCs in the pre-leukemic stage were investigated. Colony forming assay (CFC) was performed to evaluate the stemness of HSCs in vitro. The equivalent of 2×103 LT-HSCs were isolated from BM of each group and plated for 14 days. AEKI LT-HSCs gave rise to dysplastic colonies and lost replating capability compared to that of control group (colony counts: 19.33 vs 30.17, p=0.016). Moreover, a progressive decrease in PB chimerism was observed in AEKI recipients via competitive transplantation assay in vivo (15.8% vs 62.5%, p<0.0001), for which BM cellularity exhibited a significant decrease (7.24×106 vs 4.81×107, p<0.0001). But it led to an notably expansion in HSC number (LT-HSC:1.18×105 v 8.59×103, p=0.0035) which had a same tendency as the phenotype of primary mice. Based on Ki67 and DAPI staining, proportion of HSCs in G1 phase increased (59.85% vs 38.63%, p=0.0007), while that in G0 (33.58% vs 44.2%, p=0.045) and S/G2/M (4.86% vs 13.41%, p=0.0002) phases decreased. These results implied that AML1-ETO induction could reduce the quiescence and self-renewal ability of HSCs obviously in comparison with normal HSCs. Bulk RNA sequencing was performed in LT-HSCs and GMPs sorted from both groups. Genesets related to cell proliferation, cell cycle regulation and Myc targets were enriched in LT-HSCs from control group. It was also found that genesets of tricarboxylic acid (TCA) cycle, glycolysis and fatty acid metabolism were enriched in LT-HSCs from control group. To validate it, Seahorse extracellular flux assay was performed to evaluate oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on HSPC population (c-kitenriched cells). We found that OCR and ECAR both decreased significantly in AEKI group that supported the results from RNA-seq. RNA-seq data also showed that fatty acid metabolic pathway was enriched in AEKI group during the process from LT-HSCs to GMPs, which suggested that AEKI HSPCs maintained abnormal function through excessive fatty acid supply while it was not necessary during normal hematopoiesis. To confirm the above speculation, the mice were fed with fat-free or normal food after the AML-ETO induction in vivo to evaluate the impact of fatty acid on hematopoiesis. Interestingly, BM chimerism increased on the fat-free group (31.56% vs 15.68%, p=0.013), with the absolute number of LT-HSCs (5.16×105 vs 1.80×106, p=0.068), ST-HSCs (1.28×106 vs 2.72×106, p=0.026) and MPPs (4.70×104 vs 2.53×105, p=0.013) reduced obviously compared with in a normal diet. It suggested the excessive accumulation of HSCs was partly recovered. In summary, our study suggested that AML1-ETO induction could cause HSCs to lose self-renewal potential and lead to differentiation disruption during the pre-leukemic stage of AML. Limiting exogenous lipid uptake was able to rescue the phenotype of differentiation disrupt of HSPCs partly and it could be a promising therapeutic strategy.

Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold. Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production. Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34+ HSCs directly from patients with ITP and healthy controls. HSPC (CD45+ CD34+) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis. Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte). Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

Living Biomaterials to Engineer Hematopoietic Stem Cell Niches.

Living biointerfaces are a new class of biomaterials combining living cells and polymeric matrices that can act as biologically active and instructive materials that host and provide signals to surrounding cells. Here, living biomaterials based on Lactococcus lactis to control hematopoietic stem cells in 2D surfaces and 3D hydrogels are introduced. L. lactis is modified to express C-X-C motif chemokine ligand 12 (CXCL12), thrombopoietin (TPO), vascular cell adhesion protein 1 (VCAM1), and the 7th-10th type III domains of human plasma fibronectin (FN III7-10 ), in an attempt to mimic ex vivo the conditions of the human bone marrow. These results suggest that living biomaterials that incorporate bacteria expressing recombinant CXCL12, TPO, VCAM1, and FN in both 2D systems direct hematopoietic stem and progenitor cells (HSPCs)-bacteria interaction, and in 3D using hydrogels functionalized with full-length human plasma fibronectin allow for a notable expansion of the CD34+ /CD38- /CD90+ HSPC population compared to the initial population. These results provide a strong evidence based on data that suggest the possibility of using living materials based on genetically engineered bacteria for the ex-vivo expansion of HSPC with eventual practical clinical applications in HSPCs transplantation for hematological disorders.

Ubiquitin E3 ligase FBXO21 regulates cytokine-mediated signaling pathways, but is dispensable for steady-state hematopoiesis

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. By investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and exhibited low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoiesis-specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-fluorouracil injections resulted in a decrease in survival, despite these populations exhibiting minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results indicate that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine-mediated pathways.

The Role of Ubiquitin E3 Ligase, FBOX21, in Regulating Healthy and Malignant Hematopoietic Stem and Progenitor Cells (HSPC)

Hematopoietic stem cells allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. Hematopoietic cell fate decisions such as self‐renewal and differentiation are highly regulated through multiple molecular pathways and dysregulation can lead to the initiation of malignant hematopoietic disorders like acute myeloid leukemia (AML), immune deficiencies, and anemia. One pathway that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component, the protein that decides which substrate will be chosen for degradation, of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21was highly expressed in the hematopoietic stem and progenitor (HSPC) population, and was more highly expressed in this population when compared to other Fbox genes suggesting a possible role in maintaining the HSPC population. Likewise, Western blot confirmed high levels of FBXO21 in HSPCs and revealed low to no protein in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self‐renewal, and differentiation, we generated shRNAs against both Fbxo21 and FBXO21. We found that silencing Fbxo21/FBXO21 in healthy HSPCs and in human AML cell lines lead to a loss in colony formation, a decrease in proliferation, and an increase in differentiation towards the mature myeloid lineage. While overexpression of FBXO21 lead to an increase in colony formation and proliferation. This leads us to hypothesize that the ubiquitin E3 ligase, FBXO21, maintains the hematopoietic stem cell population while loss of Fbxo21/FBXO21 leads to a promotion in myeloid differentiation.We then generated the first conditional Fbxo21 knockout (KO) mouse model to delete Fbxo21 in hematopoietic cells. Our Fbxo21 KO mice exhibited no significant difference in primary sites of hematopoietic development, but display changes in hematopoietic cell populations in both young and aged mice. To further identify the substrates and pathway of FBXO21, we performed TMT Mass Spectrometry to analyze changes in protein levels upon knockdown of Fbxo21/FBXO21 in healthy HSPC and human AML cell lines. We found that both populations displayed an increase in proteins involved in the inflammatory response, the immune response primarily maintained by the myeloid cells through cytokine signaling. Which among both our Fbxo21 KO mice and human AML cell lines with FBXO21 knocked down, we see alterations in the cell’s response to certain cytokines, including IL‐6 and G‐CSF, upon Fbxo21/FBXO21 being knocked down. Therefore, deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

Hydrogel-Based 3D Culture Model for Down Syndrome Associated Transient Myeloproliferative Disorder Using Customized Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD.iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E).To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P<0.05). In colony forming unit (CFU) assay, the 3D generated HSPCs gave rise to a 2.0-fold higher number of CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colonies compared to 2D, with 2.0-fold decreased number of BFU-E (erythroid) colonies and a similar number of CFU-GM (granulocyte, macrophage) colonies (Fig. 1G). Thus, the low modulus synthetic matrix promoted hematopoietic differentiation producing higher percentage of early HSPCs as compared to the 2D culture system.We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K).In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. [Display omitted] DisclosuresBarwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.