MK Cells Research Articles

Assay optimization for the objective quantification of human multilineage colony-forming units

Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining and validating the identity of progenitor populations isolated by methods such as fluorescence-activated cell sorting (FACS). However, current methods for CFU assays are limited in their ability to robustly assay multipotent progenitors with the ability to differentiate down the myeloid, erythroid, and megakaryocytic lineages because of the lack of specific growth factors necessary for certain lineage outputs. In addition, manual counting of colony types is subjective resulting in user to user variability in assessments of cell types based on colony and cell morphologies. We demonstrate that the addition of granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF into a collagen-based MegaCult medium containing IL-3, IL-6, SCF, EPO, and TPO allows for the differentiation of common myeloid progenitors into expected proportions of colonies containing granulocytic (G), monocytic (M), erythroid (E), and megakaryocytic (Mk) cells. Additionally, we demonstrate an objective method using in situ immunofluorescence (IF) with anti-CD66b, anti-CD14, anti-CD235a, and anti-CD41 to detect G, M, E, and Mk cells, respectively. IF stained colonies can be analyzed individually at a microscope or using high-throughput microscopy. Thus, our improvements to the culture conditions and method for assay readout increase the accuracy, reproducibility, and throughput of the myeloid CFU assay.

Clinical Benefit Associated with Biomarker Changes Indicative of Disease Modification in Patients with Myelofibrosis Treated with the BET Inhibitor Pelabresib As Monotherapy or in Combination with Ruxolitinib

Bromodomain and extraterminal domain (BET) proteins play a key role in the regulation of neoplastic myeloproliferation and proinflammatory cytokine production in myelofibrosis (MF). Pelabresib (CPI-0610; PELA) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB signaling and other relevant genes involved in MF disease pathways (Mascarenhas J, et al. HemaSphere 2022;6:99-100). In the ongoing Phase 2 MANIFEST study (NCT02158858) (Kremyanskaya M, et al. Blood 2021;138:141) PELA is being evaluated as monotherapy in ruxolitinib (RUX)-intolerant, ineligible or refractory patients (pts) with MF (Arm 1) and in combination with RUX as an 'add-on' in pts with MF with suboptimal response to RUX (Arm 2) or in JAK inhibitor (JAKi)-naïve pts with MF (Arm 3). Assessment of ≥35% spleen volume reduction (SVR35) and ≥50% total symptom score reduction (TSS50) are standard clinical trial endpoints to evaluate disease response in MF. However, SVR35 and TSS50 do not fully reflect underlying disease improvement, and additional approaches are needed to understand what constitutes disease modification in MF (Pemmaraju N, et al. Cancer 2022;128:2420-2432) and to provide better insight into the mechanisms of novel drugs in development, such as PELA. Here we report results in pts enrolled in MANIFEST from exploratory analyses of treatment-induced changes at Week 24 in a range of biomarkers that are suggestive of disease modification (≥1 grade improvements in bone marrow fibrosis [BMF], reduction of Janus Kinase 2 V617F [JAK2V617F] variant allele frequency [VAF] in blood, improvements in hemoglobin and increased distance of CD61+ megakaryocytic [MK] cells in bone marrow ['declustering']) with respect to the clinical outcomes of SVR35 and TSS50. We previously demonstrated (Mascarenhas J, et al. HemaSphere 2022;6:99-100) that the increase in distance in MK cells correlated with SVR35 responses. Table 1 shows a summary of SVR35 and TSS50 response rates and changes in biomarkers associated with potential disease modification observed in the MANIFEST study in each study arm. Of note, with PELA monotherapy, 30% (7/23) of pts in a RUX-resistant/intolerant population (Arm 1) showed ≥1 grade improvement in BMF. Additional changes indicating biologic effect of PELA included declustering of MK cells (increase in distance of ≥15%) observed in 46% (5/11) of pts. Similar or better results were observed in pts treated with PELA/RUX combination. Changes in these markers of potential disease modification were associated with SVR35 and TSS50 endpoint responses. In evaluable pts with available data, splenic responses at Week 24 across the three arms were associated with ≥1 grade reduced BMF (in 18/43 pts, 42%), increased MK distance (in 20/26 pts, 77%, median change +34%) and JAK2V617F VAF reductions (in 41/50 pts, 82%, median change -13%). Of TSS50 responders at Week 24 with available data, 36% (16/44) showed ≥1 grade improvements in BMF; 71% (20/28) had an increase in MK distance (median change +22.3%); and 64% achieved reductions in JAK2V617F VAF (30/47, median change -8%). In JAKi-naïve pts (Arm 3), 68% (57/84) achieved SVR35 at Week 24; among these patients with available data, 38% (12/32) improved BMF ≥1 grade; 74% (14/19, median change +19.7%) increased MK distance; and 83% (34/41, median change -18%) reduced JAK2V617F VAF (any level of improvement). TSS50 was observed in 56% (46/82) of JAKi-naïve pts; among these patients with available data, improvements in ≥1 grade BMF, MK distance and JAK2V617F VAF (any level of improvement) were observed in 46% (11/24), 75% (12/16, median change +24%) and 78% (18/23, median change -14.3%) of pts, respectively. In nonresponders, changes in biomarkers were consistently smaller. Our data indicate differences in the association of individual markers of potential disease modification with SVR35 and TSS50. While reduction in JAK2V617F VAF and increase in MK distance appear to be associated with SVR35, a high incidence of BMF improvements were also observed in Arm 3 TSS50 responders. Extended multivariable, longitudinal, biomarker analyses for association with time-to-event endpoints (eg duration of splenic response) will be presented with a new data cutoff with the objective to investigate potential disease-modifying activity of PELA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Epigenetic Enzyme Mutations in Myeloid Malignancies Are Selected By Chromatin-Remodeling Requirements That Vary By Lineage- and Maturation-Stage

Introduction/Methods: Enzymes that modify histone H3 at lysine 27 (H3K27) to thereby regulate gene transcription (epigenetic enzymes) are recurrently inactivated by deletion and/or mutation in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemias (AML). Frustrating understanding of mechanisms is that writers and erasers of the same modification, e.g., methyltransferases that create, and demethylases that remove, H3K27 trimethylation (H3K27me3), a repression ('off') mark, are recurrently inactivated. Moreover, acetyltransferases that write H3K27 acetylation (H3K27ac), an 'on' mark mutually exclusive with H3K27me3, are also recurrently inactivated. One clue to underlying mechanisms is that MDS/MPN/AML present with diverse lineage and maturation phenotypes - perhaps these emerge from, or select for, the diverse epigenetic enzyme mutations. We therefore identified genes upregulated with specific myeloid lineage-commitment, maturation and function fates (~500 genes each) and then examined distributions of H3K27me3 and H3K27ac at these gene-loci in: (i) embryonic stem cells (ESC); (ii) hematopoietic stem and progenitor cells (HSPC); (iii) mature myeloid cells (monocyte [mono], pro-erythroblast, megakaryocyte [MK]); and (iv) AML cells.Results: Terminal-myeloid programs underwent substantial remodeling to gain H3K27ac ‘on’ mark from ESC/HSPC to mature myeloid (Fig.1A). Providing a mechanism for this, the H3K27 acetyltransferases EP300 and CREBBP were recruited into the RUNX1/SPI1 myeloid-lineage master transcription factor (MTF) hub by cooperation between their transcription activating domains. Mutated/translocated RUNX1, or mutated-NPM1 that cytoplasmically dislocated SPI1, disrupted this cooperation, and reverted hub content to default recruitment of histone deacetylases (HDAC) instead. Demonstrating cause-effect, inhibiting these HDAC renewed AML cell maturation to terminal lineage-fates. Meanwhile, MYC-target (proliferation) genes have high baseline H3K27ac in ESC and HSPC and do not require major remodeling during ontogeny (Fig.1A). An H3K27ac remodeling requirement for lineage-maturation but not proliferation/housekeeping explains selection pressure for inactivating mutations in EP300 or CREBBP, that we found in ~1.2% of MDS/MPN/AML in our (n=690) and other series. Consistent with pan-lineage-maturation needs for H3K27ac, the mutations were found in all lineage sub-types. H3K27me3 ‘off’ mark was mostly erased from myeloid programs in HSPC, but was greater at MK vs erythroid, and also at mono vs granulocyte genes (Fig.1B). This implied more need for H327me3 demethylase (KDM6A/UTX) for HSPC commitment into MK vs erythroid, or mono vs granulocyte, lineages. Accordingly, KDM6A was most upregulated in MK and mono-lineage cells (Fig.1C), and myeloid-conditional Kdm6a knockout decreased platelets and increased red cells in the spleen - reported by others: https://doi.org/10.1182/blood.V128.22.1467.1467. RUNX1-ETO has been shown to specifically impede granulocytic but not mono differentiation - https://www.nature.com/articles/2403396 and KDM6A inactivating mutations were significantly more likely to occur secondary to RUNX1-ETO (4-9% BEAT and AMLSG case series) vs other cytogenetics (0.005%). Selection for KDM6A secondary mutations could thus be to channel myeloid precursors toward lineages most efficiently impeded by primary mutations. Although H3K27me3 was substantially erased at all myeloid-commitment and terminal programs (except MK) in HSPC vs ESC, subsequent lineage-commitment and maturation entailed rewriting H3K27me3 at preceding HSPC and alternate lineage-fate programs, including at MTF genes for alternate fates (Fig.1B, D). Primary MDS/MPN/AML and AML cell lines with inactivating mutations/deletions in H3K27 methyltransferase EZH2 thus displayed aberrant co-expression of lineage MTFs and gene expression programs of normally mutually exclusive lineages (Fig.1E-G).Conclusion. Epigenetic remodeling requirements vary by myeloid lineage and maturation stage. Thus, epigenetic enzyme mutations are selected by, and cause, lineage-context of transformation. This knowledge can guide choice of specific epigenetic enzyme inhibitors to remedy the lineage-maturation defects that drive and define myeloid malignancies. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

SUMMARYMitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

The “Feedback” Regulation of Thrombopoiesis: The Role of Serotonin on Proplatelet Formation and TPO Production

We previously reported that serotonin (5-HT) is a growth factor for hematopoietic stem cells and megakaryocytic cells (Yang & Ye et al, Stem Cells, 2007; 2014). This study further proposed a possible role of serotonin on platelet formation and TPO production. Results showed that there was a stimulating effect of serotonin on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum free medium with either 5-HT (200nM) or TPO (50 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (11.33 % ± 4.93) vs. control (6 % ± 3.60), P=0.026; TPO (14.66 % ± 1.53) vs. control, P=0.043; n=3). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to TPO group. The effect of serotonin on proplatelet formation in Meg-01 cells were via 5-HT2 receptors.Meg-01 cells strongly expressed 5-HT 2A, 2B, 2C receptors by using western blot method. 5-HT also promoted proplatelet formation in these cells and this effect was reduced by 5-HT2 receptor inhibitor ketanserin (KE). Serotonin acted on cytoskeleton reorganization in human megakaryocytes via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group (serum free) than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, polymerization actin level was higher in 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced (5HT vs. Control, P=0.006; 5-HT vs.5-HT plus KE, P=0.014; n=6). Our data also demonstrated that ERK1/2 was activated in MK cells treated with 5-HT for 30 minutes (21.76 % ± 7.42). In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, the concentration of 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control. This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. It can promote the cells proliferation and reduce the apoptosis, and accelerate the maturation of megakaryocytes. The second is the indirect effect by promoting the production of TPO from bone marrow stromal cells, which is a paracrine secretion to influence thrombopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Megakaryocytic Microparticles for Gene- and Cell- Therapies

Introduction: Cell-derived microparticles (MPs) are 0.1 to 1 micron extracellular vesicles (EVs), budding off cellular plasma membranes under stress or activation. MPs play an important role in cell-to-cell communication by transferring cargo from parent to target cells. Among circulating MPs, megakaryocyte-derived MPs (MkMPs) are the most abundant MPs in circulation (Flaumenhaft et al., 2009). We have shown that, in vitro, MkMPs specifically target and are taken up by human hematopoietic stem & progenitor cells (HSPCs) via fusion or endocytosis following specific receptor recognition (Fig.1A) (Jiang et al., 2017). MkMPs transfer cargo to HSPCs and induce potent Mk differentiation of HSPCs in the absence of thrombopoietin (Jiang et al ., 2014). Here, we explore the capability of human MkMPs to transfer DNA and siRNA to HSPCs, and their ability to interact with HSPCs in vivo using a simple murine model. We aim to develop MkMP-based strategies for gene- and cell- therapies.Methods: Human MkMPs (huMkMPs) were isolated and purified from CD34+-derived Mk cells (Jiang et al., 2014). Electroporation was used for cargo loading to MkMPs. Cy5-labeled plasmid DNA (pGFPns; expressing eGFP) was loaded into MPs at two pDNA-to-MkMP ratios. siRNA (siR-MYB) targeting c-myb or Alexa 647 non-targeting siRNA were separately loaded into MkMPs. Silencing of c-myb enhances Mk differentiation of HSPCs (Bianchi et al ., 2010). Loading efficiencies were calculated after pDNA purification and quantification, or analyzed by flow cytometry (FC). Cargo delivery to HSPCs was enabled by co-culturing loaded MkMPs with HSPCs using an optimized protocol. Delivery of pGFPns was assessed by pDNA quantification, eGFP RNA levels by qRT-PCR, and eGFP expression by FC. siR-MYB-loaded MkMPs were co-cultured with HSPCs, and cells were analyzed by FC for CD41 (an Mk marker) expression at days 3, 5 and 8. To test the hypothesis that huMkMPs interact with murine HSPCs (muHSPCs) in vivo, 6×106 of MkMPs were administered into BALB/c mice intravenously via tail vein injection. At day 3, blood was collected and murine platelet (muPLT) levels measured by FC.Results: We developed a protocol to enable us to load ca. 1700 copies or 30 ng of pGFPns into MkMPs, with over 77% of MkMPs becoming Cy5+ after electroporation (Fig. 1B). In MkMP-huHSPC co-culture experiments, delivery of and expression from pGFPns was first confirmed by isolating pGFPns from and measuring eGFP mRNA in HSPCs at days 1 and 3, respectively. At day 3, over 45% of HSPCs were Cy5+ while 20% expressed eGFP (Fig. 1C), indicating effective, MkMP-mediated pGFPns delivery to HSPCs with functional biological performance. We assessed functional RNA delivery by examining the impact of siR-MYB mediated c-myb silencing in enhancing Mk differentiation of CD34+ cells beyond the enhancing impact of unloaded MkMPs. MkMP delivery of siR-MYB enabled c-myb silencing that resulted in enhanced Mk differentiation beyond that of unloaded MkMPs: the % of CD41+ and CD34-CD41+ cells increased by 29 % (Fig. 1D) and 25 %, respectively, at day 8 of co-culture versus control (unloaded MkMPs), indicating functional delivery of siR-MYB via MkMPs. In vivo murine experiments in wild-type mice were to test if huMkMPs would interact with muHSPCs, and if so, if they would induce muHSPCs into murine Mk differentiation that would result in increased muPLT numbers. Injection of huMkMPs enhanced muPLT levels by 28 % at day 3 (Fig. 1E), indicating huMkMPs target and induce muHSPCs into Mk differentiation and de novo muPLT generation. In another set of experiments, anti-CD41-antibody induced thrombocytopenia in mice (Apostolidis et al ., 2012) was rescued by huMkMP injection, further supporting the hypothesis that huMkMPs target and deliver cargo to muHSPCs.Conclusions: This is the first report showing functional MP-mediated pDNA delivery to any cell type. This is also the first report showing that HSPCs can be specifically targeted and cargo delivered using MPs. Moreover, functional siRNA delivery via these MkMPs demonstrate the potential of this delivery system for targeting the stem-cell compartment. Surprisingly, huMkMPs were able to target muHSPCs resulting in higher muPLT levels. Overall, based on our data and the unique characteristics of huMkMP, MkMPs constitute a potentially useful therapeutic delivery system for gene- and cell-therapy, with applications in regenerative and transfusion medicine. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite. Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model. Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model. Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Mouse Erythroid Cells Originate from a Megakaryocyte Precursor in Common Myeloid Progenitors

The hierarchical model of hematopoiesis posits that hematopoietic stem and progenitor cells produce common myeloid progenitors (CMP). CMP can become granulocyte/monocyte progenitors (GMP) or bipotential megakaryocyte/erythroid progenitors (MEP). MEP can produce megakaryocytic (Mk) or erythroid (Ery) cells. However, we and others have shown that early mouse and human progenitor populations express many Mk genes (Heuston, Epig. Chrom., 2016), while single cell studies have identified lineage-specific colony forming cells in progenitor populations thought to be multipotent (Psaila, Genome Biol., 2016). To identify the earliest mouse Ery and Mk cells, we performed single cell RNASeq on 10000 stem and progenitor cells (Lin-Sca1+Kit+), 12000 CMP (Lin-Sca1-Kit+CD16/32-CD34+), 6000 MEP (Lin-Sca1-Kit+CD16/32-CD34-) and 8000 GMP (Lin-Sca1-Kit+CD16/32+CD34+). TSNE analysis of expression in the 4 populations identified 33 clusters, which were correlated to biological functions using gene set enrichment analysis. In LSK, no cells with an Ery RNA profile were found, while 56% of cells co-expressed Mk-associated (e.g., Meis1, Fli1) and lymphoid genes. In CMP, 12% of the cells co-expressed Ery (e.g., Gata1, Fog1) and Mk (e.g., Pf4, Cd41) genes, while 23% had an Mk-specific profile (e.g., Fli1, Cd41) enriched for platelet biology processes (p< 3E-18). Unlike traditional models, over 94% of MEP had Ery RNA profiles enriched for ribosome synthesis and heme-biology processes (p< 4E-10). To establish developmental relationships, we performed pseudotime analysis using the Monocle and Scanpy software packages. These programs model differentiation by mapping similar transcriptomes together. Map nodes indicate lineage commitment points and cells further from a node are more differentiated. Combined analysis of LSK, CMP, and MEP generated a model with a single node and 2 trajectories. LSK with Mk and lymphoid RNA profiles diverged at the node, as did 14% of CMP. 31% of CMP with an Mk RNA profile were downstream of the node. Further downstream were cells with mixed Ery/Mk profiles, and furthest from the node were MEP with Ery profiles. A separate pseudotime analysis of CMP only 2 trajectories: one with decreasing Mk- and increasing Ery RNA profiles, and a second with an early Mk endomitotic RNA profile. Pseudotime analysis of MEP only identified a linear trajectory: cells at one end expressed early Ery RNA profiles, and cells at the other end had RNA profiles similar to those of burst-forming unit-erythroid (BFU-E). We generated a predictive set of RNAs for each TSNE cluster. We used index-sorting with 11 markers (Kit, Sca1, CD34, CD16/32, CD36, CD41, CD48, CD123, CD150, CD9, Flk2) to isolate single cells for custom high-throughput multiplex qPCR. This allowed confirmation of cell frequency within TSNE clusters while identifying surface markers for prospective isolation of cell subsets. We focused on 2 populations: CMP-E, which had an Ery RNA profile (10% of clustered CMP and 12% of CMP in the qPCR assay), and CMP-MkE, which had Mk and Ery RNA profiles (12% of clustered CMP and 13% of CMP in the qPCR assay). We prospectively isolated CMP-E and CMP-MkE to compare RNASeq profiles, ATACSeq profiles, and colony forming ability against those of bulk CMP, Ery, and Mk. In CMP-E, 54% of RNAs were expressed in both CMP and ERY, while 41% were expressed only in CMP (p < 6E-72). In contrast, 41% of CMP-E ATACSeq peaks were present in CMP and ERY, while 57% of CMP-E peaks were present only in CMP (p < 1E-3). We conclude that in CMP-E, the RNASeq profile is more erythroid than the ATACSeq profile. In CMP-MkE, 89% of RNAs were expressed in both CMP and Mk, while 7% were expressed only in CMP (p < 8E-190). Likewise, 88% of CMP-MkE ATACSeq peaks were present in both CMP and Mk, while 3% were present only in CMP (p < 1E-3). We conclude that in CMP-MkE, the RNASeq and ATACSeq profiles are equivalent. In soft agar assays, 21% of CMP-E and 3% of CMP-MkE colonies contained BFU-E, compared to 9% of control colonies. We conclude that the CMP-E and CMP-MkE populations are skewed towards the ERY and MK lineages, but are not erythro-megakaryocyte restricted. Our data support a model in which there are two megakaryocyte precursor populations and no erythroid populations in LSK. A third megakaryocyte population in CMP gives rise to erythroid cells. Finally, our data show that transcriptional changes precede chromatin accessibility changes in the earliest erythroid cells. Disclosures No relevant conflicts of interest to declare.

Keratinocyte Integrin α3β1 Promotes Secretion of IL-1α to Effect Paracrine Regulation of Fibroblast Gene Expression and Differentiation

After cutaneous injury, keratinocytes secrete paracrine factors that regulate wound cell functions; dysregulation of this signaling can lead to wound pathologies. Previously, we established that keratinocyte integrin α3β1 promotes wound angiogenesis through paracrine stimulation of endothelial cells. We hypothesize here that α3β1-dependent paracrine signaling from keratinocytes regulates the differentiation state of myofibroblasts. We report that epidermal α3-knockout mice exhibit more wound myofibroblasts and fewer cyclooxygenase 2 (Cox-2)-positive dermal cells than controls. We also found that conditioned medium from α3-expressing mouse keratinocytes (MKα3+), but not from α3-null MK cells (MKα3-), induces expression of Cox-2 in fibroblasts in a time- and dose-dependent manner and that this induction is mediated by IL-1α. Compared with MKα3- cells, MKα3+ cells secrete more IL-1α and less IL-1RA, a natural IL-1 receptor antagonist. Treatment with an IL-1α neutralizing antibody, recombinant IL-1RA, or IL-1 receptor-targeting small interfering RNA suppresses MKα3+ conditioned medium-dependent induction of Cox-2 expression in fibroblasts. Finally, active recombinant IL-1α is sufficient to induce Cox-2 in fibroblasts and to inhibit transforming growth factor-β-induced α-SMA expression. Our findings support a role for keratinocyte integrin α3β1 in controlling the secretion of IL-1α, a paracrine factor that regulates the wound myofibroblast phenotype.

Single Cell RNASeq Demonstrates That Mouse Erythroid Cells Are the Last Lineage to Emerge

The hierarchical model of hematopoiesis posits that hematopoietic stem cells (HSC) give rise to myeloid progenitors (CMP), that can become further restricted to bipotential granulocyte/monocyte progenitors (GMP) or megakaryocyte/erythroid progenitors (MEP). We and others have shown that this model may not accurately depict hematopoiesis. Recent studies have shown that shown that populations of mouse hematopoietic stem and progenitor cells (LSK) have a strong megakaryocyte (Mk) transcriptional profile (Heuston, 2018, Epig. & Chrom.), and single cell studies have identified lineage committed cells in progenitor populations thought to be multipotent. For example, we recently reported that human MEP contain 3 populations: erythroid (Ery) primed, Mk primed, and bipotential (Psaila, 2016; Gen. Bio.).To determine when Mk and Ery cells emerge during mouse hematopoiesis, we performed single cell RNASeq on 10000 LSK, 12000 CMP, 6000 MEP and 8000 GMP cells. Clustering analysis (Satija, 2018, Nat. Biotech.) of all 4 populations identified 33 transcriptionally distinct clusters. In 30 of 33 clusters, 85% of cells were from a single defined population (e.g. MEP). LSK and CMP clusters grouped closely together.We used gene set profiling (Gene Set Enrichment Analysis, GO and KEGG) to correlate transcriptional profiles of clusters with specific hematopoietic lineages and cellular activities. In LSK, the most common transcriptional profiles correlated with active cell cycling. Mk-associated genes (Meis1, Myct1, and Fli1), were co-expressed with lymphoid genes in 56% of all LSK. Consistent with previous studies, we conclude that cells with Mk transcriptional profiles are abundant in LSK. No cells with an Ery RNA signature were observed in LSK.23% of all CMP cells expressed Mk genes (e.g., Pf4, Itga2b, and Fli1) and were enriched for processes involved in platelet biology (p < 3E-18). 12% of CMP had an Ery RNA signature (low expression of Gata1, Klf1, and Nfe2) and decreased Mk gene expression (e.g., Gata2 and Gfi1b, [p < 3E-18]) compared to other CMP clusters. The high ratio of Gata2/Gata1 expression (1.90) suggests that this cluster contained immature Ery cells.More than 94% of all mouse MEP had Ery RNA signatures. Clusters could be distinguished by gene expression (e.g., Gata1, Klf1, Tfrc) and biological processes (ribosome synthesis and heme-biology processes [p < 4 E-10]). Based on the transcriptional profiles, we determined the most mature erythroid cells in MEP were late BFU-E.To compare the differentiation of Mk and Ery cells, we pooled our LSK, CMP, and MEP data for analysis using the Monocle software package. GMP contained only clusters expressing granulocytic or monocytic genes and were excluded from the analysis. Monocle arranges cells into trajectories based on their transcriptional profiles, with more differentiated cells positioned further from a common node (Xiaojie, 2017, bioRxiv). We found that LSK cells near the node had overlapping lymphoid and Mk transcriptional profiles. Closest to the node, we found 38% of CMP expressed a profile similar to LSK. An additional 45% of CMP formed one trajectory with lymphoid and granulocyte RNA signatures. Another 17% of CMP formed a second trajectory, with cells expressing an Mk signature closest to the node, cells with a mixed Ery/Mk signature further along the trajectory, and MEP cells with Ery-only signatures furthest from the node. To clarify the Mk/Ery divergence, we focused our analysis on the CMP populations expressing Mk RNAs (Figure1). We observed cells in G1/S phase with an immature Mk signature to the left of the node where the trajectories diverge. On the right, cells with immature Mk signatures were nearest the node and cells with a mixed Ery/Mk signature were at the end of the trajectory (upper right; Mk/Ery). Along the second trajectory, rapidly cycling G2/M Mk cells with an early endomitosis-associated RNA signature (e.g., Pf4, Gp1bb, Gp9, and Vwf) were located at the end of the trajectory (lower right; Mk early endomitosis).Our data are consistent with a model in which two waves of Mk differentiation begin in LSK and progresses to CMP. The Mk lineage is divided in CMP, producing cells that begin endomitosis and cells that have an Mk-repressing/Ery-activating cell program that gives rise to the Ery lineage. We conclude that the erythroid lineage is derived from an Mk-like precursor and is the last lineage to be specified in mouse hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiRNAs from Megakaryocytic Microparticles Mediating Megakaryocytic Differentiation of Hematopoietic Stem & Progenitor Cells in the Absence of Thrombopoietin

Introduction:Cell-derived microparticles (MPs) are submicron-size extracellular vesicles (EVs), playing an important role in cell-to-cell communication via cargo delivery from source to target cells. Megakaryocytic microparticles (MkMPs) are the most abundant MPs in circulation (Flaumenhaft et al., 2009). MkMPs target and deliver cargo specifically to hematopoietic stem & progenitor cells (HSPCs) through receptor-mediated fusion or endocytosis (Jiang et al., 2017), thus inducing HSPC megakaryocytic differentiation in the absence of thrombopoietin (TPO) (Jiang et al., 2014). Transfer of MkMP RNA to HSPCs is largely responsible for the observed effects (Jiang et al., 2017). Given that all MPs are highly enriched in miRNAs, we hypothesized that MkMP miRNAs mediate HSPC megakaryocytic differentiation in the absence of TPO. Thus, we used RNAseq to identify the miRNA content of MkMPs (as well as the miRNA content of control Mk cells, platelets and platelet MPs (PMPs)), and examined which MkMP miRNAs might promote HSPC megakaryocytic differentiation in the absence of TPO.Methods:Human MkMPs were isolated and purified from CD34+-derived Mk cells (Jiang et al., 2014). Total RNA from Mks, MkMPs, platelets and PMPs were extracted and small RNAs were selected for library preparation. RNAseq was performed using Ilumina HiSeq. Differential miRNA expression was analyzed via the EdgeR Bioconductor. To examine the impact of the seven most abundance miRNAs on Mk differentiation of HSPCs, individual, or combinations of, miRNA mimics were transfected into CD34+ HSPCs and cultured in the absence of TPO. Cells were analyzed by flow cytometry (FC) for CD41 (Mk marker), CD235a (erythroid marker) expression and total cell count at days 4, 7 and 10. To further examine the impact of these miRNAs, miRNA inhibitors corresponding to these seven miRNAs were loaded into MkMPs via electroporation. miRNA-inhibitor-loaded MkMPs were co-culture with CD34+ HSPCs, and CD41, CD235a, and CD42b (a late Mk marker) were analyzed by FC at days 3, 5, and 8.Results:miRNA sequencing and differential expression analysis showed that the top seven most abundant miRNAs (Fig. 1A) account for more than 57% of miRNAs in MkMPs, with 58 miRNAs being enriched in MkMPs from Mks (Fig. 1B). Similarly for differential expression vs platelet or PMP miRNAs. We hypothesized that a combination of these seven miRNAs mediates Mk differentiation of HSPCs. To test this hypothesis, miRNA mimics were transfected individually or in combinations into CD34+ HSPCs. Individual miRNA transfection showed that miR-486-5p, miR-191-5p, and let-7f-5p promote Mk differentiation by enhancing CD41 expression on days 7 & 10 (Fig. 1C), while miR-26a-5p enhanced erythropoiesis (higher CD235a expression) and suppressed megakaryopoiesis (lower CD41 expression) late in culture (Figs. 1C & 1D). While miR-22-3p promoted cell expansion (Fig. 1E), no effect on Mk differentiation was found. Transfection of multiple miRNAs demonstrated the positive role of miR-486-5p and miR-92a-3p in promoting Mk differentiation, and the importance of miR-486-5p, miR-26a-5p, miR-92a-3p, and miR-126-5p in early HSPC expansion. Furthermore, inhibition of miR-486-5p in MkMPs abolished the impact of MkMPs on programing Mk differentiation of CD34+ HSPCs by lowering CD41 expression after 8 days of co-culture.Conclusions:This is the first report of the MkMP miRNA profile. We identified seven major MkMP miRNAs mediating cell expansion and Mk differentiation of HSPCs in the absence of TPO, thus suggesting that suitable miRNA combinations can mimic TPO signaling. The latter is the focus of on-going studies in our lab by examining the impact of these miRNAs on TPO-induced signaling pathways: Jak/STAT, PI3K/Akt and MAPK. Our study elucidates the biological effect of MkMPs to HSPCs, and demonstrates the importance of combinatorial vs single miRNA studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Events Defining Human Clonal Hematopoiesis at Single Cell Resolution

For decades, hematopoiesis has been described as a cellular hierarchy maintained by self-renewing hematopoietic stem cells (HSCs) that proceed through a series of multipotent, oligopotent and then unipotent progenitors to make blood. This can be regarded as the standard "textbook" model that is widely used as the basis to interpret molecular regulatory processes, disease processes, and develop therapeutic approaches. The presence of oligopotent intermediates is crucial to the model since they define the path from multipotent cells to unipotent cells. Common myeloid progenitors (CMPs) represent the critical oligopotent progenitor from which all My (defined as granulocyte/monocyte), erythroid (Er), and megakaryocyte (Mk) cells arise. Although the standard model is still used extensively as an operational paradigm, further cell purification and functional clonal assays have led to key revisions of the model. Through the development of more efficient assays to monitor My and lymphoid fates in single cell stromal assays and an improved sorting scheme, we previously identified human multilymphoid progenitors (MLP) as the earliest lymphoid differentiation precursor with concomitant lymphoid (T, B, NK) and myelomonocytic potential, rather than common lymphoid progenitors (CLP) (Doulatov et al, Nature Immunology 2010). Murine studies came to the same conclusion. These results raise the question of whether the opposite arm of this lineage split (My, Er, and Mk fate) is also more complex than previously thought. Indeed strong evidence has emerged from murine studies for revision to the view of murine My-Mk-Er lineage development. Until recently considerable uncertainty remained concerning the myelo-erythroid branch of human hematopoiesis. Through a novel cell-sorting scheme and a more sensitive assay to assess multilineage My, Er, and Mk fate potential contemporaneously from single cells we have provided a new framework to understand normal and disease states of human hematopoiesis (Notta et al Science 2016). These studies suggest that oligopotent progenitors are a negligible component of the human hematopoietic hierarchy of adult bone marrow. Rather, multipotent cells differentiate into unipotent cells of the My-Er-Mk lineages directly support the concept of a 'two-tier' human blood hierarchy composed of two-tiers: a top-tier containing multipotent cells such as HSCs and MPPs, and a bottom-tier composed of progenitors committed directly to My, Er, or Mk lineages. Second, the blood hierarchy is not identical across development. In human fetal liver (FL), oligopotent progenitors with My-Er-Mk and Er-Mk activity were a prominent component of the hierarchy. By contrast, the adult bone marrow (BM) was dominated by unilineage progenitors with primarily My or Er potential. Finally, our study has documented multiple origins of where Mk cells arise. We found that Mk branching differs in FL and BM. In FL, Mk progenitors were enriched, but not restricted, to the stem cell compartment; while in BM, Mk fate was closely tied to multipotent cells. This result fits the two-tier model of adult hematopoiesis since branching of Mk occurs in the top tier, at the level of HSC/MPPs. To gain a deeper understanding of the molecular basis for the two tier hierarchy, we have undertaken low cell input RNA sequencing, Enhanced Reduced Representation Bisulfite Sequencing (ERRBS), and ATAC-seq to provide a comprehensive transcriptional and epigenetic roadmap of human HSPC across development.

The Human Blood Hierarchy Is Shaped By Distinct Progenitor Lineages Across Development

Background: The hematopoietic roadmap is a compilation of the various lineage differentiation routes that a stem cell takes to make blood. On several occasions over the last six decades, the murine roadmap has been reconceived due to new information overturning old dogmas. The human roadmap, which describes the extraordinary throughput of more than three hundred billion cells daily has changed little, with the classical model of hematopoiesis still prevailing. In this model, blood differentiation initiated at the level of stem cells must pass through a series of increasingly lineage-restricted oligopotent and then unipotent progenitor intermediates. As cellular differentiation flows from stem cells to unipotent cells, the oligopotent progenitors are the quintessential step where lineage choices are consolidated. One such critical oligopotent intermediate is the common myeloid progenitor (CMP), believed to be the origin of all myeloid (My), erythroid (Er) and megakaryocyte (Mk) cells. While murine studies are beginning to challenge the existence of oligopotent progenitors, a comprehensive analysis of human myelo-erythroid differentiation is lacking. Moreover, whether the pool of oligopotent intermediates is fixed across human development is unknown. The classical roadmap is the only guide that describes human hematopoiesis and consequently our view of blood homeostasis and disease is intimately tied to this conception.Rationale: The differentiation roadmap taken by human hematopoietic stem cells (HSCs) is fundamental to our understanding of blood homeostasis, hematopoietic malignancies and regenerative medicine.Results: We mapped the cellular origins of My, Er and Mk lineages across three timepoints in human blood development: fetal liver (FL), neonatal cord blood (CB) and adult bone marrow (BM). Using a new cell-sorting scheme based on markers linked to Er and Mk lineage specification (CD71 and BAH1), we found that previously described populations of multipotent progenitors (MPPs), CMPs, and megakaryocyte-erythroid progenitors (MEPs) were considerably heterogeneous and could be further purified beyond current definitions. Nearly 3000 single cells from 11 cellular subsets from the CD34+ compartment of FL, CB and BM (33 subsets in total) were evaluated for their My, Er and Mk lineage potential using an optimized single-cell assay.In FL, the ratio of cells with multilineage versus unilineage potential remained constant in both the stem cell (CD34+CD38-) and progenitor cell (CD34+CD38+) enriched compartments. By contrast in BM, nearly all multipotent cells were restricted to the stem cell compartment, whereas unilineage progenitors dominated the progenitor cell compartment. Oligopotent progenitors were only a negligible component of the human blood hierarchy of adult BM leading to the conclusion that multipotent cells differentiate into unipotent cells directly.Mk-Er activity predominately arose in the stem cell compartment at all developmental timepoints. In CB and BM, most Mks emerged as part of mixed clones from HSCs/MPPs, indicating that Mks directly branch from a multipotent cell and not from oligopotent progenitors like CMP. In FL, a striking 80% of single-cell clones with Mk activity were derived from a novel progenitor in the stem cell compartment, although less potent Mk progenitors were also present in the progenitor compartment. Interestingly, in a hematological condition of HSC loss (aplastic anemia), Mk-Er but not My progenitors were more severely depleted, pinpointing a close physiological connection between HSC and the Mk-Er lineage.Conclusion: Our data indicate that there are distinct roadmaps of blood differentiation across human development. Prenatally, Mk-Er lineage branching occurs throughout the cellular hierarchy. By adulthood, both Mk-Er activity and multipotency are restricted to the stem cell compartment, whereas the progenitor compartment is composed of unilineage progenitors forming a 'two-tier' system, with few intervening oligopotent intermediates. DisclosuresNo relevant conflicts of interest to declare.

Trisomy 21-Associated Abnormalities in IGF Signalling and the Fetal Microenvironment Both Contribute to Disruption of Fetal Hematopoiesis in Down Syndrome

In Down syndrome (DS), trisomy 21 (T21) causes perturbation of fetal liver (FL) hematopoiesis leading to expansion of megakaryocyte-erythroid (MK-E) progenitors. On this background, FL hematopoietic stem/progenitor cells (HSPC) may acquire GATA1 gene mutations which usually present as a neonatal preleukemic condition, Transient Myeloproliferative Disorder (TMD). TMD develops only in fetal life and most cases spontaneously regress after birth, suggesting unique features of the T21 fetal microenvironment may be important in driving both abnormal DS FL hematopoiesis and mutant GATA1 clones in TMD. Previous work showed activation of insulin-like growth factor (IGF) signalling in TMD and DS-AMKL and fetal-specific IGF dependence of murine MK development implicating T21 in altered fetal IGF signalling.To investigate the role of IGF signalling in fetal MK-E development in DS FL without GATA1 mutations, we performed gene expression profiling (GEP) of DS FL (n=5) and normal FL CD34+ cells (n=3). GSEA showed significant enrichment of IGF targets. We therefore measured expression of the principal IGF receptor, IGF1R, on DS FL HSPC (n=6). Surface IGF1R was uniformly expressed on all DS FL HSPC subpopulations. However, there was no difference in IGF1R surface or gene expression between DS and normal FL HSPC (n=8) suggesting that increased IGF1R expression on TMD and DS-AMKL cells is an early event and may reflect their fetal rather than T21 origin. To determine if the enrichment of IGF targets in DS FL HSPC might reflect increased IGF production in the FL microenvironment, we compared IGF expression in DS FL mesenchymal stromal cells (MSC)(n=4) and hepatocytes with normal FL MSC (n=4). Both DS and normal FL MSC expressed large amounts of IGF2, but little or no IGF1 with no difference between DS and normal MSC. Similarly, IGF2 was expressed by IHC at similar levels in both DS and normal fetal hepatocytes and MSC whereas IGF1 was barely detectable. Normal and DS fetal bone marrow (BM) MSC also expressed high levels of IGF2, but not IGF1 in contrast to adult BM MSC. Thus, IGF2 is the main IGF produced in the human fetal hematopoietic environment. Consistent with a specific role in DS FL hematopoiesis, IGF2 caused marked proliferation of DS FL clonogenic MK and MK-E cells (n=3) and stimulated MK proliferation in liquid culture while in normal FL, IGF2 stimulated EPO-independent BFU-E and Pre-BFU-E but had no effect on MK cells. Since differential effects of IGF2 on DS/normal FL HSPC might be due to altered expression of the Insulin Receptor (InsR), which binds IGF2 with low affinity, we compared InsR surface and gene expression in DS and normal FL HSPC but found no significant difference. IGF2 also binds to IGF2R, a negative regulator of IGF2, however there was no difference in IGF2R expression between DS and normal FL HSPC. Co-culture of normal FL CD34+ cells with DS or normal FL MSC (n=3) promoted MK and erythroid cell proliferation and differentiation. Since the effect of DS FL MSC on normal FL CD34+ cells was significantly greater (~4-fold) than that of normal FL MSC, the data above suggest this is unlikely to be due to IGF2 alone. To investigate whether T21 alters the MSC secretome, we performed GEP of DS FL MSC (n=3) and compared this with normal FL MSC (n=5). Both normal FL and DS FL expressed known hematopoietic cytokine genes, including SCF, CXCL12, G-CSF, M-CSF, Flt3 and IL-6, as well as IGF2 but not IGF1, EPO or TPO. Of the top 80 differentially expressed genes (Fold Change>2), ~40% encode secreted proteins, including several which affect IGF availability.Conclusion: T21 perturbs FL hematopoiesis through both HSPC-intrinsic mechanisms, including changes in IGF responsiveness, and through alterations to the FL microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Alternative Methods Of Thrombocytopenia: Patients With ITP Have Increased Megakaryocyte/T Cell Interactions In The Bone Marrow and Higher Serum TRAIL Levels

AbstractThe pathology of immune thrombocytopenia (ITP) is not fully understood. Although antiplatelet antibodies (APAs) are thought to be causative, these are not detected in up to 30% of patients. In addition, T cell directed therapy is frequently successful. Patients with ITP display cytotoxic activity towards platelets, and abnormalities in T cell repertoire. However, there is incomplete understanding of the interactions between T cells and megakaryocytes within the bone marrow. We hypothesized that if T cell or NK cell cytotoxicity against megakaryocytes contributes to thrombocytopenia, then T and/or NK cells should be in direct contact with the megakaryocyte in the bone marrow. We therefore further explored the interactions between T cells and megakaryocytes in bone marrow trephine biopsies - taken at or near to diagnosis - in patients with ITP. To further explore apoptotic pathways, serum TRAIL levels were also assessed at different time points in disease. MethodsBone marrow trephine samples were taken as clinically indicated at or around the time of diagnosis and processed as per department protocol. Samples from patients with known ITP (n=9) were further processed and stained with CD3, CD8, CD4 and CD56. Immunohistochemistry was performed using Leica Bond-max automated immunohistochemistry system. The density of MK cells, CD3, CD8 and CD4 was recorded and the numbers of MK in contact with T or NK cells were recorded on multiple sites on the trephine. Control bone marrows from staging marrows for lymphoma were used for comparison (n=6). For further analysis of the non-antibody component of ITP, serum TRAIL levels were measured in 63 samples from 43 patients at different stages of disease. Patients had all consented for research. ResultsResults of the bone marrow examination are shown in the table. Bone marrow samples from patients with ITP had higher density of megakaryocytes than control bone marrows (median 41/mm3 compared to 20/mm3 - p=0.03). Megakaryocytes appeared normal in morphology. CD3 density was also higher in patients with ITP, although this was not significant (p=0.17). There was however increased interaction between CD3 and MK cells in patients with ITP compared to controls (median 50% of MK cells in contact with CD3 cells in ITP compared to 32% in controls - p=0.02) – figure 1a. There was no correlation between MK cell density, T cell density and MK-T cell interaction, making this finding unlikely to have occurred by chance. In ITP patient samples further analysis of T cell type was undertaken and shown in the table showing interactions occur between both CD4s and CD8s and the MK cells (figure 1b). Four patients had reverse CD4:8 ratios in the bone marrow. Interaction between MK and CD56+ cells were less frequent (0 to 8%). There was no association with response to treatment and MK or T cell findings, although patients who responded to rituximab appeared to have lower MK density. Patients who responded to thrombopoietic (TPO) agonists - and by virtue of their use, had not responded to other treatments – had a high density of MK cells at diagnosis (bone marrows were taken at diagnosis, and before TPO agonists were started). Serum TRAIL levels were higher in patients with chronic ITP and with platelets <50 when compared to patients in remission (platelets > 150) (p<0.1). In patients with serial samples, there was an inverse relationship between TRAIL and platelet count.ITP PatientsControlsMK Density (per mm2)41.2 [13.2 – 76.7]19.6 [10.8 – 33.3]CD3 Density (per mm2)511.8 [297 – 805.9]317.5 [209 – 609]T-Cell (CD3)/MK Cell Interaction (%)50 [29 – 75]32 [17 – 41]CD4/MK Cell Interaction (%)52 [25 – 71]N/ACD8/MK Cell Interaction (%)25 [13 – 33]N/ANK Cell/MK Cell Interaction (%)6 [0 – 8]N/A ConclusionPreliminary evidence suggests increased interactions between T cells and megakaryocytes in the bone marrow of patients with ITP. The demonstration of interactions between CD3 cells and MK cells in both patients and controls shows the importance of the MK cell in the immune system. Further investigation is under way to explore whether CD8 or NK cell interaction is only found in patients with ITP, and whether these interactions result in MK cell death, or reduction in platelet production. High TRAIL levels in patients with chronic disease, refractory to first and second line therapy further suggests that increased apoptosis plays an important role in the ongoing thrombocytopenia in chronic disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Generation of Megakaryocytic Progenitors from Human Embryonic Stem Cells in a Feeder- and Serum-Free Medium

BackgroundThe production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a ‘spin embryoid body’ method in serum-free differentiation medium.Methodology and Principal FindingsImmunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a+ cells sorted from these differentiation cultures produced 100–800 Mk progenitors at day 13 and 25–160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a+ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34+ and CD45+ cells.ConclusionsWe describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

The Importance of Matrix Elasticity and Shear Force on Megakaryocytic Differentiation

Abstract 1329 Introduction.Environmental (matrix) elasticity (Engler et al., Cell 126: 677 (2006)) and biomechanical forces (Baruch et al., Blood 114:1975 (2009)) are important differentiation signals for stem cells. In this study, we investigated how these biomechanical/biophysical signals affect megakaryocytic (Mk) differentiation, aiming to uncover signaling mechanisms behind these effects, and establish better culture environment for Mk differentiation and platelet biogenesis. CHRF cell (Fuhrken et al., Exp. Hematol. 35: 476 (2006)), a well- established Mk cell line, was used in this study. Upon PMA stimulation, CHRF cells undergo Mk-like differentiation, including endomitosis and projection of cytoplasmic extensions. Methods and Results.Application of shear stress (2.5 dyne/cm2; physiological range: 1.3∼4.1 dyne/cm2) to CHRF cells attached to fibronectin-coated surfaces for two hours resulted in retraction, shedding and formation of cytoplasmic extensions (proplatelet-like structures), whereas there were no morphological changes in static culture. Shear force also accelerated endomitosis as assessed by flow-cytometry based analysis of BrdU incorporation: CHRF cells exposed for two hours to shear flow had 16% more BrdU positive cells compared to cells under static conditions. The stress responsive p53 is a regulator in both cell-cycle arrest by laminar flow (Zeng et al., JBC 278: 24594 (2003)) and Mk differentiation (Fuhrken et al., JBC 283: 15589 (2008)). To determine if p53 is involved in the response of CHRF cells to shear stress, western analysis was used to show that two hours of exposure to shear (2.5 dyne/cm2) increased the ratio of K382-acetylated p53 to total p53 by more than 188% (n=3, p<0.05) while total p53 protein expression did not change significantly. These data suggest that p53 acetylation is mediating, partially at least, the response of Mk cells to biomechanical forces.In order to examine the effect of matrix elasticity on Mk differentiation, polyacrylamide gels were used to control elasticity. We coated the gel surface with fibronectin to provide holding points for CHRF cells to sense gel elasticity. As matrix elasticity increased, the % of CHRF cells with cytoplasmic extensions increased first and decreased later (Fig. 1, n=3, Error bar: SEM). The highest percentage (17%) of CHRF cells displaying cytoplasmic extensions was observed on a gel with elasticity (ca. 8.2 kPa) mimicking the in vivo vascular niche. Non-muscle myosin IIA (NMMIIA) has been shown to serve as elasticity sensor in mesenchymal stem cells. Thus, we aimed to investigate the role of NMMIIA in the response of CHRF cells to matrix elasticity. Blebbistatin, an inhibitor of NMMIIA, minimized the differences of the percentage of cells with cytoplasmic extensions between gels with different elasticity (Fig. 2), thus suggesting that NMMIIA is involved in the response of CHRF cells to matrix elasticity and may also serve as a biological elasticity sensor. As shown by fluorescent microscopy, the cellular distribution of NMMIIA was directed by matrix elasticity (Fig. 3). On soft gels (75 Pa; n=3), NMMIIA was concentrated in the center of the cytoplasm. On a moderately stiff gel (5.6 kPa; n=3), NMMIIA was distributed across the whole cell, while on glass (the stiffest condition tested; n=3), NMMIIA moved into the nucleus and the tips of cytoplasmic extensions. Others have shown NMMIIA is involved in regulation of gene expression when it moves into the nucleus, so our observations that NMMIIA translocates into the nucleus as gel elasticity increases strongly suggests that NMMIIA may regulate gene transcription during proplatelet formation and platelet biogenesis. In future we want to investigate and prove this hypothesis. [Display omitted] Conclusions and Significance.Our data shed light on the largely unexplored role of biomechanical and biophysical parameters (of physiological significance) on Mk differentiation. The impact of these parameters is both of physiological and practical significance in the context of platelet biogenesis and ex vivo platelet generation. Disclosures:No relevant conflicts of interest to declare.

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

Influence of Residual Normal Metaphases In Patients with Monosomal Karyotype.

AbstractAbstract 1671▪Breems et al.(J Clin Oncol 2008;26:4791-97) observed that a monosomal karyotype(MK: ≥ 3 cells with ≥2 autosomal monosomies or with 1 autosomal monosomy plus a structural abnormality) conveyed a worse prognosis in untreated AML than a complex karyotype (CC ≥ 4 distinct clonal abnormalities),although there was considerable overlap between MK and CC. The SWOG (Medeiros et al. Blood e pub June, 2010) has confirmed these findings and extended them to patients age 60 or above. Previous data (Estey et al. Haematologica 2000;85:246-49) suggested that the presence of residual normal cells improved prognosis in patients with monosomy 7, prompting us to see if the same was true in patients with MK. Our analysis focused on survival and used the same 176 MK patients identified by Mederios et al. from 1,344 patients enrolled in one of 10 successive SWOG clinical trials. The MK patients had a median age of 61 years and 94% had a CC (≥ 3 distinct clonal abnormalities); 68% received induction with standard dose ara-C, 14% with other ara-C doses, and 18% without ara-C. A univariate proportional hazards model indicated that survival improved as the % of normal cells increased and accordingly as the % of abnormal cells (MK and non MK) decreased. Further investigation indicated that this reflected the number of normal, rather than the number of abnormal, cells. Thus having one or more normal cells was associated with a hazard ratio (HR) of 0.95 (p = 0.02), while the presence of 5 or more normal cells decreased the HR to 0.76. In contrast, there was no association between the number of abnormal MK cells and survival (p=0.15). Accounting for age and treatment (as noted above) did not alter these conclusions (HR for having 1 or more normal cells 0.95, p = 0.03).Despite the p-values these findings have limited medical significance for MK patients; since even the aforementioned decrease in HR to 0.76 would result in a median survival of only 5.3 months vs. the median of 4 months for all 176 MK patients. Nonetheless the results indicate that it might be worthwhile to examine the prognostic effect of residual normal metaphases in patients with better prognosis karyotypes. Disclosures:No relevant conflicts of interest to declare.