scholarly journals Endocytosis of the Thrombopoietin Receptor Mpl Regulates Both Megakaryocyte and Erythroid Progenitors and Is Critical for Myelofibrosis Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3714-3714
Author(s):  
Melissa M Lee-Sundlov ◽  
Haley E Ramsey ◽  
Robert J Dickey ◽  
Martha Sola-Visner ◽  
Karin M Hoffmeister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Jak2 V617f ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Phenotypic Analysis ◽  
Hematopoietic Stem ◽  
Erythroid Maturation ◽  
Over Time

Abstract Somatic mutations in the tyrosine kinase JAK2, the thrombopoietin (TPO) receptor Mpl and the chaperone calreticulin cause myelofibrosis due to constitutive TPO/Mpl signaling in abnormal hematopoietic stem cells (HSCs). Impaired Mpl-mediated endocytosis has been reported in myelofibrosis patients carrying the most frequent JAK2-V617F mutation. Mpl-mediated endocytosis is also impaired in Dnm2fl/fl Pf4-Cre (Dnm2Plt-/-) mice specifically lacking the highly conserved endocytic GTPase dynamin 2 (DNM2) in the megakaryocyte (MK) lineage. Consequently, Dnm2Plt-/-mice develop hallmarks of myelofibrosis such as elevated circulating TPO levels, constitutive JAK2 phosphorylation, marked expansion of HSCs, massive MK hyperplasia, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. To determine whether the phenotype is due to unrestrained TPO/Mpl signaling in HSCs, Dnm2Plt-/- mice were crossed with Mpl-/-mice. Mpl-/- Dnm2Plt-/- mice were obtained with a normal Mendelian distribution at weaning, and bone marrow HSC and MK numbers were significantly reduced in Mpl-/- Dnm2Plt-/- mice, similar to those of Mpl-/- mice. Surprisingly, Mpl-/- Dnm2Plt-/- mice died at a median age of 26 days postnatal and presented a severe splenomegaly, similar to that of Dnm2Plt-/- mice. Complete blood counts were analyzed from birth to 3 weeks postnatal. Blood platelet counts increased over time in control mice, reaching maximal values at 3 weeks postnatal, and remained low in Mpl-/-, Dnm2Plt-/- and Mpl-/- Dnm2Plt-/- mice. Blood erythrocyte counts increased over time in control mice, were significantly slower in single Mpl-/- and Dnm2Plt-/- mice, and failed to increase in Mpl-/- Dnm2Plt-/-mice, resulting in severe anemia. Erythroid maturation in Mpl-/- Dnm2Plt-/- spleens was investigated by flow cytometry analysis using CD71 and Ter119 as erythroid markers, where immature erythroblasts are defined as CD71high/Ter119low and mature erythroblasts as CD71low/Ter119high. Approximately 75% of erythroid cells in control spleens were mature erythroblasts, and the distribution significantly decreased to 40% and 20% in single Mpl-/- and Dnm2Plt-/- spleens, respectively. In Mpl-/- Dnm2Plt-/- spleens, only 5% of erythroid cells were mature erythroblasts, consistent with severely impaired erythroid maturation. Flow cytometry phenotypic analysis of bone marrow MK and eryrthroid progenitors revealed a significant increase of the distribution of progenitors with both MK and erythroid potential (Pre-Meg-E) in Mpl-/- Dnm2Plt-/-mice. The data shows that TPO/Mpl signaling and endocytosis orchestrate erythropoiesis and thrombopoiesis to control the bone marrow environment. Mpl regulates both MK and erythroid progenitors, highlighting clinically relevant interactions between these two blood cell compartments in myelofibrosis development. Disclosures No relevant conflicts of interest to declare.

scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

JAK2 Signaling Specifies Phenotypic Pleiotropy In Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1608-1608
Author(s):  
Lily Huang ◽  
Huiyu Yao ◽  
Yue Ma
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Phenotypic Diversity ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Wild Type ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Phenotypic Differences ◽  
V617f Mutation

Abstract Myeloproliferative neoplasms (MPNs) are a phenotypically diverse group of pre-leukemic diseases characterized by overproduction of one or more of the myeloid cell lineages. Gain-of -function mutations in the Janus tyrosine kinase 2 (JAK2) are major determinants in MPNs, These include the V617F mutation and mutations in exon 12. Interestingly, MPN phenotype in patients with exon 12 mutations is distinct from that of patients with the V617F mutation. Mechanisms underlying the phenotypic differences are not well understood. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function mutations. Interestingly, three of them with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only. To determine the mechanisms underlying the phenotypic differences by different JAK2 mutants, we characterized hematopoietic progenitors and precursor subsets in these mice for their proliferation, apoptosis and differentiation. Quantification of the hematopoietic stem and progenitor population showed an increased percentage of granulocyte-monocyte progenitors (GMP) and skewing of differentiation towards the granulocytic lineage in JAK2(V617F) and JAK2(N622I) mice compared to JAK2(K539I) or wild-type JAK2 mice. Because no difference was observed in the proliferation or apoptosis of bone marrow progenitors from JAK2 mutant mice, differentiation of the common myeloid progenitors (CMP) was likely skewed towards GMP by JAK2(V617F) and JAK2(N622I). Consistent with this hypothesis, similar results were observed in colony forming assays from sorted CMP populations. In the spleen, a decrease in GMP apoptosis and an increase in apoptosis of the megakaryocyte-erythrocyte progenitors (MEP) also contributed to the skewing towards the granulocytic lineage in JAK2(N622I) mice. Similar to MPN patients, mice expressing JAK2 mutants exhibited splenomegaly. We found that JAK2 mutants caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. As a result, more differentiated precursors were expanded in the spleens of JAK2 mutants mice compared to mice expressing wild-type JAK2. Consistent with their phenotypes, the percentage of Annexin V＋7AAD－erythroblasts in JAK2(K539I) and JAK2(V617F) mice was significantly less than in JAK2(N622I) or wild-type JAK2 mice. On the other hand, both proliferation and apoptosis contribute to the differential degrees of granulocytosis among mice expressing different JAK2 mutants. In line with the different effects elicited by different JAK2 mutants in progenitor and precursor cells, signal transduction pathways were differentially activated downstream of different JAK2 mutants. In summary, our results showed that JAK2 mutants differentially skew differentiation in early stem and progenitor compartments, and also regulate apoptosis and proliferation of distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide the mechanistic basis for the phenotypic diversity observed in MPNs with different JAK2 mutants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Erythro-Myeloblastic Island (EMBI): A Hematopoietic Niche Balancing Erythropoiesis and Myelopoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 842-842
Author(s):  
Katie Giger Seu ◽  
Laurel Romano ◽  
Julien Papoin ◽  
Edward David Muench ◽  
Diamantis Konstantinidis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Multispectral Imaging ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Erythroid Cells ◽  
Murine Models ◽  
Native Bone ◽  
Hematopoietic Niche

Abstract Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.

JAK2 Signaling Specifies Phenotypic Pleiotropy in Myeloproliferative Neoplasms.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2868-2868
Author(s):  
Lily Huang ◽  
Huiyu Yao ◽  
Yue Ma
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Phenotypic Diversity ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Hematological Toxicity ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Tyrosine Kinase 2

Abstract Abstract 2868 The Janus tyrosine kinase 2 (JAK2) plays an important role in hematopoiesis of multiple lineages. A gain-of-function JAK2 mutation, V617F, is the major determinant in myeloproliferative neoplasms (MPNs), a phenotypically diverse group of hematological diseases in which cells of the myelo-erythroid lineage are overproduced. JAK2 kinase inhibitors showed hematological toxicity in treating MPNs, calling for novel therapeutics that can target only the affected lineage while sparing others. This task is hindered by lack of understanding in how JAK2 signaling differentially regulates the generation of different blood cells. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function JAK2 mutations in addition to V617F (1). Surprisingly, three activating JAK2 mutants with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only (2). Quantification of the hematopoietic stem and progenitor populations in mice expressing wild-type JAK2 or JAK2 mutants showed significant granulocytic skewing by JAK2(V617F) and JAK2(N622I) both in the bone marrow and spleen. In contrast, erythroid skewing by JAK2(K539I) was observed. Consistent with these results, qualitative and quantitative differences were observed in signaling events downstream of JAK2 in stem and progenitor cells from mice expressing different JAK2 mutants. JAK2 mutants also caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. In later more differentiated compartments, JAK2(K539I) and JAK2(V617F) expanded erythroid precursor cells, including proerythroblasts and later precursors, to cause erythrocytosis, while JAK2(V617F) and JAK2(N622I) expanded myeloid precursors to cause granulocytosis. The expansion of these later compartments was at least in part due to a decrease in apoptosis. Together, our results showed that JAK2 mutants differentially skew early stem and progenitor compartments toward the erythroid or granulocytic lineage, and expand distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide mechanistic basis for the phenotypic diversity observed in mice expressing different JAK2 mutants. Our results show that differential JAK2 signaling regulates hierarchically early and late progenitor compartments to drive erythropoiesis vs. granulopoiesis. These results shed light on MPN biology and may facilitate the design of novel and more effective therapeutic agents that specifically target affected lineage without compromising other lineages. Disclosures: No relevant conflicts of interest to declare.

RUNX1C-Mediated Reprogramming Of Human Bone Marrow Stromal Cells into Early Blood Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2463-2463
Author(s):  
Weihong Yin ◽  
Christopher D Porada ◽  
Stephen Walker ◽  
Colin Bishop ◽  
Graca Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Dermal Fibroblasts ◽  
Hematopoietic Stem ◽  
Surface Marker Expression ◽  
Somatic Cell Reprogramming ◽  
Hematopoietic Lineage

Abstract Somatic cell reprogramming to the hematopoietic lineage, either through a pluripotent state or directly, opens the possibility of production of a ready source of autologous hematopoietic stem cells (HSC) that can be used to treat/cure a wide variety of blood disorders. While it has previously been shown that dermal fibroblasts (HFF) can be directly reprogrammed to the hematopoietic lineage, the efficiency was relatively low and the resultant hematopoietic cells lacked multilineage differentiative potential. Stro1(+) isolated stromal progenitors (SIPs) can easily be isolated from the bone marrow (BM) and expanded ex-vivo to obtain clinically significant numbers of cells. In similarity to HSC, SIPs are derived from the mesoderm, and are intimately linked with HSC specification during ontogeny. As such, they are likely to be epigenetically closer to HSC than HFF, and therefore good candidates for reprogramming into hematopoietic cells. To verify the uniqueness of SIPs for reprogramming, we transduced SIPs and HFF with OCT4 and/or RUNX1C, a master transcription factor (TF) that triggers the developmental onset of definitive hematopoiesis, in the following combinations: 1) OCT4 alone; 2) RUNX1C alone; or 3) OCT4+RUNX1C. We then performed a timeline of gene/cell surface marker expression (using microarray, qRT-PCR, and flow cytometry) from day 3-16 post-transduction. Visual inspection of the cultures showed that, while reprogrammed colonies began to appear in SIPs cultures at day 9, no colonies were seen during this time period in HFF cultures. Flow cytometry and molecular analyses of colonies obtained from OCT4+RUNX1C combination demonstrated that expression of CD41, the earliest marker of commitment to the hematopoietic lineage, commenced within only 3-4 days and peaked at day 5-6, by which time ∼20% of SIPs expressed this marker. This peak in CD41 expression coincided with commencement of expression of CD34 and CD45, and maximal induction of several hematopoiesis-specific TFs and phenotypic markers such as PU.1, HOXB4, GATA2, MIXL, WNT3, KDR, CDX4, which occurred at 1-3 logs higher levels in SIPs than HFF. Further studies demonstrated that the chromatin remodeling function of OCT4 could be replaced with the histone methyltransferase inhibitor Bix-01294, with the combination of RUNX1C and Bix-01294 inducing levels of CD34 and CD41 expression by day 5 that were similar to those achieved with RUNX1C plus OCT4. The present studies thus take several important steps towards making the promise of producing autologous hematopoietic cells for transplantation via direct reprogramming a reality. Disclosures: No relevant conflicts of interest to declare.

Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 945-945
Author(s):  
Rialnat Adebisi Lawal ◽  
Kathleen E. McGrath ◽  
Laura M. Calvi
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Erythroid Differentiation ◽  
Enzymatic Digestion ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Osteolineage Cells

Abstract Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells. We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool. To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites. Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures: No relevant conflicts of interest to declare.

Modeling Myelodysplastic Syndromes In Mice By Altered Hoxa1 Spliceform Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 97-97
Author(s):  
Chacko Joseph ◽  
Jean Hendy ◽  
Stewart A Fabb ◽  
Emma K Baker ◽  
Alistair M Chalk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Models ◽  
Myelodysplastic Syndromes ◽  
Conflicts Of Interest ◽  
Integration Site ◽  
Survival Curve ◽  
Macrocytic Anemia ◽  
Small Subset ◽  
Erythroid Cells ◽  
Hematopoietic Stem

Abstract Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cell diseases with 30% of the patients developing acute myeloid leukemia (AML). The etiology of MDS is largely unknown and as a result there are no curative therapies. Mouse models that faithfully represent MDS are based on rare genetic abnormalities and currently represent only a very small subset of MDS patients. Using in silico analysis we have identified that homeobox A1 (HOXA1) mRNA is significantly upregulated in 50% of MDS patients (1.8-fold increase, n=183 MDS, 17 controls, P<0.05). Importantly, the upregulation was observed across all subsets of patients, including those with a normal karyotype. Both human HOXA1 and mouse Hoxa1 are expressed as two different spliceforms generated by alternative splicing within exon 1 of the wildtype Hoxa1 (WT-Hoxa1). These spliceforms encode a full-length (Hoxa1-FL) and a truncated form (Hoxa1-T), the latter lacking the homeobox domain. Given that mutations in splicing machinery have been identified in up to 85% of MDS patients we hypothesised that deregulated HOXA1 spliceforms may contribute to MDS. We identified that the Hoxa1 isoforms were differentially expressed in murine hematopoietic stem cells (HSCs) and progenitors. Retroviral overexpression of either WT-Hoxa1 (which generates both Hoxa1-FL and Hoxa1-T) or Hoxa1-T in murine bone marrow (BM) cells showed opposing effects on in vitro cell proliferation and colony forming cell (CFC) production respectively, suggesting that Hoxa1-T may negatively regulate Hoxa1-FL. We therefore generated a mutant Hoxa1 (MUT-Hoxa1), which expresses normal Hoxa1-FL but not Hoxa1-T, by oligomutagenesis at the splice site of Hoxa1-T. We transplanted recipient mice (n>28 per group) with bone marrow (BM) overexpressing either MUT-Hoxa1, WT-Hoxa1 or empty vector (MXIE) control. All recipients of MUT-Hoxa1 BM developed peripheral blood (PB) macrocytic anemia (mean ± SEM: Hb (g/L): MUT-Hoxa1: 126±2.9**; WT-Hoxa1: 134±2.7; MXIE: 135±1.4; **P<0.01 vs MXIE), without leukocytosis. Furthermore, thrombocytopenia was also observed in MUT-Hoxa1 and WT-Hoxa1 recipients (PB platelets (x 106/ml): MUT-Hoxa1: 738±88*; WT-Hoxa1: 777±57*; MXIE: 922 ± 37, *P<0.05 vs MXIE). Overexpression of MUT-Hoxa1 or WT-Hoxa1 was associated with significant apoptosis in BM erythroid cells (% apoptosis in GFP+ Ter119+ cells: MUT-Hoxa1: 20.7±4.9*; WT-Hoxa1: 25.3±5.2** MXIE: 13.6±2.5, *P<0.05, **P<0.01 vs MXIE). We also observed numerous dysplastic erythroid cells and micromegakaryocytes in the MUT-Hoxa1-overexpressing cells. Strikingly, 88% of recipients of MUT-Hoxa1 BM, but no WT-Hoxa1 recipients, developed AML with a median time of 10.5 months post-transplant, independent of retroviral integration site (Kaplan-Meier survival curve, P<0.05 vs MXIE). Therefore, overexpression of MUT-Hoxa1 or WT-Hoxa1 in BM cells results in clinical features of MDS, with MUT-Hoxa1 overexpression resulting in a more aggressive MDS that spontaneously progresses to AML. Analysis of BM from non-leukemic mice identified that MUT-Hoxa1 and WT-Hoxa1 recipients had significantly decreased HSC-containing lineage -ve, c-kit+, Sca-1+ (LKS+) cells (% of lin- GFP+ BM: MUT-Hoxa1 1.72 ± 0.28#; WT-Hoxa1: 2.08 ± 0.37#; MXIE: 5.09 ± 0.56%; #P<0.0001 vs MXIE, n>12). This was accompanied by increased progenitor cell-containing LKS- (% of lin- GFP+ BM: MUT-Hoxa1: 53.32 ± 5.65*; WT-Hoxa1: 47.42 ± 4.50; MXIE: 38.37 ± 4.13; *P<0.05 vs MXIE, n>12). Strikingly, granulocyte/monocyte progenitors were significantly reduced in the MUT-Hoxa1 LKS- cells, accompanied by a marked accumulation of megakaryocyte/erythroid progenitors (MEPs). We have identified deregulated gene pathways in the MUT-Hoxa1 BM MEPs by microarray analyses. We are currently testing drug candidates identified from these screens for their effects on BM obtained from our MUT-Hoxa1 and WT-Hoxa1 mouse models and MDS patients. Taken together, these results suggest that overexpression of Hoxa1-FL results in MDS, and that concurrently inhibiting splicing of Hoxa1-T (MUT-Hoxa1) forms a more aggressive MDS phenotype. Preliminary analysis of CD34+ BM cells from MDS patients reveal significant numbers of patients with elevated HOXA1-FL mRNA compared to lower or absent levels of HOXA1-T. Our MUT-Hoxa1 and WT-Hoxa1 mouse models will therefore be highly valuable in identifying better therapies for a significant subset of MDS patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1174-1174
Author(s):  
Darlene Monlish ◽  
Angela Herman ◽  
Molly Romine ◽  
Sima Bhatt ◽  
Laura G. Schuettpelz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Immune Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Tlr2 Agonist ◽  
Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM3CSK4, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM3CSK4 led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2-/- + Tlr2+/+ bone marrow transplanted into Tlr2+/+ or Tlr2-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM3CSK4, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM3CSK4-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 81-81
Author(s):  
Silvana Di Giandomenico ◽  
Pouneh Kermani ◽  
Nicole Molle ◽  
Mia Yabut ◽  
Fabienne Brenet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Progenitor Cells ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Erythroid Precursors ◽  
Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1274-1274
Author(s):  
Elisabeth F Heuston ◽  
Bethan Psaila ◽  
Stacie M Anderson ◽  
NISC Comparative Sequencing Program ◽  
David M. Bodine
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Transcriptional Profiles ◽  
Gene Set ◽  
Rna Signature

Abstract 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. Disclosures No relevant conflicts of interest to declare.

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