ALBERT

Description: Langmuir DOI: 10.1021/la5008022

Description: Stable and permanent hematopoiesis is established from the most primitive long-term self-renewing hematopoietic stem cells (LT-HSC), which can give rise to more differentiated short-term (ST-HSC) and multi-potent progenitors (MPP). Progenitors further differentiate into more committed cells that can generate the mature lymphoid and myeloid lineages. In order to maintain a normal hematopoietic system, HSCs must be properly regulated. We previously cloned Ubiquitin Specific Protease 18 (USP18/UBP43) during analysis of hematopoietic cells of t(8;21) AML fusion protein AML1-ETO knock-in mice (Liu et al, 1999 Mol Cell Biol 19:3029-3038; Schwer et al, 2000 Genomics 65, 44-52). However, its function in hematopoiesis, especially in hematopoietic stem cells, has not been carefully examined. We show here that depletion of Usp18 in C57/BL6 mice leads to death at embryonic days 13.5-14.5 with less fetal liver cellularity. To examine the precise role of Usp18 in vivo, we generated Usp18 conditional knockout mice (Usp18f/f). Survival analyses of Usp18f/- crossed with Usp18f/+Vav-iCre revealed that the embryonic lethality of Usp18 -deficient mice is due to defects in hematopoiesis. To examine the hematopoietic potential of fetal liver cells of Usp18-deficient mice, we conducted a colony forming assay using the E12.5 fetal livers. All types of colonies as well as the number of total cells from colonies were substantially reduced in Usp18-/- fetal liver compared to control, indicating that the blood progenitor cells of Usp18-/- fetal liver are not fully functional. To assess whether Usp18 is required for fetal liver HSC maintenance, we determined the frequency of HSCs in the fetal liver of Usp18+/+, Usp18+/-, and Usp18-/-. We detected the Lin- Sca-1+ c-Kit+ (LSK) cell population, which is HSC-enriched population in fetal livers, in mice of all three genotypes. Recent studies indicate that the most primitive LT-HSC population in fetal livers includes ESAM positive (LSK CD48- CD150+ ESAM+) stem cells (Ooi et al, 2009 Stem Cells 27:653-661; Pietras et al, 2014 JEM 211:245-262). Both the frequency and absolute numbers of the LT-HSC population in Usp18 -/- fetal livers were appreciably reduced compared to wild-type. Taken together, we conclude that Usp18 is indispensable for fetal liver HSC maintenance. We then addressed whether Usp18 is required for the HSC maintenance in adult mice by analyzing the frequency of HSCs in UBCER-Cre negative or positive Usp18 f/- bone marrow cells. After tamoxifen injections, we observed a significant reduction in the frequency of the LT-HSC population in Usp18f/-UBCER-Cre positive bone marrow cells compared to Usp18 f/-UBCER-Cre negative ones. Consistent with these results, Usp18 f/-UBCER-Cre positive bone marrow cells were much less competitive than Cre negative cells by competitive bone marrow transplantation assay. Finally, to examine whether the suppression of Usp18 in the leukemic cells provides a survival benefit, we used secondary-transplanted mice receiving Usp18f/fUBCER-Cre positive AML1-ETO9a leukemia cells (5 × 10 5 EGFP+ cells) isolated from primary transplanted mice. The tamoxifen treatment was initiated 3 weeks after transplantation. All the mice in the vehicle injected group (n = 7) succumbed to leukemia within a week after treatment started. However, mice treated with tamoxifen (n = 7) showed a longer survival time. Five of seven mice are still alive after 5 weeks of bone marrow transplantation, demonstrating the critical role of USP18 in maintenance of leukemia stem cells. Collectively, we conclude that Usp18 is essential for hematopoietic stem cell maintenance, and specific modulating activity of USP18 in leukemic cells may be considered as an effective therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Description: Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation in embryonic stem cells and many cell line models. Therefore, we hypothesized that ‘stem-ness’ of hematopoietic stem/progenitor cells (HSPCs) are programmed by epigenetic mechanisms and attempted to reveal the molecular mechanisms in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic components of chromatin, has at least three variants: H3.1, H3.2, and H3.3. Previous studies have shown that one of the H3 variants, H3.3, was consistent with open chromatin structure. Here we found that the incorporation of histone variant H3.3 initiates on hematopoietic genes in HSPCs prior to differentiation. HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequencing (ChIPSeq) analysis was performed using newly-established monoclonal antibodies that specifically recognize endogenous H3.3. Although previous conventional studies have demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our sensitive ChIPSeq analysis revealed that more than half of the H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., virtually all genes were marked with H3.3 in embryonic stem cells, while all hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked when cells are differentiated. Furthermore, our analysis visualized that within the regions incorporated with H3.3, transcriptionally active regions marked by H3K4me3 and repressed regions marked by H3K27me3 are mutually exclusive. These data suggest that in hematopoietic differentiation, H3.3 incorporation initiates around relatively wide ranges of hematopoietic genes, and then either of active or repressive histone modification sequentially occurs. Interestingly, in leukemic cells, such selective H3.3 incorporation appeared to be disorganized. To identify factors that induce H3.3 incorporation defect in leukemic cells, we used a public database provided by the ENCODE project. We have constructed a system to manage all these datasets and to comprehensively explore the factors closely related to H3.3. Interestingly, correlations of our H3.3 ChIPSeq data with the ENCODE transcription factors’ binding site data were significantly different between analyses of AML and normal cells. By this approach, we identified hematopoietic transcription factors such as CEBPB and YY1 were associated with impaired H3.3 incorporation in AML. In addition, by comparing these transcription factors and single nucleotide variants (SNVs) obtained from Exome-Sequence, we found links between these transcription factors and particular SNVs in common pathways. These data suggest that this H3.3 ChIPSeq analysis should also be useful to extract oncogenic variants from many SNVs obtained by conventional Exome-Sequence analysis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2231 Poster Board II-208 Primary B cell development from hematopoietic stem cells occurs in the bone marrow (BM). During their development, B cell precursors progress through a series of distinct developmental stages; early-B cells, pro-B cells, and pre-B cells. The number of these B cell precursors is gradually declined with aging. Benign B cell precursors, originally termed hematogones (HG), can be observed in the BM during hematopoietic recovery phase in some patients who received chemotherapy or allogeneic bone marrow transplantation (allo-BMT). However, little is known about HG following cord blood stem cell transplantation (CBT). We retrospectively analyzed populations of B cell precursors of BM samples from 115 patients who received allogeneic stem cell transplantation (SCT). Patients included 49 women and 66 men, with a median age of 49 years (19-66 years). The underlying diseases of 115 patients varied; 43 acute myelogenous leukemia, 18 acute lymphocytic leukemia, 4 chronic myelogenous leukemia, 15 myelodysplastic syndrome, 17 malignant lymphoma, and 18 others. 65 patients underwent allo-BMT and 50 underwent CBT. Mean number of the infused cells amounted of 2.80 × 108 cells/kg (0.92-4.02 × 108) for allo-BMT and 2.78 × 107 cells/kg (1.77-5.00 × 107) for CBT. 68 patients received myeloablative conditioning regimen and 47 received reduced intensity conditioning regimen. GVHD prophylaxis included 3 cyclosporine (CSP) alone, 16 CSP and mycophenolate mofetil, 33 CSP and methotrexate (MTX), 5 tacrolimus (Tc) alone, and 58 Tc and MTX. Median time between day 0 of SCT and days performed on evaluation of HG by BM aspiration was 38 days (14-140 days). At that time, engraftment of donor cells was confirmed by chimerism analysis, indicating HG were proven to be donor-origins. HG were detected averagely in 1.56% of BM cells (0.01-12.27%) for allo-BMT recipients and 7.04% (0.03-55.56%) for CBT recipients, respectively. These results indicated CBT recipients presented much higher frequency and prominent reconstitution of HG compared to allo-BMT recipients (p

Description: Abstract 2301 To evaluate stem cell potential of human cells in xenotransplant models, a variety of immunodeficient mouse lines have been developed. Depletion of lymphoid cells including T, B and NK cells by introducing with Il2rgnull, and SCID or RAGnull mutations is necessary to avoid rejection of human cells in these models. Interestingly, in mice having these immunodeficiencies, the NOD strain shows even better engraftment of human cells as compared to C57BL/6 or Balb/c strains. Recently, we found that in xenograft rejection, the innate phagocytic reaction of mouse macrophages could occur because murine signal regulatory protein alpha (mSIRPA) on macrophages cannot bind to human CD47 (hCD47). However, NOD-specific polymorphism of mSIRPA allows NOD-type SIRPA to bind hCD47, resulting in inhibition of phagocytic reaction against human cells in this strain at least in vitro. Here, we have established a new immunodeficient BRGS mouse line, C57BL/6.Rag2nullIl2rgnull mice with NOD-type SIRPA. To test the reconstitution activity of human hematopoiesis in vivo, we transplanted 5 × 103 CD34+CD38− human cord blood cells intrafemorally into C57BL/6.Rag2nullIl2rgnull (C57BL/6-RG), BRGS or NOD.Rag1nullIl2rgnull (NOD-RG) mice. At 8 weeks after transplantation, human CD45+ cells were not detectable in C57BL/6-RG mice in the bone marrow. Both BRGS and NOD-RG showed successful reconstitution, and their frequency of human CD45+ cells in the bone marrow was 59.9 % and 55.8% in average, respectively. The frequency of human CD45+ cells was maintained at least until 24 weeks after transplantation. Percentages of CD19+ B cells, CD33+ myeloid cells and CD34+CD38− cells that contain the majority of human hematopoietic stem cells (HSCs) were almost equal between the BRGS and the NOD-RG strains. In the spleen, the majority of human cells were CD19+ B cells expressed surface immunoglobulin light chain λ/κ, reflecting their normal maturation. In the thymus, CD4+CD8+ thymic precursors, and CD4+ and CD8+ single positive T cells were present, and they expressed surface T cell receptor (TCR)-ab or TCR-gd. These data show that replacement of the C57BL/6-Sirpa with the NOD-Sirpa is sufficient for the C57BL/6-RG strain to gain the human cell engraftment ability equal to the NOD-RG strain. In addition, the BRGS strain has normal complement activity, in contrast to the NOD strain that does not have C5, a component necessary for complement-dependent cytotoxic (CDC) activity. We injected 8 × 105 cells of Raji cells into BRGS or NOD-RG mice, and administered rituximab, an anti-CD20 antibody that has both CDC and ADCC activities, to test the in vivo efficacy of rituximab on transplanted Raji cells. After injection of rituximab, percentages of human CD45+ Raji cells were significantly decreased in BRGS mice (15.1 %), whereas percentages of Raji cells in NOD-RG mice were only slightly reduced by rituximab treatment (79.2 %). These data clearly show that the CDC activity of antibodies can operate in the BRGS strain. Collectively, this study formally proves that the excellent transplantability of human grafts in the NOD strain is explained simply by a single gene mutation, NOD-specific polymorphism of SIRPA, and that the BRGS strain should be very useful in future xenotransplant experiments of human stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: [Introduction] Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma (PTCL) with a dismal prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment in ATL patients. Mogamulizumab, a humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, is a novel immunotherapeutic agent, effective in treating patients with PTCL such as ATL, PTCL-not specified, and cutaneous T-cell lymphoma. However, in allo-HSCT setting, we should be careful to use mogamulizumab because CCR4 is expressed in regulatory T cells: The mogamulizumab treatment may accelerate GVHD by eradicating regulatory T cells in allo-HSCT patients. Here, we retrospectively analyzed the effect of mogamulizumab on GVHD development in ATL patients treated with mogamulizumab prior to allo-HSCT. [Patients and Methods] Data from the Fukuoka Bone Marrow Transplantation Group were retrospectively analyzed after the approval of mogamulizumab use in Japan. [Results] A total of 24 patients with ATL received mogamulizumab prior to allo-HSCT between April 2012 and April 2015 in our group. The median age at allo-HSCT was 58.5 years (range, 32-72). The median intervals from the last administration of mogamulizumab to allo-HSCT were 25 days (range, 9-126). The median total dose of mogamulizumab was 3 mg/kg (range, 1-8 mg/kg). After treatment with mogamulizumab, 18 patients (75%) had achieved in remission (CR in 4 patients and PR in 14) at allo-HSCT. Ten patients received unrelated bone marrow, 5 received related peripheral blood, and 9 received cord blood as stem cell sources. Eleven patients were treated with full-intensity conditioning and 13 received reduced-intensity conditioning. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitors (cyclosporine or tacrolimus) with short-term methotrexate in 14 patients and mycophenolate mofetil in 9. The cumulative incidence (CI) of acute GVHD at 100 days was 66.6% in grade 2-4 and 33.3% in grade 3-4. The involved organs of acute GVHD were skin in 14 patients, gut in 10, and liver in 4. Among 14 patients who developed grade 2-4 acute GVHD, 5 had severe fluid retention such as pleural effusion or ascites associated with GVHD. Chronic GVHD was observed in 6 patients, and 5 of them were extensive disease. The CI of transplant-related mortality (TRM) and relapse at 1-year were 53.2% (95%CI, 29.3-72.3%) and 29.6% (95%CI, 12.6-48.9%), respectively. The leading cause of death was GVHD (n = 7). The 1-year overall survival and progression-free survival were 19.2% (95%CI, 5.7-38.8%) and 17.2% (95%CI, 4.9-35.7%), respectively. [Discussion] Use of mogamulizumab prior to transplantation in allo-HSCT patients has a merit to decrease the burden of ATL cells. However, it was associated with an increase of TRM due to severe GVHD. Although most of ATL patients achieved better disease status at allo-HSCT through mogamulizumab and the survival rate was expected to be 50% based on the previous data, the survival in the present study was ~20%. These data suggest that mogamulizumab administered before transplantation may have retained until an early phase of post-transplantation, and the donor or host-derived regulatory T cells might be eliminated, allowing the GVHD T-cell clone to expand. Since mogalizumab is a potent anti-ATL agent, we need to develop new treatment protocols integrating mogalizumab at a suitable dose or administration timing, to minimize the unwanted GVHD development in future studies. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: Abstract 2983 Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs). TIM-3+ AML cells reconstituted human AML in immunodeficient mice, whereas TIM3− AML cells did not, suggesting that the TIM-3+ population contains all functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden, and eliminated LSCs capable of reconstituting human AML in secondary recipients (Kikushige et al, Cell Stem Cell, 2010).We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38− LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although TIM-3 was not expressed in CD34+CD38− stem cell fraction in normal bone marrow cells, TIM-3 was progressively up-regulated in this population of MDS, along with disease progression into leukemia: The average percentages of TIM-3+ cells in the CD34+CD38− population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). Thus, TIM-3 might be useful to isolate malignant stem cells responsible for progression into AML in MDS patients. The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. Previous reports have shown that galectin-9 and HMGB-1 are the ligand of TIM-3 in lymphocytes and dendritic cells. TIM-3 is reported to signal differently in lymphocytes and myeloid cells, because TIM-3 ligation results in different patterns of tyrosine phosphorylation in these cell types, suggesting that TIM-3 has lineage- or cellular context-dependent signal transduction pathways or functions. Therefore, we considered that it should be critical to identify the function of TIM-3 in primary AML cells. We cultured TIM-3+ AML cells in the presence or absence of galectin-9 or HMGB-1, and performed cDNA microarray analysis to find genes activated in response to TIM-3 ligation. Interestingly, pro-apoptotic genes such as BAX and SIVA were significantly down-regulated in the presence of galectin-9 or HMGB-1, suggesting that TIM-3 signaling could promote survival of TIM-3-expressing LSCs. These data suggest that TIM-3 is a surface marker useful to track malignant LSCs in progression from MDS to AML, and TIM-3 may function for maintenance of LSC through inducing survival-promoting signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the surface of LSCs in most types of AML, and that TIM-3-targeted therapy could eradicate AML LSCs, based on in vivo xenograft experiments using patients’ AML samples (Kikushige et al, Cell Stem Cell, 2010). Since only the TIM-3+ but not the TIM-3- fraction of human AML cells can reconstitute human AML in immunodeficient mice, we hypothesized that the TIM-3 plays indispensable function to maintain AML LSCs. Galectin-9 is a ligand for TIM-3, and ligation of TIM-3 by galectin-9 has been shown to phosphorylate tyrosine residues of TIM-3 and activate Src family kinases through its SH2 domain in T cells and monocytes. We found that serum galectin-9 concentration was significantly elevated in AML patients (431.1+58.6pg/ml, n=13) but not in the healthy individuals (25.2+6.8pg/ml, n=7) or patients with B cell malignancies (36.1+15.5pg/ml, n=10). Primitive CD34+ AML cells had abundant galectin-9 protein in their cytoplasms. We then transplanted human CD34+ primitive AML cells into irradiated immunodeficient mice. Strikingly, only in mice reconstituted with human AML, but not in those with normal cord blood or human ALL cells had elevated serum levels of human galectin-9: Serum galectin-9 levels were 234.7+69.0pg/ml (n=8) in mice reconstituted with primary human AML cells, whereas 4.64+4.64pg/ml (n=12) in mice with normal human hematopoiesis. These results collectively suggested that AML cells secreted galectin-9 in vivo in an autocrine manner. We then performed transcriptome analyses of primary CD34+TIM-3+ AML cells after galectin-9 ligation. Results were further evaluated by a pathway enrichment analysis, which demonstrated that both NF-κB and β-catenin pathways were activated. In fact, galectin-9 ligation of TIM-3+ human AML cells induced activation of the NF-κB pathway via ERK and AKT phosphorylation. Activation of ERK and AKT pathways are known to inhibit the GSK3β activity, and to promote the nucleus translocation of β-catenin in several cancers. To demonstrate significant nucleus translocation of β-catenin of primary AML cells, we established a quantitation system by utilizing the Array Scan VTI system. By this system, we could formally prove that TIM-3 ligation by galectin-9 significantly promoted the nucleus translocation of β-catenin in primary AML cells. We also have extensively analyzed TIM-3 and galectin-9 interaction in cells from patients with myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelogeneous leukemia. Strikingly, in all cases, frequencies of CD34+CD38-TIM-3+ cells dramatically increased along with disease progression from early/chronic phase to overt leukemias. Furthermore, serum levels of galectin-9 were also dramatically elevated after leukemic transformation. Significant nucleus translocation of β-catenin by galectin-9 ligation was also found in these diseases after leukemic transformation. A recent study has shown that NF-κB and β-catenin pathways could play a cooperative role in conferring the cancer-stem-cell properties to non stem cells in intestinal cancer model. These data collectively suggest that TIM-3 and galectin-9 constitutes a pan-myeloid autocrine loop to develop malignant stem cells in the vast majority of human myeloid malignancies. Thus, signaling molecules downstream of TIM-3 and galectin-9 ligation, as well as surface TIM-3 itself might be good candidates for cancer stem cell-target therapy common to most myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P

Description: Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies. Disclosures: Miyamoto: Kyushu University Hospital: Employment.