Toll-Like Receptor (TLR) Signaling Adaptor Protein MYD88 in Myelodysplastic Syndromes (MDS)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 556-556
Author(s):  
Sophie Dimicoli ◽  
Yue Wei ◽  
Rui Chen ◽  
Carlos E. Bueso-Ramos ◽  
Sherry A. Pierce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Innate Immunity ◽  
Hematopoietic Stem Cells ◽  
Myelodysplastic Syndromes ◽  
Adaptor Protein ◽  
B Cell Lymphoma ◽  
Toll Like Receptor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 556 We have documented that deregulation of the Toll-like Receptor 2 (TLR2) centered innate immunity signals contribute to the pathogenesis of myelodysplastic syndromes (MDS). Of relevance, oncogenically active mutations of MYD88, a signal adaptor protein for TLR signal, have recently been identified as recurrent genetic lesions in both B-cell lymphoma and in chronic lymphocytic leukemia (CLL) (Vu N et al. Nature 2010 and Puente et al. Nature 2011). This information further supports the importance of innate immunity deregulation in leukemogenesis. To further characterize this pathway in MDS, we analyzed potential genetic alteration and expression level of MYD88 in patients of MDS. In a cohort of 40 MDS whole bone marrow mononuclear cell DNA, we first performed pyrosequencing analysis focusing on a list of previously reported MYD88 mutations (V217, W218, S219, I220, S222, M232, S243, L265, and T294). We did not detect mutation of any these hotspots on MYD88 in MDS. We then expanded the sequencing efforts to the entire coding region of MYD88 using an approach that combines PCR amplification and massive parallel sequencing. Still, no mutation of MYD88 was detected using this technique. We then examined the expression of MYD88 in CD34+ hematopoietic stem cells from 65 patients with MDS. In comparison to healthy donors, 26% of MDS patients (N=17) presented a more than 2 fold increase of MYD88 RNA, and 15% (N=10) had a 30%–90% increase. In average, MYD88 RNA level was 1.7 fold increased compared to control. Of potential clinical relevance, patients with higher MYD88 RNA expression in bone marrow CD34+ cells (above median value) (N=33) had a propensity of shorter period (24.4 mo) of overall survival (OS) in comparison to patients with lower levels of MYD88 expression (N=32) (32 mo)(P=0.05). We also found that patients with higher levels of MYD88 expression (split at 0.8 fold to controls) tended to have higher WBC (P=0.02). We have previously shown that blockade of the TLR2 mediated innate immunity signaling in MDS CD34+ cells could positively regulate erythroid lineage differentiation. To evaluate the potential of MYD88 blockade, we applied a 26 AA MYD88 inhibitory peptide that blocks its homodimerization (Invivogen, San Diego, CA) on primary CD34+ cells isolated from patients with lower-risk MDS (N=5). Methylcellulose medium supported colony formation assays indicated that the presence of MYD88 inhibitor led to an average of 60% increase for the numbers of erythroid colonies and a 30% increase for the numbers of total colonies. At the same time, we did not observe these effects of MYD88 blockade on the CD34+ cells isolated from the patients of higher-risk MDS (N=3). IL-8 is one of the key downstream transcriptional targets of the TLR-MYD88-NFkB innate immunity signaling that was documented to be elevated in bone marrow plasma of MDS. ELISA assays also indicated that blockade MYD88 in cultured MDS CD34+ cells led to a decrease of IL-8 concentration in medium. Taken together, these results indicated that MYD88 is overexpressed in hematopoietic stem cells of MDS and that blockade of MYD88 mediated innate immunity signaling may have therapeutic potential in treating patients with MDS. Disclosures: Kantarjian: Genzyme: Research Funding.

Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3106-3106
Author(s):  
Pietro Sodani ◽  
Buket Erer ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
Andrea Roveda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Marrow Transplant ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17527-17527
Author(s):  
H. Lannert ◽  
T. Able ◽  
S. Leicht ◽  
R. Saffrich ◽  
V. Eckstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Proteome Analysis ◽  
Becton Dickinson ◽  
Hematopoietic Stem ◽  
Cd34 Cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals ARID1A Is Critical for Maintaining Normal Hematopoiesis in Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3833-3833
Author(s):  
Lin Han ◽  
Vikas Madan ◽  
Anand Mayakonda ◽  
Pushkar Dakle ◽  
Weoi Woon Teoh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Chromatin Remodeling ◽  
B Cell Lymphoma ◽  
Chromatin Accessibility ◽  
Brdu Incorporation ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Hematopoietic Development

Abstract ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development. Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs. Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation. To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

2021 ◽  
Author(s):  
Carlos Carrascoso-Rubio ◽  
Hidde A. Zittersteijn ◽  
Laura Pintado-Berninches ◽  
Beatriz Fernández-Varas ◽  
M. Luz Lozano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Dyskeratosis Congenita ◽  
Bone Marrow Failure Syndrome ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34 + cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1 -interfered CD34 + cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence and apoptosis. Moreover, DKC1 -interfered human CD34 + cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome, and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

Retroviral-Mediated Gene Transfer of CD18 into Hematopoietic Stem Cells in Dogs with Canine Leukocyte Adhesion Deficiency Reverses the Severe Deficiency Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3169-3169
Author(s):  
Mehreen Hai ◽  
Thomas R. Bauer ◽  
Yu-chen Gu ◽  
Laura M. Tuschong ◽  
Robert A. Sokolic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Post Infusion ◽  
Low Levels

Abstract Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.

Use of the NOD/SCID/γcnull Mouse Model To Assess the Hepatocyte-Producing Ability of Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1695-1695
Author(s):  
Hisanori Fujino ◽  
Hidefumi Hiramatsu ◽  
Atsunori Tsuchiya ◽  
Haruyoshi Noma ◽  
Mitsutaka Shiota ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Hematopoietic Cells ◽  
Human Albumin ◽  
Hematopoietic Stem ◽  
Hepatic Cells ◽  
Cd34 Cells

Abstract Hematopoietic cells have been shown to generate nonhematopoietic cells, although the true plasticity of stem cells has been questioned. Here we used the NOD/SCID/γcnull mouse model, which permits efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells, to investigate whether human hematopoietic stem cells can differentiate into human hepatocytes. Freshly collected cord blood was depleted of phagocytes with Silica® followed by CD34 positive selection using auto MACS®. These cells were intravenously transplanted into irradiated mice, after which the liver was either undamaged or damaged by chemicals. The livers of these mice contained hepatocyte-specific (albumin, CYP family, TAT, alpha1AT, CPSI, prealbumin, transferrin and RBP4), cholangiocyte-specific (CK19) and vascular endothelial cell-specific (eNOS) human mRNAs. Immunohistochemistry detected the human hepatocyte specific antigens, albumin and alpha-1-antitrypsin-positive hepatocytes, cholangiocytes and CD68+ Kupffer cells. We also found human albumin in the murine bloodstream. Human albumin levels in the peripheral blood of transplanted mice correlate with the degree of PB chimerism and increase with time after transplantation. Furthermore, after obtaining liver cells by collagenase perfusion, flow cytometry revealed the presence of human albumin-positive cells that bear both human and murine MHC molecules, suggesting cell fusion occurs. All of the above phenomena were found in both liver-damaged and undamaged mice. In addition, we found human CD34+ cells are recruited from the murine bone marrow to the liver only in the case of acute liver injury but do not acquire hepatic stem/progenitor characteristics. Our observation suggests there are two pathways that yield hepatic cells from hematopoietic stem cells. The first requires liver damage that recruits CD34+ cells from the bone marrow via the circulation while the second pathway does not involve liver damage and appears to represent a constitutive default pathway of hematopoietic to nonhematopoietic transition. Our model is thus a versatile tool for investigating the development of functional human hepatic cells from hematopoietic cells and the feasibility of using hematopoietic cells in clinical situations.

Expression of Endomucin, a CD34-Like Sialomucin, Marks Definitive Hematopoietic Stem Cells throughout Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 563-563
Author(s):  
Azusa Maeda ◽  
Atsushi Iwama ◽  
Koji Eto ◽  
Hideo Ema ◽  
Toshio Kitamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Signal Sequence ◽  
High Yield ◽  
Specific Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract In order to identify cell surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was applied to mouse bone marrow (BM) CD34− c-Kit+ sca-1+ lineage− (CD34−KSL) cells which is highly enriched for HSCs. Among the identified genes, mRNA expression of Endomucin, an endothelium-specific gene encoding a CD34-like sialomucin, appeared highly specific to CD34-KSL HSCs. To further investigate the expression of Endomucin, we generated two rat anti-mouse Endomucin monoclonal antibodies that recognize different epitopes (AE2D4, AE7F2). Taking advantage of these and another monoclonal antibody, V7c7 (1999, Blood, 93; 1; 165), detailed expression analysis was performed. Endomucin expression was largely confined to lineage markers-negative (Lin−) cells. Approximately 7 % of Lin− cells were Endomucin-positive. Cells strongly expressing Endomucin represented 30% of c-kit+ sca-1+ cells. Gating out CD34+ cells from Lin− Endomucin+ population resulted in high yield of KSL cells. High correlation between Lin− Endomucin+CD34− cells and KSL cells was confirmed by in vivo bone marrow transplantation. When Lin− cells were fractionated by their expression of CD34 and Endomucin, only Lin− Endomucin+CD34− cells contributed to long-term repopulation (LTR), and as few as 100 cells were enough to obtain engraftment. Furthermore, the majority of CD34−KSL cells were Endomucin+, and again, only CD34−KSL-Endomucin+ cells had LTR activity. These data indicate two facts: 1) A single positive marker, Endomucin can substitute for c-kit+ sca-1+, 2) All LTR -HSCs express Endomucin. We then analyzed the expression of Endomucin during embryonic development of the hematopoietic system. Definitive HSCs arise from the hemogenic endothelium lining the wall of the dorsal aorta in embryonic aorta-gonads-mesonephros (AGM) region, then seed to the fetal liver. E10.5 AGM CD45− cells were segregated into subpopulations by their expression of Endomucin and CD41, an early marker of embryonic hematopoiesis. In vitro coculture system with a stromal cell line, OP9, was applied to detect the ability of hematopoietic potential. Hematopoietic activity was exclusively found in the CD41+Endomucin+ population, that represents 24% of CD41+ cells. Taken together, these data indicate that Endomucin marks both embryonic and adult HSCs, providing a novel useful cell surface marker for definitive HSCs throughout development. Figure Figure

Robo4/Magic Roundabout Is a Novel Surface Marker for Murine and Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 682-682
Author(s):  
Fumi Shibata ◽  
Yuko Goto-Koshino ◽  
Miyuki Ito ◽  
Yumi Fukuchi ◽  
Yoshihiro Morikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cd34 Cells ◽  
Human Hscs

Abstract A variety of cell surface markers such as c-Kit, Sca-1, CD34 and Flt-3 have been utilized to prospectively isolate murine or human hematopoietic stem cells (HSCs). While murine HSCs were shown to be highly enriched in CD34−c-Kit+Sca-1+Lineage- (CD34−KSL) fraction, this population is still not homogeneous for long-term HSCs. In human, CD34+ cells are regarded as crude HSC fraction and used for clinical applications. However, quiescent human HSCs are also found in CD34− fraction, indicating that CD34 is not a bona fide marker for human HSC. Thus, novel surface markers that can be used to purify human or murine HSCs to homogeneity need to be identified. Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are predominantly expressed in nervous system. Slit2, a ligand for Robo, is a large leucine-rich repeat-containing secreted protein that is also expressed in brain. By binding with Robo, Slit2 acts as a repellant for axon guidance of developing neurons and they are critical for correct wiring of neuronal network. Robo family comprises four family members, Robo1 – Robo4, and Robo4 is distinct in that it is expressed specifically in endothelial cells, but not in brain. In this study, we investigated Robo4 for its possible application for HSC identification in murine and human hematopoietic system. By RT-PCR, Robo4 was specifically expressed in murine KSL fraction, and was not expressed in lineage positive cells and various progenitors such as common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP) and common lymphoid progenitor (CLP). Moreover, the expression of Robo4 was highest in side population of KSL cells (KSL-SP), and moderate in KSL-main population (KSL-MP) cells. Monoclonal antibody raised against Robo4 identified its high expression in KSL cells by FACS. FACS analysis of human cord blood cells revealed that Robo4 is highly expressed in CD34+ cells, and CD34+Robo4high population fell into CD38− fraction, which enriches human HSCs. Bone marrow transplantation experiments revealed that Robo4+ fraction of murine KSL cells had long-term repopulating activity, while Robo4−KSL cells not. Although both Robo4+ and Robo4− CD34−KSL cells repopulated murine hematopoietic system for long-term, Robo4+CD34−KSL cells achieved higher chimerism after repopulation compared with Robo4−CD34−KSL. To investigate the physiological role of Robo4 in HSC homeostasis, we next examined the expression of Slit2 in hematopoietic system. Interestingly, Slit2 is specifically expressed in bone marrow stromal cells, but not in hematopoietic cells. Moreover, Slit2 is induced in osteoblasts, a critical cellular component composing HSC niche, in response to myelosuppressive stress such as 5FU treatment. These results indicate that Robo4 is expressed in murine and human hematopoietic HSCs and useful for HSC purification, and Robo4 - Slit2 system may play a role in HSC physiology in niche environment under hematopoietic stress.

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