scholarly journals MicroRNAs as Haematopoiesis Regulators

2013 ◽  
Vol 2013 ◽  
pp. 1-20 ◽  
Author(s):  
Ram Babu Undi ◽  
Ravinder Kandi ◽  
Ravi Kumar Gutti
Keyword(s):  
Blood Cells ◽  
Erythroid Differentiation ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Complex Process ◽  
Development And Differentiation ◽  
Multiple Myelomas

The production of different types of blood cells including their formation, development, and differentiation is collectively known as haematopoiesis. Blood cells are divided into three lineages erythriod (erythrocytes), lymphoid (B and T cells), and myeloid (granulocytes, megakaryocytes, and macrophages). Haematopoiesis is a complex process regulated by several mechanisms including microRNAs (miRNAs). miRNAs are small RNAs which regulate the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells. Evidence shows that miRNAs play an important role in haematopoiesis; for example, myeloid and erythroid differentiation is blocked by the overexpression of miR-15a. miR-221, miR-222, and miR-24 inhibit the erythropoiesis, whereas miR-150 plays a role in B and T cell differentiation. miR-146 and miR-10a are downregulated in megakaryopoiesis. Aberrant expression of miRNAs was observed in hematological malignancies including chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelomas, and B cell lymphomas. In this review we have focused on discussing the role of miRNA in haematopoiesis.

scholarly journals The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3874-3874
Author(s):  
Tanabe Mikoto ◽  
Noriharu Nakagawa ◽  
Kohei Hosokawa ◽  
Luis Espinoza ◽  
Kana Maruyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Healthy Individuals ◽  
Hematopoietic Stem ◽  
Immune Mediated

Abstract [Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. Disclosures Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

scholarly journals A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3736-3736
Author(s):  
Tanabe Mikoto ◽  
Nguyen Hoang Maianh ◽  
Kohei Hosokawa ◽  
Noriharu Nakagawa ◽  
Luis Espinoza ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Leukemia Cell Line ◽  
Serum Free Medium ◽  
Free Medium ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Company Limited ◽  
Immune Mediated

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

Differences in oncogenic potency but not target cell specificity distinguish the two forms of the BCR/ABL oncogene

1991 ◽  
Vol 11 (9) ◽  
pp. 4710-4716
Author(s):  
M Kelliher ◽  
A Knott ◽  
J McLaughlin ◽  
O N Witte ◽  
N Rosenberg
Keyword(s):  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Hematological Diseases ◽  
Aggressive Disease ◽  
Disease Patterns ◽  
Cell Specificity ◽  
Different Types ◽  
Indolent Disease

Two forms of activated BCR/ABL proteins, P210 and P185, that differ in BCR-derived sequences, are associated with Philadelphia chromosome-positive leukemias. One of these diseases is chronic myelogenous leukemia, an indolent disease arising in hematopoietic stem cells that is almost always associated with the P210 form of BCR/ABL. Acute lymphocytic leukemia, a more aggressive malignancy, can be associated with both forms of BCR/ABL. While it is virtually certain that BCR/ABL plays a central role in both of these diseases, the features that determine the association of a particular form with a given disease have not been elucidated. We have used the bone marrow reconstitution leukemogenesis model to test the hypothesis that BCR sequences influence the ability of activated ABL to transform different types of hematopoietic cells. Our studies reveal that both P185 and P210 induce a similar spectrum of hematological diseases, including granulocytic, myelomonocytic, and lymphocytic leukemias. Despite the similarity of the disease patterns, animals given P185-infected marrow developed a more aggressive disease after a shorter latent period than those given P210-infected marrow. These data demonstrate that the structure of the BCR/ABL oncoprotein does not affect the type of disease induced by each form of the oncogene but does control the potency of the oncogenic signal.

scholarly journals Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3758-3779 ◽  
Author(s):  
N Uchida ◽  
HL Aguila ◽  
WH Fleming ◽  
L Jerabek ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Critical Role ◽  
White Blood Cells ◽  
Cell Types ◽  
Dose Dependence ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

Essential Roles for Mef2c in Lymphoid Commitment and B-Cell Function.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 377-377
Author(s):  
Sandra Stehling-Sun ◽  
Rebecca Jimenez ◽  
Andrew Hu ◽  
Fernando D. Camargo
Keyword(s):  
B Cells ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Muscle Development ◽  
Cell Function ◽  
Physiological Role ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Interleukin 7

Abstract MEF2 transcription factors are well-established regulators of muscle development. Recently, work in murine models has identified one of these factors, Mef2c, as an important regulator in the pathogenesis and the development of acute myeloid leukemia (AML). However, little is know about the molecular mechanism and physiological role of Mef2c in hematopoiesis. Using conditional gene ablation, we have discovered an unexpected role for MEF2c in hematopoietic stem cells (HSCs), where it is required for pan-lymphoid commitment. Competitive repopulation experiments using Mef2c-null HSCs deleted by means of the Mx1-Cre/poly(IC) approach, revealed completely normal monocytic, granulocytic and erythroid differentiation capacities by mutant cells. Generation and renewal of myeloid progenitors and HSCs was also normal. However, contribution to lymphoid lineages (T-cells, B-cells and natural killer cells) was dramatically reduced. Mef2c-deleted HSCs were able to generate lymphoid primed multipotent progenitors (LMPPs) and expressed normal levels of Flt-3 and the master lymphoid regulator ikaros. However, expression of the interleukin-7 receptor (IL-7R) and the number of phenotypically defined common lymphoid progenitors (CLPs) were substantially reduced. We have found two conserved Mef2c-binding sites in the promoter of the Il-7R gene, indicating that Mef2c could directly regulate Il-7R transcription. This and other potential molecular mechanisms of Mef2c-mediated lymphoid commitment will be discussed. We have also studied the effects of lineage-specific deletion of Mef2c in both myeloid and lymphoid populations. Whereas deletion in myelomonocytic cells using the LysM-Cre strain resulted in no anomalies, B-cell specific ablation with the CD19-Cre line revealed major phenotypical and functional abnormalities. CD19-Cre:Mef2cf/f mice show impaired germinal center formation and reduced antibody production in response to T-cell dependent antigens. In addition Mef2c-null mature B-cells fail to express the mature marker CD23, the low affinity receptor for IgE, which we show is a direct transcriptional target. As a consequence of CD23 reduction, CD19-Cre:Mef2cf/f mice have increased IgE production, thus indicating a potential role of Mef2c in allergic disease. Our work here sheds new light on the molecular mechanisms of lymphopoiesis and identifies MEF2 factors as critical hematopoietic transcriptional regulators.

Impairment of Erythropoiesis In Inbred Cellular Prion Protein Deficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2032-2032
Author(s):  
Hana Glierova ◽  
Martin Panigaj ◽  
Jana Semberova ◽  
Olga Janouskova ◽  
Eva Dvorakova ◽  
...  
Keyword(s):  
Prion Protein ◽  
Mouse Models ◽  
Blood Cells ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Erythroid Cell ◽  
Hematopoietic Stem ◽  
Acute Anemia ◽  
The Difference

Abstract Abstract 2032 Cellular prion protein (PrPc) plays a key role in pathogenesis of prion diseases, however, its physiologic function remains unclear. The involvement of PrPc in hematopoiesis was suggested by importance of its expression for self renewal and survival of long term repopulating hematopoietic stem cells. Prion diseases were shown to deregulate transcription of several erythroid genes and we have demonstrated reduction of erythroid cell and erythropoietin production in FVB PrP-/- (Zurich I) mice in response to acute anemia (Zivny J. et al. Blood Cells Mol Dis. 2008;40:302-307). In this study, we exploited different mouse models with manipulated level of PrPc expression to verify the role of PrPc in erythropoiesis. First set of experiments was carried out on PrP-/- (Zurich I) and Tga20 PrP over-expressing mice on a mixed C57Bl6/129Sv genetic background. Inbred C57Bl6 mice served as a wild type control (WT). Induction of acute anemia by phenylhydrazine (PHZ) in PrP-/- and WT mice (n=18) led to drop in the hematocrit (HCT) from 52.5±1.5 and 49.8±2.5% to 37.9± 1.0 and 41.9±3.0% after 24 h, respectively. The course of anemia was significantly deeper in PrP-/- mice at 72 h, 96 h and 120 h (p < 0.01) after PHZ administration. Plasma levels of erythropoietin (Epo) in response to anemia reached higher maximum levels in PrP-/- than WT mice (2250 vs. 1810 pg/mL) although rose more slowly. The level of Epo mRNA in kidneys increased approximately 30-fold in both, WT and PrP-/- mice, however, in WT mice peaked at 24 h whereas in KO mice at 96 h. We repeated the study with smaller groups of PrP-/- and Tga20 mice (n=9) and analysed samples 24 h and 96 h post anemia induction. Random PrP gene re-introduction in Tga20 mice rescued the animals from severe anemia. Decrease in HCT after PHZ administration was significantly lower in Tga20 comparing to PrP-/- mice and was accompanied by less elevated reticulocyte (RTC) count, plasma Epo level and level of Epo mRNA in kidneys. Next we studied the dynamics of unchallenged erythropoiesis in PrP-/-, Tga20 and WT mice by in vivo labelling of blood cells with NHS-biotin and subsequent flow cytometric analysis of relative numbers of newly produced non-labelled RBC. WT mice had significantly enhanced turnover of RBC with higher counts of non-labelled RBC comparing to PrP-/- during 46 days of chase (p < 0.05). Half- life of labeled RBC in WT mice was 20 days, but 32 and 30 days in PrP-/- and Tga20 mice, respectively. Tga20 mice displayed tendency to increased RBC turnover over PrP-/- mice, but the difference was significant only 2 and 33 days after initiation of the experiment. Having in mind possible limitations of experiments conducted in genetically modified inbred mice we have designed second set of experiments in more stringent animal models. We mated C57Bl6/129Sv PrP-/- mice with inbred C57Bl6 and outbred CD-1 mice. Heterozygotes in F1 generation were mated and their PrP -/-, PrP -/+ and PrP +/+ offspring used in the experiments. Anemia was induced by PHZ and blood was sampled from tail vein at defined time points and HCT and RTC count were analysed. In C57Bl6 crossbreeds we observed significantly higher starting HCT level in PrP-/- mice (p < 0.05) compared to PrP-/+ and PrP+/+ mice reaching 53.2±2.3, 50.0±2.1 and 49±2.9%, respectively. Similar decrease in HCT was observed for all PrP groups 24 h after PHZ administration, however, significant differences between PrP-/- and PrP+/+ mice were recorded at 48 h and 72 h. The recovery to normal HCT was again retarded in PrP-/- mice. RTC counts rose more rapidly in PrP+/+ mice after PHZ administration and declined to basal levels faster than in PrP-/- mice, the difference reached significance at 24 h, 48 h and 96 h. Dynamics of unchallenged erythropoiesis in C57Bl6 crossbreeds was similar in all three PrP genotypes with no significant differences in numbers of newly produced RBC during 57 days of the experiment. In CD-1 crossbreed mice no significant differences in HCT and RTC counts were detected after PHZ induced anemia among PrP-/-, PrP-/+ and PrP+/+ siblings. Also the dynamics of unchallenged erythropoiesis was similar in all PrP genotypes. To sum up, our data confirmed the role of PrPc in stress erythropoiesis in studied inbred mouse models. In outbred model the effect of PrP deletion on erythropoiesis seems to be compensated. (GACR310/08/0878, GAUK86408) Disclosures: No relevant conflicts of interest to declare.

MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3871-3871
Author(s):  
Li-Sheng Wang ◽  
Ling LI ◽  
Liang Li ◽  
Keh-Dong Shiang ◽  
Min Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myeloid Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Control Vector ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Direct Target

Abstract Abstract 3871 Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures: No relevant conflicts of interest to declare.

Leukemia Cell Infiltration Causes Defective Erythropoiesis Partially through MIP-1alpha

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1185-1185
Author(s):  
Yajie Wang ◽  
Sha Hao ◽  
Ping Lu ◽  
Hui Cheng ◽  
Yuemin Gong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Inhibitory Effect ◽  
Gene Set Enrichment Analysis ◽  
Cell Infiltration ◽  
Down Regulation ◽  
Data Set ◽  
Western Blots ◽  
Hematopoietic Stem

Abstract Leukemia often results in severe anemia, which may significantly contributes to mortality and morbidity of the patients. However, the mechanisms underlying the insufficient erythropoiesis in leukemia have been poorly understood. In this study, with our recently established non-irradiated MLL-AF9 acute myeloid leukemia (AML) murine model (Cheng H et al, Blood 2015), we observed a significant decrease in hemoglobin and red blood cells (RBCs) of Peripheral blood (PB) in the leukemic mice (n=6 per group, p=0.0122 vs p=0.0003). The absolute numbers of the erythroblasts at different stages (Pro Es, Ery.A, Ery.B, Ery.C) in bone marrow (BM) were also reduced. Consistently, by gene set enrichment analysis (GSEA) of microarray data of LKS+ cells (GSE52506 ) from leukemic mice, we found significant down-regulation of erythroid differentiation related genes such as GATA1, FOG-1, LMO2 and KLF1. These genes were more significantly inhibited in megakaryocytic-erythroid progenitors (MEPs) and Pro Es from the leukemic mice. Notably, the MEPs were the most reduced subset among all the committed hematopoietic progenitor cells (HPCs) during leukemia progression (90% decrease compared to control, p = 0.0007). MEPs were gradually accumulated in the G0 phase (from 22% to 70%, p<0.001). In contrast, erythroblasts (Pro Es, Ery.A, Ery.B) were more cycling (G1/S/G2/M) in leukemic mice and the proportions of Annexin V+ cells in erythroblasts but not in MEPs were also increased during leukemia development. Colony-forming cell (CFC) assays revealed that BM plasma of leukemia mice exerted an inhibitory effect on both BFU-Es and CFU-Es of BM mononuclear cells (BMMNCs) but not on other types of colonies (40% decrease for CFU-Es, 60% decrease for BFU-Es, p<0.001). Consistently, BM plasma of AML patients could also reduce the yield of BFU-Es and CFU-Es from CD34+ cord blood cells (n=7, p=0.006). To determine which cytokines may be responsible for the inhibitory effect, we collected serum and BM plasma from control and leukemic mice for cytokine array analysis. Among the elevated cytokines, MIP-1alpha was previously reported to be up-regulated in leukemic stem cells and its higher expression was found in the majority of patients with leukemia and a subset of patients with lymphoma and myeloma according to the Oncomine data set. We also confirmed it in a cohort of untreated AML patients (n=32). Importantly, AML patients with higher expression of MIP-1alpha showed reduced survival time (median=13.08 months) compared with the patients with lower expression (median=25.86 months) based on the leukemia-gene-atlas (LGA) analysis (n=72). By the CFC assay and single cell culture with different subsets of hematopoietic stem cells (HSCs) and HPCs, MIP-1alpha was able to largely mimic the inhibitory effect on the erythroid differentiation at both stem cell and progenitor cell levels. Mechanistically, we observed higher expression of MIP-1alpha receptor CCR1 in HSCs, MEPs and erythroblasts than CCR5. Administration of CCR1 antagonist, BX471 could partially recover the yield of erythroid colonies after treatment of MIP-1alpha or leukemia BM plasma. An increase of phosphorylation of p38 (phos p38) and resulted down-regulation of GATA1 after MIP-1alpha treatment were documented by Western blots and immunostaining. In summary, our results demonstrate that leukemic cell infiltration causes severe inhibition of erythropoiesis largely at different erythroid precursor levels and this inhibitory effect is at least partially medicated by elevated MIP-1alpha level via CCR1-p38 activation in the leukemic microenvironment. Disclosures No relevant conflicts of interest to declare.

Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4055-4063 ◽  
Author(s):  
Vanta J. Jokubaitis ◽  
Lidia Sinka ◽  
Rebecca Driessen ◽  
Genevieve Whitty ◽  
David N. Haylock ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Blood Cells ◽  
Fetal Liver ◽  
Converting Enzyme ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Nonobese Diabetic ◽  
Human Hscs ◽  
Ventral Wall

Abstract Previous studies revealed that mAb BB9 reacts with a subset of CD34+ human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9+. Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9+CD34+ UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+BB9− counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

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