SCL/TAL1 Regulates Erythropoietin Receptor Expression.

Abstract The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.

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Abstract 255: The Kidney and Heart are Hemogenic Organs During Embryonic Life.

Hypertension ◽

10.1161/hyp.60.suppl_1.a255 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Yan Hu ◽

Minghong Li ◽

R. Ariel Gomez ◽

Maria Luisa S Sequeira Lopez

Keyword(s):

General Circulation ◽

Heart Function ◽

Erythropoietin Receptor ◽

Erythroid Cells ◽

Scientific Controversy ◽

Hematopoietic Stem ◽

Adult Mice ◽

Circulating Cells ◽

Receptor Locus ◽

Embryonic Life

During early embryonic life, the heart starts to beat before an effective circulation is established, and the kidney starts to form its vasculature before it connects to the general circulation. We and others have shown a close lineage relationship between endothelial cells (ECs) and hematopoietic cells. In fact, during embryonic development erythroblasts bud from the endothelium of developing vessels, a process we termed hemovasculogenesis. Those studies suggested the possibility that embryonic organs may have hemogenic potential. To test this hypothesis, we performed lineage studies and colony forming unit (CFC) assays to trace the fate of hematopoietic stem cells (HSCs), erythroid cells, and ECs in heart and kidney from embryonic mice. Using ER-GFPcre mice that express both GFP and cre under control of the erythropoietin receptor locus in the erythroid cells, we identified hematopoietic progenitors (Hb+Nanog+) within nascent vessels in the early embryonic kidney and heart. Using EC-SCL-Cre-ERT transgenic mice that specifically express tamoxifen inducible Cre in EC progenitors, we found both circulating and non-circulating cells from the EC lineage in the early embryonic heart and kidney. CFC assays using HSC-SCL-Cre-ERT; mTmG mice (which express GFP in the cells from the HSC lineage upon tamoxifen induction) showed that both the embryonic kidney and heart possess HSCs. Further, transplantation studies of pre-vascular embryonic kidneys from EC-SCL-Cre-ERT;R26R mice under the kidney capsule of WT adult mice showed blood cells derived from the embryonic kidney suggesting that the embryonic kidney also possesses HSCs that originate in situ. These studies indicate that the embryonic kidney and heart function as hematopoietic organs during early embryogenesis. In addition to solve an important scientific controversy in our understanding of lineage/fate relationships in the developing embryo, these findings are relevant for tissue repair/regeneration and may help explain why under pathological circumstances, hematopoiesis occurs in extramedullary organs.

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b1 Integrin Deficiency in Both Erythroid Cells and Their Microenvironment Does Not Affect Basal Erythropoiesis but Critically Impairs Survival and Erythroid Response to Phenylhydrazine-Induced Stress in Adult Mice

Blood ◽

10.1182/blood.v112.11.3567.3567 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3567-3567

Author(s):

Tatiana Ulyanova ◽

Gregory V. Priestley ◽

Yi Jiang ◽

Stephen Padilla ◽

Thalia Papayannopoulou

Keyword(s):

Critical Role ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

In Vivo Experiments ◽

Adult Mice ◽

Erythroid Precursors ◽

And Control ◽

Deficient Mice

Abstract Previous experiments in vitro have emphasized the important role of a5b1 integrin/fibronectin interactions in terminal stages of erythroid differentiation (JCB1987, 105:3105), whereas in vivo experiments with genetically deficient mice (JI2000, 165:4667) and recent in vitro ones emphasized the important contribution of a4b1 integrin in the expansion of fetal erythroid progenitors (JCB2007, 177:871) or for optimal responses post stress in adult animals (MCB2003, 23:9349). However, no abnormalities in erythropoiesis were reported in a model of conditional ablation of b1 integrins post-transplantation (Blood2006, 108:1857). Therefore, it has not been clear to what extent each of the two major b1 integrins (a4b1 and a5b1) alone or in combination is critical for expansion and/or terminal erythroid differentiation of adult cells at homeostasis and/or after stress. We have made detailed and parallel observations comparing erythropoiesis in two genetic models with conditional ablation of b1 or a4 integrins at homeostasis and after phenylhydrazine (PHZ)-mediated stress. Basal erythropoiesis in b1-, a4-deficient and control mice as assessed by hematocrit levels and total nucleated erythroid cells (Ter119+) in BM and spleen was similar. Furthermore, both b1 and a4-deficient mice showed an increase in circulating progenitors (1275±230 CFC/ml PB, 2446±256 CFC/ml PB, respectively) over controls (338±113 CFC/ml PB). However, post PHZ-induced hemolytic stress there was a dramatic difference in outcomes of b1-deficient, but modest differences in a4-deficient mice compared to controls. Survival of b1-deficient mice by day 6 post PHZ was 33% compared to 100% in a4-deficient and control groups. In b1-deficient animals, no significant increase in spleen cellularity (153±26×106 and194±64×106 cells/spleen at day 0 and 6 post PHZ, respectively) was detected and the expansion of total erythroid precursors (CD71hi,Ter119+) in the spleen was minimal (from 2.08×106 to 10.8×106 cells/spleen at day 6). In contrast, in a4-deficient and control mice by the same time spleen cellularity increased respectively by 3 and 8 fold, and erythroid precursors expanded by 400 and 2,500 fold. Of interest, BM response to PHZ was not significantly different among all groups. To test whether the splenic response was cell-autonomous or environmentally controlled we compared PHZ response in wild type recipients reconstituted with b1-ablated (Cre+b1D/D) or with control (Cre-b1f/f) BM cells. Recipients of b1-ablated cells had an impaired response compared to recipients of control cells, which was somewhat intermediate to that seen in non-transplanted b1-deficient animals; by day 6 post PHZ, spleen cellularity was 300±24×106 cells/spleen and erythroid precursors expanded by 130 fold in recipients of b1-ablated BM cells compared to 859±159×106 cells/spleen and 900 fold precursor increase in control recipients. These data suggest that both erythroid and their environmental cells were responsible for the reduced survival and poor spleen response in b1-deficient mice. The target environmental cells (fibroblasts, endothelial cells, macrophages) and/or matrix involved will be the focus of future studies. It is of interest that in contrast to splenic response, the increased release of progenitors from BM seen in animals reconstituted with b1D/D cells was as high as that seen in non-transplanted b1- deficient animals and with the same qualitative characteristics, suggesting this alteration in biodistribution of progenitors is cell autonomous. Taken together, our data suggest that a combined expression of b1 integrins in erythroid and cells in their microenvironment is critical for survival and optimal splenic response to a PHZ-induced stress in adult mice; release of progenitors seen at homeostasis in both b1 and a4 models is cell autonomous with a preferential erythroid progenitor release from BM seen only in b1-deficient but not in a4-deficient mice; in contrast to results with fetal liver cells showing a critical role of a4b1 but not a5b1 integrin for proliferative expansion of erythroid cells, in adults a5b1 expression in erythroid and environmental cells in the spleen assumes a more critical role. Our data expand the current knowledge on the distinct dependency of a4b1 vs a5b1 integrins in basal vs stress erythropoiesis and bridge previously divergent information from in vitro and in vivo experiments.

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Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20082038 ◽

2019 ◽

Vol 20 (8) ◽

pp. 2038 ◽

Cited By ~ 2

Author(s):

Zahra Masoumi ◽

Gregory E. Maes ◽

Koen Herten ◽

Álvaro Cortés-Calabuig ◽

Abdul Ghani Alattar ◽

...

Keyword(s):

Mammalian Target Of Rapamycin ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Surface Adhesion ◽

Hematopoietic Stem ◽

Ampk Signaling ◽

Placental Dysfunction ◽

Transcriptional Changes ◽

Amp Activated Protein Kinase

Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.

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Erythropoietin Receptor Expression: A Role for the Tal1/SCL and GATA-1 Complex

Blood ◽

10.1182/blood.v112.11.3583.3583 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3583-3583

Author(s):

Heather Marie Rogers ◽

Li Wang ◽

Xiaobing Yu ◽

Constance Tom Noguchi

Keyword(s):

Binding Site ◽

Erythroid Differentiation ◽

Receptor Expression ◽

Proximal Promoter ◽

Open Chromatin ◽

Erythroid Cells ◽

E Box ◽

High Level ◽

E Boxes ◽

Epor Expression

Abstract Tal1/SCL is required for hematopoietic stem cell development and is essential for normal erythropoiesis. Although Tal1-DNA binding is not required for hematopoietic development, Tal1 participates in a complex with other binding partners GATA-1, LMO2, Ldb1 and possibly Sp1. During erythropoiesis, Tal1 can activate expression via direct association with E-box-GATA DNA sequence motifs for lineage specific genes such as Protein 4.2. In mice, loss of Tal1 in adult erythropoiesis also affects TER119 expression and BFU-E growth resulting in anemia. We found that knock down of Tal1 expression in primary erythropoietin (EPO) stimulated hematopoietic progenitor cells in culture inhibited EPO receptor (EPOR) expression and erythroid differentiation, consistent with the anemia observed in mice with targeted deletion of adult Tal1 expression. In contrast, overexpression of Tal1 in erythroid cells increased both EPOR expression and erythroid differentiation. In fact, overexpression of EPOR was sufficient to increase differentiation in erythroid progenitor cell cultures. Increased EPOR expression by Tal1 was mediated by 3 conserved E-boxes in the 5′ UTR. In reporter gene assays in K562 erythroid cells, EPOR promoter activity was lost with the mutation of these E-boxes, which are located 75 bp downstream of the required GATA-1 binding motif in the human EPOR proximal promoter. Tal1/E2A dimer binding to the E-box region was demonstrated by gel mobility shift assay. Tal1 transactivated EPOR mRNA and chromatin immunoprecipitation (ChIP) analysis confirmed that Tal1 bound directly to the E-box region in intact erythroid cells. We previously showed that GATA-1 also transactivates EPOR gene expression and is required for high level of EPOR transcription activity. In ChIP assays, an antibody to Tal1 also pulled down chromatin containing the GATA-1 binding site as well as the E-box region and conversely, antibodies to GATA-1 pulled down chromatin corresponding to the GATA-1 binding site and the E-box region. These data show that the complex containing Tal1 and GATA-1 bound to the E-box region and to the GATA-1 site in the EPOR proximal promoter and that the complex has greater occupancy at the GATA-1 site in the proximal promoter. High-level Tal1 expression increased chromatin containing Tal1 or GATA-1 associated with the GATA-1 binding site as well as the down stream E-box region, with a greater proportionate increase in the E-box region. Hence, in addition to the GATA-1 binding site, the downstream E-box region is necessary for high-level EPOR expression. These data suggest that transactivation of the EPOR promoter via these sites is mediated by a Tal1 and GATA-1 containing complex and that during erythropoiesis EPO induction of both Tal1 and GATA-1 regulates the high expression of its own receptor. We hypothesize that the high binding of GATA-1 and Tal1 at the GATA site opens the upstream chromatin structure. The over-expression of Tal1 promotes the open status and facilitates the spreading of open chromatin along the chromatin to the downstream E-box region. Subsequent E-box binding by Tal1 and its interaction with GATA-1 maintains the open chromatin and activates transcription.

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Requirements for survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells

Journal of Experimental Medicine ◽

10.1084/jem.20062395 ◽

2007 ◽

Vol 204 (7) ◽

pp. 1603-1611 ◽

Cited By ~ 73

Author(s):

Cindy G. Leung ◽

Yanfei Xu ◽

Bretton Mularski ◽

Hui Liu ◽

Sandeep Gurbuxani ◽

...

Keyword(s):

Steady State ◽

Spindle Assembly Checkpoint ◽

Survivin Expression ◽

Erythroid Differentiation ◽

Inhibitor Of Apoptosis ◽

Specific Gene ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Apoptosis Protein ◽

High Level

Survivin, which is the smallest member of the inhibitor of apoptosis protein (IAP) family, is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis, and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues, survivin has been proposed as an attractive target for anticancer therapies and, in some cases, has even been touted as a cancer-specific gene. Survivin is, however, expressed in proliferating adult cells, including human hematopoietic stem cells, T-lymphocytes, and erythroid cells throughout their maturation. Therefore, it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question, we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow, with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly, heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals, with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult, and further, that a high level of survivin expression is critical for proper erythroid differentiation.

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Complete Myeloid Potential Arises First in the Mammalian Yolk Sac

Blood ◽

10.1182/blood.v112.11.730.730 ◽

2008 ◽

Vol 112 (11) ◽

pp. 730-730 ◽

Cited By ~ 1

Author(s):

Kathleen E. McGrath ◽

Jenna M. Cacciatori ◽

Anne D. Koniski ◽

James Palis

Keyword(s):

Mast Cells ◽

Fetal Liver ◽

Yolk Sac ◽

Receptor Expression ◽

Erythroid Cells ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Second Wave ◽

Definitive Hematopoiesis

Abstract In the mouse embryo, hematopoietic function is required by E10.5 (embryonic day 10.5) before adult-repopulating hematopoietic stem cells (HSC) exist. The earliest erythroid function is provided by a wave of primitive erythroid progenitors that arise at E7.5, in association with megakaryocyte and macrophage progenitors. Intriguingly, a second wave of hematopoietic potential arises between the first primitive hematopoietic wave and functional HSC formation. This second progenitor wave also forms in the yolk sac but is distinguished from the primitive wave by its slightly later onset (E8.25), generation of definitive erythroid cells, and its additional association with granulocyte and mast cell progenitors. The proposed function of these “wave 2” progenitors is to colonize the newly formed fetal liver (beginning at E10) and differentiate into the first mature definitive erythroid cells observed in circulation at E12. However, it is unclear how much definitive hematopoiesis arising in the yolk sac recapitulates the paradigm of later HSC-derived myeloid potential, progenitor hierarchy and immunophenotype. To investigate this question, we examined markers of adult myeloid progenitor maturation in the yolk sac and early fetal liver. As previously described by others, all definitive hematopoietic progenitors in the yolk sac, unlike those in the bone marrow, express CD41, which we found associated with Fc gamma receptor expression (FcγR, CD16/32) beginning at E8.5. By E9.5, definitive hematopoietic progenitors can be identified by their surface co-expression of ckit, CD41, FcγR, as well as endoglin. When cultured in vitro, these cells can differentiate into all myeloid lineages, including neutrophils, eosinophils, basophils and mast cells as identified by morphology, immunophenotype and gene expression. Preliminary clonal analysis confirms that a common erythroid/granulocyte progenitor exists in this population. Consistent with adult myelopoiesis, we found a qualitative association of higher FcγR expression with granulocyte fate and higher endoglin expression associated with erythroid fate. However, the unusual co-expression of these four markers and the prevalence of erythroid fate, even in FcγRhi cells, suggest the definitive hematopoietic progenitors in the yolk sac may be quite plastic and highly predisposed to an erythroid fate. Consistent with the concept that these “wave 2” progenitors colonize the fetal liver, we also found similar ckit+CD41+FcγR+endoglin+ cells in the early liver (E11.5) with the potential to produce a variety of myeloid cells when cultured in vitro. The emergence of enucleated definitive erythrocytes by E12, within 24 hours HSC fetal liver colonization, implies that these first erythrocytes are derived from the yolk sac definitive progenitors found in the liver by E10.5. We therefore asked whether the multiple myeloid potentials associated with “wave 2” progenitors are similarily realized the early fetal liver. Beginning at E11.5 we found a population of Gr1+Mac1+ cells in the liver with morphological and histological characteristics of neutrophils, and increase 100-fold in number between E12.5 and E14.5. In contrast, we did not observe eosinophils, basophils or mast cells by morphology, immunophenotype or by RT-PCR for lineage-specific messages. We conclude that complete definitive myeloid potential first arises in the yolk sac from a unique population of ckit+FcγR+CD41+endoglin+ progenitors. Our data suggest that these progenitors then enter the fetal liver and differentiate into a subset of their potential fates producing the first mature definitive erythromyeloid cells. This second wave of hematopoietic progenitors emerging from the yolk sac thus serves as a novel model of mulitpotential definitive hematopoiesis.

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Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Blood ◽

10.1182/blood.v61.4.751.751 ◽

1983 ◽

Vol 61 (4) ◽

pp. 751-758 ◽

Cited By ~ 16

Author(s):

M Bondurant ◽

M Koury ◽

SB Krantz ◽

T Blevins ◽

DT Duncan

Keyword(s):

Terminal Differentiation ◽

Erythroid Differentiation ◽

Precursor Cells ◽

Erythroid Cells ◽

Friend Virus ◽

Isolated Cells ◽

Two Dimensional Gel Electrophoresis ◽

Erythroid Precursor ◽

Short Time

Abstract Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.

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Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽

10.1182/blood.v64.4.930.bloodjournal644930 ◽

1984 ◽

Vol 64 (4) ◽

pp. 930-936 ◽

Cited By ~ 1

Author(s):

I Max-Audit ◽

U Testa ◽

D Kechemir ◽

M Titeux ◽

W Vainchenker ◽

...

Keyword(s):

Pyruvate Kinase ◽

Cell Lines ◽

Fetal Liver ◽

Erythroid Cells ◽

Low Concentrations ◽

Erythroleukemia Cell ◽

Erythroleukemic Cell ◽

High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

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Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-142963 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Gordon G. L. Wong ◽

Gabriela Krivdova ◽

Olga I. Gan ◽

Jessica L. McLeod ◽

John E. Dick ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Myeloid Differentiation ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Lineage Differentiation ◽

Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.8.2548.420k38_2548_2554 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2548-2554 ◽

Cited By ~ 291

Author(s):

Takashi Sato ◽

Joseph H. Laver ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Populations ◽

Hematopoietic Stem ◽

Adult Mice ◽

Cd34 Expression ◽

Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

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