ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3′-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3′ untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

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Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

Blood ◽

10.1182/blood-2009-10-250894 ◽

2010 ◽

Vol 115 (22) ◽

pp. 4367-4376 ◽

Cited By ~ 16

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Target Genes ◽

Lentiviral Vector ◽

Erythroid Differentiation ◽

Down Regulation ◽

Hematopoietic Stem ◽

Progenitor Cell Proliferation ◽

Cord Blood Cells

Abstract Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in long-term proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34+ cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent.

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

Blood ◽

10.1182/blood.v126.23.1185.1185 ◽

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.

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Hematopoietic Stem Cells, but Not Progenitors, Are the Exclusive Target for STAT5-Induced Long-Term Self-Renewal.

Blood ◽

10.1182/blood.v112.11.1347.1347 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1347-1347

Author(s):

Szabolcs Fatrai ◽

Albertus T.J. Wierenga ◽

Edo Vellenga ◽

Simon M. G. J. Daenen ◽

Jan J. Schuringa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Erythroid Differentiation ◽

Retroviral Vectors ◽

Independent Manner ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Constitutive activation of STAT5 has been associated with leukemic transformation. Previously, we demonstrated that overexpression of activated STAT5 in human cord blood (CB) cells results in increased stem cell self-renewal and long-term expansion which coincided with the induction of erythropoiesis. In the present study we investigated whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. We generated retroviral expression systems where STAT5 is fused to the estrogen receptor ligand binding domain (ER) allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Human CB cells were transduced with control and STAT5-ER retroviral vectors followed by MoFlo sorting into four populations: hematopoietic stem cells (HSC, defined as CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA−), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123−CD45RA−). Sorted populations were plated on MS5 bone marrow stromal cocultures and STAT5 activity was induced by 4-OHT treatment. HSC cells expressing activated STAT5 displayed a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed in cocultures initiated with CMPs and MEPs. Also, CFC numbers dropped significantly after 2 weeks and neither of the cultures could be maintained longer than 3 weeks regardless of STAT5 activity. FACS measurements and cytospins showed a block in myelopoiesis and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations, while the differentiation potential of the GMP remained unaffected. Next, we aimed to identify STAT5 target genes which were upregulated in the STAT5 HSC population that were not responsible for erythroid differentiation and played a role in STAT5-induced self-renewal and long-term expansion. GATA1 was down-modulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarray was performed on both GATA1 downmodulated STAT5-transduced CB cells as well as on STAT5-transduced HSC and progenitor populations. Micoarray data from the two experiments were compared and 39 GATA1- independent STAT5 target genes were identified in the STAT5 HSC population. Many of these genes encoded for membrane proteins or proteins involved in adhesion, migration and signal transduction, and these are currently under investigation. In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5- induced long-term self-renewal. We identified a set of genes that is upregulated in the STAT5-transduced HSC population in a GATA1-independent manner which is potentially responsible for STAT5-induced self-renewal and long-term expansion.

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The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells

Blood ◽

10.1182/blood.v100.6.2063.h81802002063_2063_2070 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2063-2070

Author(s):

Cécile Challier ◽

Laurence Cocault ◽

Rolande Berthier ◽

Nadine Binart ◽

Isabelle Dusanter-Fourt ◽

...

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Es Cells ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Chimeric Receptor ◽

Hematopoietic Differentiation ◽

Es Cell ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

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Deciphering Molecular Control of VEGFR2 Regulation in Hematopoietic Progenitors: GATA1-Mediated Repression of VEGFR2 Promotes Optimum Erythropoiesis

Blood ◽

10.1182/blood.v126.23.1172.1172 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1172-1172

Author(s):

Avishek Ganguly ◽

Omar S. Aljitawi ◽

Soumen Paul

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Fetal Liver ◽

Embryonic Stem ◽

Erythroid Differentiation ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Molecular Control ◽

Endothelial Development

Abstract VEGFR2 (also known as Flk1) is expressed in hemetopoietic precursors and is essential for both hematopoietic and vascular development. Interestingly, development of differentiated hematopoietic cell from hematopoietic stem cells (HSCs) is associated with VEGFR2 repression, whereas VEGFR2 expression is maintained throughout endothelial development. This differential regulation of VEGFR2 has been implicated as a key step to successfully branch out hematopoietic vs. endothelial development. However, molecular mechanisms that regulate transcriptionally active vs. repressive Vegfr2 chromatin domains in hematopoietic stem/progenitor cells (HSPCs) vs. differentiated hematopoietic cells are incompletely understood. Here, we report that transcription factor GATA1, a master-regulator of erythroid differentiation, is essential to repress VEGFR2 expression in erythroid progenitors. Genetic complementation analysis demonstrated that VEGFR2 expression in maintained in GATA1-null erythroid progenitors and rescue of GATA1-function induces VEGFR2 repression. Mechanistic studies in primary hematopoietic progenitors from mouse fetal liver and differentiating mouse embryonic stem cells (ESCs) identified a repressor element at the (-)88 kb region of the Vegfr2 locus from which GATA1 represses Vegfr2 transcription in erythroid progenitors. Furthermore, CRISPR/Cas9-mediated deletion of the Vegfr2(-)88 kb region results in reduced erythroid differentiation during fetal liver hematopoiesis. These results indicate that GATA1-mediated repression of VEGFR2 could be a determinant of optimum erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells

Blood ◽

10.1182/blood.v100.6.2063 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2063-2070 ◽

Cited By ~ 2

Author(s):

Cécile Challier ◽

Laurence Cocault ◽

Rolande Berthier ◽

Nadine Binart ◽

Isabelle Dusanter-Fourt ◽

...

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Es Cells ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Chimeric Receptor ◽

Hematopoietic Differentiation ◽

Es Cell ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

Abstract The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

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Graded Repression of PU.1 Expression by GATA Factors Influence Hematopoietic Progenitor Cell Fate

Blood ◽

10.1182/blood.v112.11.2469.2469 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2469-2469

Author(s):

Stella T. Chou ◽

Charles Bailey ◽

Yu Yao ◽

Zan Huang ◽

Kim Nichols ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Target Genes ◽

Fetal Liver ◽

Embryonic Stem ◽

Hematopoietic Cell ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Differentiation ◽

Gata Factor ◽

Hematopoietic Progenitors

Abstract Hematopoietic cell fate is determined by combinatorial interactions between nuclear proteins that activate and repress transcription. This principle is illustrated by the transcription factors GATA-1 and PU.1, which promote erythro-megakaryocytic and granulocyte-macrophage development respectively. These two proteins interact physically to cross-antagonize each other’s activities, creating regulatory loops for hematopoietic differentiation. Previously, we showed that loss of GATA-1 causes expansion of bipotental megakaryocyte erythroid progenitors (MEPs) from embryonic stem cells or fetal liver derived hematopoietic progenitors. These cells, termed G1ME, for GATA-1-null megakaryocyte-erythroid, proliferate continuously in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. G1ME cells express GATA-2, a GATA-1-related protein normally found in multipotential hematopoietic progenitors and stem cells. These cells also express moderate levels of PU.1 mRNA, approximately 1/3 of that expressed in the myeloid cell line 416B. Upon retroviral restoration of GATA-1, GATA-2 is downregulated and PU.1 mRNA decreases rapidly. Microarray analysis of GATA-1-rescued G1ME cells revealed repression of PU.1 and many of its downstream target genes, raising the possibility of direct PU.1/Sfpi1 gene repression by GATA-1. Chromatin immunoprecipitation (ChIP) studies identified two GATA factor-binding sites at the PU.1/Sfpi1 locus. In the absence of GATA-1, when the PU.1/Sfpi1 gene is active, these sites are occupied by GATA-2. Retrovirally expressed GATA-1 replaces GATA-2 at these sites, repressing PU.1 transcription during concomitant erythro-megakaryocytic maturation. These findings resemble the “GATA-factor switch” described at other loci such as Gata2 and Kit where GATA-2 and GATA-1 compete for the same cis elements to activate and repress transcription respectively. To test this, we used siRNA to repress GATA-2 expression in G1ME cells by about 60%. Strikingly, this caused PU.1 to be upregulated 4-fold, indicating that GATA-2 also represses PU.1/Sfpi1, but to a lesser extent than GATA-1. Moreover, G1ME cells expressing GATA-2 siRNA differentiated into macrophages, as evidenced by morphology, expression of numerous cell-type specific markers and massive induction of macrophage specific genes including myeloperoxidase, Mac-1, and C/EBPα. Our findings illustrate two new insights into the transcriptional control of hematopoietic cell differentiation: First, cross-antagonism between GATA-1 and PU.1 not only occurs at the level of protein-protein interaction, but also through direct transcriptional repression. Second, in addition to having opposite effects on transcription of the same target gene as described previously, GATA-2 and GATA-1 can act cooperatively and successively to exert repressive effects of different magnitudes that gradually restrict gene expression during hematopoietic development. In this model, hematopoietic progenitors express GATA-2 and low levels of PU.1 that maintain the multipotential state but are not sufficient for myelopoiesis. Repression of GATA-2 in the absence of GATA-1 raises PU.1 levels to stimulate granulocyte-macrophage development. In contrast, activation of GATA-1 causes PU.1 to be fully repressed, promoting erythrocyte and megakaryocyte differentiation. Our data illustrate how lineage fate and hematopoietic differentiation are influenced by the stoichiometry between GATA-1, GATA-2, and PU.1 in multipotential progenitors.

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Angiotensin-Converting Enzyme (ACE) Expression Defines the Earliest Primitive and Definitive Lympho-Hematopoietic Progenitors Derived from Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v108.11.1662.1662 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1662-1662

Author(s):

Elias T. Zambidis ◽

Venta J. Jokubaitis ◽

Ada Tam ◽

Lidia Sinka ◽

Curt I. Civin ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

In Vitro Assays ◽

Hematopoietic Differentiation ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Abstract Pluripotent human embryonic stem cells (hESC) provide unprecedented opportunities for studying obscure human developmental events, such as those required for the genesis of hematopoietic stem cells (HSC). A poorly characterized aspect of embryonic and adult HSC development is the role of the renin-angiotensin system (RAS), which is implicated in regulating HSC proliferation at yolk sac (YS), fetal, and adult stages. We have recently described somatic ACE surface expression not only in adult HSC, but also at the earliest stages of emergent hemato-endotheliogenesis using a novel monoclonal antibody (BB9). ACE expression identifies primitive subsets of adult CD34+ bone marrow HSC, but more intriguingly, marks emergent hematopoietic cells from both CD34− and CD34+ areas of human YS, intraembryonic subaortic patches, and hemogenic endothelial layers of the aorta-gonad-mesonephros (AGM) region. The pattern of human embryonic ACE expression is consistent with the hypothesis of ACE+CD34− hemangioblasts emigrating dorsally from the para-aortic splanchnopleura, and subsequently colonizing the ventral aorta to give rise to CD34+ hemogenic endothelium. We tested the hypothesis that ACE expression would similarly identify emerging hemato-endothelial progenitors derived from our recently-described hESC-based hematopoietic differentiation system. This embryoid body (hEB)-based system recapitulates hemato-endothelial, primitive, and definitive stages of human embryonic blood development. ACE expression kinetics during hEB differentiation correlated well with the onset of hemato-endothelial differentiation and gene expression (e.g., SCL/tal1, CDX4, CD31, and CD34). Furthermore, using improved, novel methods of hEB hematopoietic differentiation that dramatically augment multilineage progenitors for both primitive and definitive hematopoiesis, we observed that levels of hEB ACE expression were directly correlated to increased hematopoietic potency. Subsequent FACS purification of these hEB cells demonstrated that the earliest detectable multilineage lympho-hematopoietic competency was contained entirely within both ACE+CD34−CD45− hEB and ACE+CD34+CD45− populations. These early ACE+CD45− hEB populations were heterogenous, and co-expressed abundant levels of known hemato-endothelial markers such as CD31, KDR, CD164, CD43, and CD71. Using novel in vitro assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ hEB contained common progenitors for both primitive and definitive hematopoiesis, with ACE+CD34−CD45− hEB being enriched for the highest number of progenitors. We were also able to demonstrate that further maturation of these ACE+ hEB cells in an in vitro AGM-like stromal environment produced definitive hematopoietic progenitors that resembled those obtained from cord blood CD34+ cells. The regulatory effects of angiotensin agonist/antagonist peptides on hEB-derived hematopoietic ACE+ progenitors, and their in vivo correlation to ACE+ cells obtained from early human YS and AGM tissue is currently in progress. Furthermore, single-cell analysis is underway to delineate an ACE+ hEB-derived hemangioblastic precursor of not only endothelium, but also primitive and definitive lympho-hematopoiesis.

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Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice

Blood ◽

10.1182/blood.v78.6.1448.1448 ◽

1991 ◽

Vol 78 (6) ◽

pp. 1448-1451 ◽

Cited By ~ 81

Author(s):

M Musashi ◽

SC Clark ◽

T Sudo ◽

DL Urdal ◽

M Ogawa

Keyword(s):

Stem Cells ◽

Colony Formation ◽

Interleukin 4 ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Synergistic Interactions ◽

Two Factors ◽

Stromal Cell Line ◽

Interleukin 11

Abstract Interleukin-11 (IL-11) is a newly identified lymphohematopoietic cytokine originally derived from the primate bone marrow stromal cell line, PU-34. Separately, we reported that IL-11 augments IL-3-dependent proliferation of primitive murine hematopoietic progenitors in culture. We have now examined the synergistic interactions between IL-11 and IL- 4 in support of colony formation from marrow cells of mice treated 2 days before with 150 mg/kg 5-fluorouracil. Neither recombinant human IL- 11 nor murine IL-4 alone was effective in the support of colony formation. When the two factors were combined, there was major enhancement of colony formation, including that of multilineage colony- forming cells. Serial observations (mapping studies) of development of multipotential blast cell colonies indicated that the synergy between IL-11 and IL-4 is due in part to shortening of the dormant period of the stem cells, an effect very similar to that of IL-6 and granulocyte colony-stimulating factor. The combination of IL-11 and IL-4 may be useful in the stimulation of dormant hematopoietic stem cells in vivo.

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Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

10.1101/202010 ◽

2017 ◽

Cited By ~ 1

Author(s):

Philippe E. Mangeot ◽

Valérie Risson ◽

Floriane Fusil ◽

Aline Marnef ◽

Emilie Laurent ◽

...

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Target Genes ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Target Cells ◽

Mouse Bone Marrow ◽

Primary Cells ◽

Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

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