Rcor1 Deficiency Disrupts Myeloerythroid Lineage Differentiation and Causes Severe Myelodysplasia

Abstract Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Blood ◽

10.1182/blood.v114.22.551.551 ◽

2009 ◽

Vol 114 (22) ◽

pp. 551-551

Author(s):

Suparna Nanua ◽

Jun Xia ◽

Mark Murakami ◽

Jill Woloszynek ◽

Daniel C. Link

Keyword(s):

Er Stress ◽

Protein Misfolding ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Clinical Samples ◽

Missense Mutations ◽

Loss Of Function ◽

Granulocytic Differentiation ◽

Disease Pathogenesis ◽

Chemical Inducers

Abstract Abstract 551 Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Blood ◽

10.1182/blood.v122.21.3669.3669 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3669-3669

Author(s):

Stephan Emmrich ◽

Franziska Schmidt ◽

Ramesh Chandra Pandey ◽

Aliaksandra Maroz ◽

Dirk Reinhardt ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Gene Set Enrichment Analysis ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Lncrna Expression ◽

Fold Reduction ◽

Non Coding Rnas

Abstract Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs. The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis. Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs. Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA). For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays. Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

Blood ◽

10.1182/blood.v120.21.2811.2811 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2811-2811

Author(s):

Xiaomei Yan ◽

Yue Zhang ◽

Goro Sashida ◽

Aili Chen ◽

Xinghui Zhao ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Fetal Liver ◽

Loss Of Function ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

De Novo Aml

Abstract Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Role of Long Coding RNAs in Epigenetic Modulation of Hematopoiesis

Blood ◽

10.1182/blood.v122.21.sci-28.sci-28 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-28-SCI-28

Author(s):

Mitchell J. Weiss

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Protein Localization ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Similar Degree ◽

Erythroid Progenitors

Abstract Long noncoding (Lnc) RNAs are RNA transcripts greater than 200 nucleotides (nt) that regulate gene expression independent of protein coding potential (1-3). It is estimated that thousands of lncRNAs play vital roles in diverse cellular processes. LncRNAs modulate many stages of gene expression by regulating transcription, epigenetics, splicing, translation, and protein localization. We hypothesize that multiple lncRNAs are expressed specifically during erythrocyte and megakaryocyte differentiation, and are likely to have important roles. To identify lncRNAs in erythro-megakaryopoiesis, we performed strand-specific, paired-end deep sequencing (RNA-Seq) to a depth of 200 million reads per sample on two replicates each of murine Ter119+erythroblasts, CD41+ megakaryocytes and bipotential megakaryocyte-erythroid progenitors (MEPs) [lin- Kit+ Sca1- CD16/32- CD34-], and used bioinformatic filtering tools to identify approximately 1,100 candidate lncRNAs. Over 60 percent of these lncRNAs are novel unannotated transcripts with exquisite lineage-specific expression. Using erythroid and megakaryocytic primary cell ChIP-Seq for key transcription factors (TFs) GATA1, TAL1, GATA2,and FLI1, we found that the loci of lncRNAs show similar degree of TF binding as coding genes. We used the erythroid line G1E-ER4 (which expresses estrogen-activated GATA1) to confirm that lncRNAs bound by GATA1 are also directly regulated by it. Furthermore, we used histone methylation ChIP-Seq to show that most lncRNAs arise from classical “promoters” with high H3K4me3 levels and low H3K4me1 levels. Thus, we find that lncRNAs show epigenetic features similar to the promoters of coding genes and are directly regulated by similar TF networks. Comparison of the transcriptomes of mouse fetal liver and human cord blood erythroblasts demonstrated that lncRNAs are expressed in a highly species-specific fashion, i.e., most lncRNAs identifiable in one species are not transcribed in the other, even though the corresponding genomic region is present in both species. Numerous non-conserved but functional lncRNAs are reported in the literature, and the significance of conservation in lncRNA biology is greatly debated. In order to identify functional lncRNAs, we are currently performing RNAi knockdown on numerous candidates to assess how loss of function affects erythroid maturation. We are also performing HITS-CLIP of key chromatin modifying complexes and erythroid transcription factors to identify lncRNAs bound to them. Our studies are beginning to define new layers of gene regulation in normal erythro-megakaryopoiesis, which may be relevant to the pathophysiology of related disorders including various anemias, myeloproliferative and myelodysplastic syndromes and leukemias. 1. Wang K.C., Chang H.Y. Molecular mechanisms of long noncoding RNAs. Molecular Cell. 2011;43(6):904-914. Prepublished on 2011/09/20 as DOI 10.1016/j.molcel.2011.08.018. 2. Hu W., Alvarez-Dominguez J.R., Lodish H.F. Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO reports. 2012;13(11):971-983. Prepublished on 2012/10/17 as DOI 10.1038/embor.2012.145. 3. Paralkar V.R., Weiss M.J. Long noncoding RNAs in biology and hematopoiesis. Blood. 2013;121(24):4842-4846. Prepublished on 2013/05/07 as DOI 10.1182/blood-2013-03-456111. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Blood ◽

10.1182/blood.v124.21.3846.3846 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3846-3846

Author(s):

Ji-Yoon Noh ◽

Shilpa Gandre-Babbe ◽

Yuhuan Wang ◽

Vincent Hayes ◽

Yu Yao ◽

...

Keyword(s):

Transcription Factor ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Embryonic Stem ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Transfusion Therapy ◽

Ips Cell

Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

Download Full-text

LFA-1-Mediated Adhesion to ICAM-2 Is Critical for Stromal Cell-Dependent Early T-Lymphoid Differentiation from Human Hematopoietic Precursors

Blood ◽

10.1182/blood.v126.23.2397.2397 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2397-2397

Author(s):

Hirohito Minami ◽

Kohshi Ohishi ◽

Masahiro Masuya ◽

Naoyuki Katayama

Keyword(s):

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Monocytic Cells ◽

Blocking Antibody ◽

Lineage Differentiation ◽

B Lineage ◽

B Lymphoid

Abstract The regulatory mechanism of human early T-lymphoid differentiation remains less defined. We previously reported that human telomerized bone marrow stromal cells support the generation of CD7+ CD56- early T- as well as CD10+ CD19+ early B-lymphoid precursors from human hematopoietic precursors. Here we examined whether and how early T lymphopoiesis is regulated by interaction with stromal cells. Low or no levels of LFA-1 were expressed on CD34+ CD38- CD45RA- immature hematopoietic precursors. However, high levels of LFA-1 were detected on CD34+ CD38- CD45RA+ CD10+ CD7+/- CD19- immature lymphoid precursors, while those of LFA-1 were diminished on CD34+ CD38+ CD45RA+ CD10+ CD19+ more mature pro B cells. On the other hand, ICAM-1 and ICAM-2 were expressed in a portion of the telomerized stromal cells. ICAM-3 was not detected. Various levels of ICAM-1, ICAM-2, or ICAM-3 were also expressed on hematopoietic and lymphoid precursors. To examine the role of LFA-1-mediated adhesion to stromal cells or adjacent hematopoietic precccursors in early lymphoid differentiation, we examined the effect of anti-LFA-1 blocking antibody (Ab) on the differentiation of CD34+ CD45RA- CD7- CD10- CD38lo/- hematopoietic precursors in the cultures on stromal cells or with conditioned medium (CM) obtained from cultures of stromal cells. In the cultures on stromal cells, anti-LFA-1 Ab strongly inhibited the generation of CD7+ CD10- CD45RA+ and CD7- CD10+ CD45RA+ lymphoid precursors from hematopoietic precursors after 21 days of culture. Significant number of CD14+ monocytic cells was generated with or without anti-LFA-1 Ab. In the cultures with CM, anti-LFA-1 Ab showed marginable effect on lymphoid differentiation. To elucidate the effect of anti-LFA-1 Ab on more mature lymphoid precursors, CD7+ CD10- CD45RA+ or CD7- CD10+ CD45RA+ cells were isolated after culture of hematopoietic cells for 14 days and cultured on stromal cells in the presence or absence of anti-LFA-1 Ab. Anti-LFA-1 Ab remarkably inhibited the generation of CD7+ and CD10+ CD19+ lymphoid cells from the CD7+ CD10- CD45RA+ early T-lymphoid precursors. Notably, few or no CD45RA+ CD14- lymphoid cells were detected. Anti-LFA-1 Ab did not affect B-lineage differentiation from CD10+ CD19- CD45RA+ early B-lymphoid precursors to CD10+ CD19+ CD45RA+ proB cells. We next examined which ICAM ligand is responsible for the observed effects by anti-LFA-1 Ab. Anti-ICAM-2 Ab inhibited the generation of CD7+ CD45RA+ and CD10+ CD45RA+ lymphoid precursors from CD34+ CD45RA- CD7- CD10- CD38lo/- hematopoietic precursors on stromal cells, as observed with anti-LFA-1 Ab. No effect was observed with anti-ICAM-1 Ab. Anti-ICAM-2 Ab further suppressed the generation of CD7+ and CD10+ lymphoid cells from the cultured CD7+ CD10- CD45RA+ early T-lymphoid precursors, but did not depress B-lymphoid differentiation from the cultured CD7- CD10+ CD45RA+ early B-lymphoid precursors. Taken together, these data indicate that LFA-1-mediated adhesion to ICAM-2 is essential for stromal cell-dependent early lymphoid differentiation of hematopoietic and CD7+ early T-lymphoid precursors. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Identification of Pre-Gmps and Pre-Meps: Two Novel Types of Committed Progenitor Cells in Bone Marrow

Blood ◽

10.1182/blood-2021-153800 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3266-3266

Author(s):

Ryan Mack ◽

Lei Zhang ◽

Kanak Joshi ◽

Shanhui Liu ◽

Mark Sellin ◽

...

Keyword(s):

Conflicts Of Interest ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Effector Cells ◽

Functional Studies ◽

Lineage Differentiation ◽

Reliable Marker ◽

Relationship Of

Abstract Elucidating the stepwise differentiation processes that leads from multipotent hematopoietic stem cells to mature effector cells is critical for understanding both normal and neoplastic hematopoiesis. Early studies suggested that common myeloid progenitors (CMPs) are oligo-lineage hematopoietic progenitors that produce all lineages of myeloid cells, including granulocytes, monocytes, erythrocytes and megakaryocytes. CMPs do so by first giving rise to megakaryocyte-erythroid progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs), two types of bi-lineage progenitors. However, this concept was challenged by several recent studies where single cell techniques demonstrated that CMPs, GMPs and MEPs are highly heterogenic. The existence of lineage-restricted subsets within the CMP population leads to questions about whether erythroid and megakaryocytic lineage commitment is actually initiated at the multipotent progenitor or CMP stage. During the past 15 years, several lineage-restricted subsets of progenitors have been separated out from CMP population, including monocyte-dendritic progenitors, megakaryocyte progenitors, and erythroid progenitors based on expression of CD115/Flt3, CD41/CD150, and CD105/CD150, respectively. However, the remaining CMP population is still highly heterogenic. Thus, further separation of functional subsets within the CMP compartment is required. By screening cell surface markers that can further separate CMPs, we have identified CD27 as a reliable marker to separate all megakaryocyte/erythrocyte-committed progenitors from granulocyte/monocyte-committed progenitors. In addition, we found that CD62L is only expressed on granulocyte/monocyte-committed progenitors. CD27 and CD62L co-staining can separate CMP into CD27 +CD62L +, CD27 +CD62L - and CD27 -CD62L - subsets. Biology and morphology study showed that CD27 +CD62L - cells are closely associated with GMPs, whereas CD27 -CD62L - cells are closely associated with MEPs. In vitro culture and in vivo transplantation functional studies demonstrated that 1) CD27 +CD62L + cells are pre-GMPs that give rise to FcGRII/III + GMPs and only produce granulocytes and monocytes; 2) CD27 -CD62L - cells are pre-MEPs that give rise to MEPs and primarily produce erythrocytes and megakaryocytes with minimal contribution to granulocytes and monocytes; 3) CD27 +CD62L - subset enriches cells with genuine CMP potential capable of producing GMPs, MEPs, and subsequent progeny. Taken together, we have identified two novel populations of committed progenitors that serve as intermediates between CMP-GMP and CMP-MEP commitment pathways. Identification of pre-GMPs and pre-MEPs fills in the gap between CMPs-GMPs and CMPs-MEPs, supporting the hierarchal relationship of myeloid lineage differentiation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Abnormal Expression of Human Mitoferrin (SLC25A37) Is Associated with a Variant of Erythropoietic Protoporphyria.

Blood ◽

10.1182/blood.v108.11.3.3 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3-3 ◽

Cited By ~ 2

Author(s):

George C. Shaw ◽

Nathaniel B. Langer ◽

Yongming Wang ◽

Liangtao Li ◽

Jerry Kaplan ◽

...

Keyword(s):

Amino Acid ◽

Fetal Liver ◽

Amino Acid Level ◽

Ectopic Expression ◽

Disease Process ◽

Hypochromic Anemia ◽

K562 Cells ◽

Loss Of Function ◽

Erythropoietic Protoporphyria ◽

Iron Deficient

Abstract Mitoferrin (mfrn, slc25a37) is the high-affinity transporter of iron into mitochondria of developing erythroblasts and is essential for normal erythropoiesis. Recently, we described the cloning and characterization of mfrn in the zebrafish frascati (frs) mutant, in which homozygous null mutation leads to profound hypochromic anemia and embryonic lethality (Shaw GC, et al. 2006 Nature 440:96–100). Here we characterize the human MFRN orthologue and implicate a specific disease process purportedly caused by defects in MFRN expression. We studied human MFRN expression by a) analysis of human-tissue northern blots, b) fluorescent immunohistochemistry and confocal microscopy for subcellular localization, c) genetic complementation in MRS3/4 deficient yeast strains, and d) microinjection and rescue efficiency of frs mutant zebrafish. Human MFRN, mapping to chromosome 8p21, shows 82% and 64% similarity at the amino acid level to orthologous mouse and zebrafish Mfrn proteins respectively, and 60% similarity to a paralogous MFRN2 (SLC25A28). MFRN mRNA is highly expressed in fetal and adult hematopoietic tissues, including fetal liver and bone marrow, similar to zebrafish and mice. The MFRN protein localizes to the mitochondria like other SLC25 solute carriers. Ectopic expression of human MFRN capably restores hemoglobinized cells in anemic frs embryos, programs increased 55Fe-labeled heme synthesis in K562 cells, and rescues MRS3/4 mutant yeast for growth on iron-deficient media, demonstrating conservation of MFRN function. We evaluated 6 unrelated probands with a variant of erythropoietic protoporphyria (EPP) who harbor no mutations in ferrochelatase, the genetic cause of classic EPP. Five of the 6 index cases had severe hepatic disease necessitating liver transplantation. Our analysis of all 6 patients revealed the presence of an aberrantly spliced MFRN transcript, which was absent in normal individuals and patients with classic EPP. This aberrant spliceoform results in insertion of a 477 bp fragment from intron 2 and leads to an early nonsense codon after amino acid 156, prematurely truncating the MFRN protein. In contrast to normal MFRN cDNA, the variant MFRN cDNA from EPP patients fails to complement MRS3/4 mutant yeasts in low-iron media, demonstrating its loss-of-function activity. Our data demonstrate conserved function of human MFRN in erythroid mitochondrial iron metabolism and an association between a loss-of-function MFRN transcript and a variant of erythropoietic protoporphyria.

Download Full-text

Trib2 Pseudokinase Marks Early CMP Progenitors, and Promotes Granulocytic Plus Erythroid Progenitor Cell Formation

Blood ◽

10.1182/blood.v122.21.2448.2448 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2448-2448

Author(s):

Lei Li ◽

David Kuhrt ◽

Arvind Dev ◽

Edward Jachimowicz ◽

Koichi Akashi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Expression Profiles ◽

Ectopic Expression ◽

Cell Formation ◽

Relative Increase ◽

Erythroid Progenitors ◽

Monocytic Cell ◽

Erythroid Progenitor ◽

Altered Expression ◽

Gm Csf

Abstract Altered expression of Trib2 pseudokinase has been associated with AML (M2, M4 subtypes) (Cancer Cell, 10: 401), and ectopic expression of Trib2 in hematopoietic progenitors drives myeloid leukemogenesis (Blood, 116: 1321). In addition, inhibition of Trib2 expression by EPO in erythroid progenitors has been demonstrated (Blood, 111: 5390). Beyond this, little is known about Trib2’s expression profiles and roles during normal myelopoiesis. To critically address functional roles, we have generated and characterized a novel Trib2-KO mouse model. Within bone marrow CFU-GM formation faltered, and Trib2-KO Linneg progenitors exhibited deficit granulocyte production (with a relative increase in monocytes). This was not associated with significant differences in survival or cell cycle features among developing granulomonocytic cells. Instead, Trib2 levels proved to be heightened among Linneg progenitors (and inhibited in GM cells following GMCSF, GCSF or ATRA exposure). This prompted analyses of CMP and early-CMP (eCMP) populations in which RNAseq demonstrated peak Trib2 expression. Furthermore, eCMP and CMP pools were elevated in Trib2-KO mice. Within progenitors of the erythroid lineage, Trib2 expression levels also selectively increased, and in Trib2-KO mice BFUe and CFUe levels were decreased (and RBC plus HGB were diminished). Intriguingly, when Trib2-KO mice were challenged with Flt3L plus GMCSF, BFUe (but not CFU-GM or GEMM) were mobilized to peripheral blood at levels 13.3-fold above wild-type controls. This indicates candidate effects of Trib2 on a proposed BFUe niche. Overall, novel multi-lineage roles for Trib2 are revealed in regulating eCMP pools, GM-CSF populations, balanced granulocytic vs monocytic cell formation, and the production of erythroid progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Direct Contact with Stromal Cells in Association with SDF-1 Is Important for B-Lineage Differentiation Toward CD19+ ProB Cells from Human Hematopoietic Precursors, but Dispensable for Generation of CD7+CD45RA+ Multipotent Lymphoid Precursors

Blood ◽

10.1182/blood.v124.21.5138.5138 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5138-5138

Author(s):

Hirohito Minami ◽

Kohshi Ohishi ◽

Yoshiki Nakamori ◽

Masahiro Masuya ◽

Naoyuki Katayama

Keyword(s):

Stromal Cells ◽

Direct Contact ◽

Conflicts Of Interest ◽

Neutralizing Antibody ◽

Hematopoietic Progenitors ◽

Monocytic Cells ◽

Differentiation Potential ◽

Human Cd34 ◽

Lineage Differentiation ◽

B Lineage

Abstract The regulatory mechanism of human early B- and T- lymphoid differentiation has not been well studied. Coculture on telomerized human bone marrow stromal cells supported the generation of CD7+CD45RA+ multipotent precursors with differentiation potential for T-, B-, NK-lineage, and monocytic cells, CD10+CD19-CD45RA+ B-biased precursors, and CD10+CD19+CD45RA+ proB cells from human CD34+lin- hematopoietic progenitors. CD7+CD45RA+ and CD10+CD19- lymphoid precursors can be developed from hematopoietic progenitors by soluble factors produced from stromal cells, but the generation of CD19+ proB cells was reduced. Replating analysis showed that direct contact with stromal cells promoted B-lineage differentiation toward CD19+ proB cells from CD34+lin- cell-derived CD7+CD45RA+ and CD10+CD19- lymphoid precursors. SDF-1 is shown to be critical for B-lineage differentiation from studies of mice. SDF-1 was produced from the human telomerized stromal cells. Inhibition of binding to SDF-1 with neutralizing antibody against CXCR4 suppressed B-lineage differentiation to CD19+ proB cells from hematopoietic precursors, while the generation of CD7+CD45RA+ cells was not significantly affected. By replating analysis, anti-CXCR4 Ab inhibited the differentiation to CD19+ proB cells from CD7+CD45RA+ and CD10+CD19- lymphoid precursors on stromal cells. These data indicate that direct contact with stromal cells in association with SDF-1 produced from stromal cells is important for B-lineage differentiation toward CD19+ proB cells from CD7+CD45RA+ and CD10+CD19- lymphoid precursors but dispensable for differentiation toward CD7+CD45RA+ multipotent lymphoid precursors. Disclosures No relevant conflicts of interest to declare.

Download Full-text