Decay-accelerating factor is present on paroxysmal nocturnal hemoglobinuria erythroid progenitors and lost during erythropoiesis in vitro.

A glycoprotein that regulates the deposition of C3b on the erythrocyte surface, called decay-accelerating factor or DAF, is absent from the red blood cells (RBC) of patients with paroxysmal nocturnal hemoglobinuria (PNH), explaining in part their abnormal sensitivity to complement. We used a specific antiserum to DAF, flow microfluorometry, and clonogenic assays for erythroid progenitor cells to study PNH erythropoiesis in vitro. By fluorescence-activated cell sorter analysis, all RBC from normal individuals are DAF+. In contrast, the RBC of six patients with PNH showed discrete populations of DAF- cells (10-44%; x +/- SEM = 31 +/- 6%). The DAF- RBC population was partly eliminated by prior acidified serum lysis. To determine whether erythropoietic progenitors expressed DAF, bone marrow cells were sorted by flow microfluorometry and the separated DAF+ and DAF- populations then cultured in vitro. In two normal individuals, but also in six patients with PNH, erythroid colonies formed only from cells in the DAF+ fraction. However, a variable proportion of the normoblast progeny of these DAF+ progenitor cells from patients with PNH was DAF-. Individual bursts removed from cultures of PNH bone marrow showed two discrete populations by fluorescence; the majority of normoblasts were DAF-, only 3 of 27 individual bursts had greater than 50% DAF+ cells, and in three patients, DAF- normoblasts averaged 79%. In contrast, the progeny of individual bursts from normal individuals comprised a unimodal DAF+ population. In each PNH patient, one normal burst (greater than 80% DAF+ normoblasts) was detected, possibly reflecting a normal residual population of erythroid progenitors. By the criterion of DAF expression, there was no evidence of separate populations of normal and PNH type progenitor cells. The phenotypically normal erythroid progenitors of PNH bone marrow acquire the PNH characteristics during differentiation in vitro.

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Heterozygous Mutations in DDX41 Cause Erythroid Progenitor Cell Defects

Blood ◽

10.1182/blood-2021-153544 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 148-148

Author(s):

Timothy M Chlon ◽

Emily Stepanchick ◽

Analise Sulentic ◽

Kathleen Hueneman ◽

Daniel Starczynowski

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Progenitor Cells ◽

Liquid Culture ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Colony Assays

Abstract Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3147 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3147-3153 ◽

Cited By ~ 88

Author(s):

G A Blobel ◽

C A Sieff ◽

S H Orkin

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Transcription Factor ◽

Progenitor Cells ◽

Erythroid Cell ◽

High Dose ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

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Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

Blood ◽

10.1182/blood.v92.10.3658 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3658-3668 ◽

Cited By ~ 128

Author(s):

Birgit Panzenböck ◽

Petr Bartunek ◽

Markus Y. Mapara ◽

Martin Zenke

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Stem Cell Factor ◽

Large Cell ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Peripheral Blood Stem Cells ◽

Erythroid Progenitor ◽

Cell Numbers

Abstract Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

Journal of Experimental Medicine ◽

10.1084/jem.20200164 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 3

Author(s):

James W. Swann ◽

Lada A. Koneva ◽

Daniel Regan-Komito ◽

Stephen N. Sansom ◽

Fiona Powrie ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Chronic Inflammation ◽

Inflammatory Disease ◽

Erythropoietin Receptor ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

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Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation

Blood ◽

10.1182/blood.v75.3.603.603 ◽

1990 ◽

Vol 75 (3) ◽

pp. 603-610 ◽

Cited By ~ 91

Author(s):

T Takahashi ◽

K Ozawa ◽

K Takahashi ◽

S Asano ◽

F Takaku

Keyword(s):

Bone Marrow ◽

Virus Infection ◽

Progenitor Cells ◽

Virus Replication ◽

Erythroid Progenitor ◽

Aplastic Crisis ◽

Bone Marrow Cultures ◽

Transient Aplastic Crisis ◽

Marrow Cultures

Abstract B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus- induced transient aplastic crisis.

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Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽

10.1182/blood.v130.suppl_1.81.81 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 81-81

Author(s):

Silvana Di Giandomenico ◽

Pouneh Kermani ◽

Nicole Molle ◽

Mia Yabut ◽

Fabienne Brenet ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Progenitor Cells ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Effector Cells ◽

Erythroid Precursors ◽

Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia

Blood ◽

10.1182/blood.v67.4.962.bloodjournal674962 ◽

1986 ◽

Vol 67 (4) ◽

pp. 962-968 ◽

Cited By ~ 1

Author(s):

JM Lipton ◽

M Kudisch ◽

R Gross ◽

DG Nathan

Keyword(s):

Culture Technique ◽

Growth Characteristics ◽

Erythroid Progenitors ◽

Plasma Clot ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Normal Individuals

To explore the etiology of congenital hypoplastic or Diamond-Blackfan anemia (DBA) we investigated in vitro erythropoiesis in nine patients. Of the nine, seven were clinically responsive to prednisone. Four were infants evaluated at the time of diagnosis. Six were never or were only minimally transfused. Those for whom prednisone had been prescribed had discontinued the drug a minimum of five months prior to study. The bone marrows of these nine patients were compared with those of hematologically normal individuals and with those of four patients with transient erythroblastopenia of childhood (TEC) whose erythroid aplasia was as severe as that of the patients with DBA. Using the plasma clot semisolid culture technique to enumerate erythroid progenitors and to evaluate the growth characteristics of the colonies to which they give rise, we concluded that at the onset of DBA: (a) erythroid progenitor frequency does not correlate with the degree of anemia and erythroblastopenia; (b) erythroid progenitor differentiation may in some cases be abnormally insensitive to crude preparations of erythropoietin; and (c) progenitor erythropoietin insensitivity in vitro does not necessarily indicate prednisone insensitivity in vivo. Thus, DBA does not appear to be solely the result of deficient formation of erythroid progenitors but is, in addition, a disorder that is due to defective progenitor differentiation in vivo.

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Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

Blood ◽

10.1182/blood.v92.10.3658.422k18_3658_3668 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3658-3668 ◽

Cited By ~ 9

Author(s):

Birgit Panzenböck ◽

Petr Bartunek ◽

Markus Y. Mapara ◽

Martin Zenke

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Stem Cell Factor ◽

Large Cell ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Peripheral Blood Stem Cells ◽

Erythroid Progenitor ◽

Cell Numbers

Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

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Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia

Blood ◽

10.1182/blood.v67.4.962.962 ◽

1986 ◽

Vol 67 (4) ◽

pp. 962-968 ◽

Cited By ~ 60

Author(s):

JM Lipton ◽

M Kudisch ◽

R Gross ◽

DG Nathan

Keyword(s):

Culture Technique ◽

Growth Characteristics ◽

Erythroid Progenitors ◽

Plasma Clot ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Normal Individuals

Abstract To explore the etiology of congenital hypoplastic or Diamond-Blackfan anemia (DBA) we investigated in vitro erythropoiesis in nine patients. Of the nine, seven were clinically responsive to prednisone. Four were infants evaluated at the time of diagnosis. Six were never or were only minimally transfused. Those for whom prednisone had been prescribed had discontinued the drug a minimum of five months prior to study. The bone marrows of these nine patients were compared with those of hematologically normal individuals and with those of four patients with transient erythroblastopenia of childhood (TEC) whose erythroid aplasia was as severe as that of the patients with DBA. Using the plasma clot semisolid culture technique to enumerate erythroid progenitors and to evaluate the growth characteristics of the colonies to which they give rise, we concluded that at the onset of DBA: (a) erythroid progenitor frequency does not correlate with the degree of anemia and erythroblastopenia; (b) erythroid progenitor differentiation may in some cases be abnormally insensitive to crude preparations of erythropoietin; and (c) progenitor erythropoietin insensitivity in vitro does not necessarily indicate prednisone insensitivity in vivo. Thus, DBA does not appear to be solely the result of deficient formation of erythroid progenitors but is, in addition, a disorder that is due to defective progenitor differentiation in vivo.

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