Large Numbers Of Erythroid Cells Research Articles

A Diamond-Blackfan Anemia Patient's Response to Eltrombopag and Genomic Analysis in Different Lineages

Diamond-Blackfan Anemia (DBA) is a rare, inherited bone marrow (BM) failure syndrome characterized by pure red cell aplasia generally presenting within the first year of life. The majority of cases are due to heterozygous mutations in ribosomal genes, either autosomal dominant inherited or sporadicde novoevents. Despite the recognition of the importance of ribosomal biology, the underlying mechanisms are still poorly understood. The only curative treatment is hematopoietic stem cell transplantation, otherwise steroids or transfusions will cause significant long-term morbidity. There is an unmet need for new therapies. We describe the unique clinical course and molecular analyses of a 32-year-old Hispanic woman, provide insights into disease pathophysiology as well as hope for an effective new treatment. She was diagnosed with DBA at the age of one month with isolated severe normochromic anemia. She failed various therapies, including corticosteroids, danazol, growth factors, and additional immunosuppression and developed significant iron overload despite desferal chelation. She was enrolled in a phase II clinical trial investigating the safety and efficiency of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or unilineage cytopenias (NCT01328587). EPAG was administered in an escalating manner reaching 300mg daily. She achieved transfusion-independence by 16 weeks. This dose was continued for an additional 6 months and discontinued for protocol-defined robust response, ie Hb >10g/dL. After discontinuation, her hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated, her Hb again rapidly improved and the EPAG dose was slowly tapered. Seven years since enrollment, the patient has remained on drug, currently at a dose of 25mg daily with normal blood cell counts and no EPAG related toxicity. BM cytogenetics and morphology have remained normal throughout follow up, as has a myeloid somatic mutation panel. Next generation sequencing has revealed a novel, pathogenic mutation in intron 4(c.356+3>C) of theRPS19gene affecting a splice donor site previously implicated in DBA. Testing of both parents was negative, documenting this to be ade novomutation. With digital droplet PCR (ddPCR), we have monitored the variant allele frequency (VAF) in fibroblasts (germline) and different hematopoietic populations (Figure 1). Fibroblasts showed a VAF 50% (heterozygous), while her whole BM and whole blood show a lower VAF between 25% and 37%, both before and on EPAG treatment. Across lineages were analyzed when she was on EPAG, lymphocytes showed the highest VAF (42% to 45%), followed by HSPCs and granulocytes at 24% to 32%. Of note, the erythrocytes progenitors (CD45-CD71+) had the lowest VAF of 8% to 14%. Sequencing of myeloid and erythroid colony forming units (CFU) with both pre and on EPAG BM samples confirmed only heterozygous mutations or wild-type (WT) CFUs in percentages comparable to their respective VAF detected by bulk ddPCR. These results suggest the emergence of WT clones within the BM either through mosaicism or a reversion event. The discrepancy in VAF between peripheral and BM samples is consistent with a proliferative advantage for WT clones, especially in the erythroid and myeloid lineages. Yet despite the presence of WT HSPCs, profound transfusion-dependent anemia persisted in this patient long-term. An imbalance between globin chain and heme production has been documented to result from ribosomal dysfunction in DBA, leading to increased iron-mediated cellular stress and death of erythroid progenitors. We hypothesize the residual mutant cells suppress the growth of WT cells, given the close proximity of large numbers of erythroid cells aggregated by macrophages in erythroid islands, potentially via passage of free iron. These observations have implications for the development of gene addition or gene correction therapies for DBA, suggesting that correcting a fraction of HSPCs will not revert the phenotype. Given the potent intracellular chelation activity of EPAG, and the lack of the EPAG target MPL receptor on erythroid progenitors, we hypothesize that iron chelation is a potential mechanism for improvement of red blood cell production by EPAG in DBA. The results in our patient have encouraged the opening of a prospective clinical trial (NCT04269889) to further explore the potential of EPAG for DBA patients. Figure Disclosures Young: Pharos I&BT Co., Ltd.:Research Funding.Pakbaz:Amgen:Membership on an entity's Board of Directors or advisory committees;Dova:Membership on an entity's Board of Directors or advisory committees;Novartis:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Terumo BCT:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Global Blood Therapeutics:Speakers Bureau.Dunbar:Novartis:Research Funding.

A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules.

This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.

The Glucocorticoid Receptor Plays an Important Role in Controlling Lipid Metabolism During Erythroid Maturation

AbstractAbstract 3195The role of glucocorticoids in erythropoiesis is substanciated by the erythrocytosis observed in Cushing’s syndrome and failure in glucocorticoid receptor (GR) knockouts to respond to anaemia. Hydrocortisone, the natural GR agonist, synergizes with erythropoietin (EPO) to sustain the generation of erythroid bursts in cultures. This combined approach can be used to generate ex-vivo large numbers of erythroid cells from marginal stem cell sources [mononuclear cells (MNC) from low volume cord blood or blood donations] in liquid cultures, defined as Human Erythroid Massive Amplification (HEMA) cultures, stimulated with SCF, IL-3, EPO (GFs), estradiol (ES) and dexamethasone (DXM), a synthetic GR agonist more potent than hydrocortisone. Experiments in mice and human cells have indicated that GR activation mediates erythroid expansion by blocking terminal maturation of erythroid precursors and inducing these cells into a reversible self-replication state. By gene expression profiling, we have identified that erythroid precursors obtained in vitro with DXM express higher levels of genes controlling lipid metabolism than those obtained without DXM (see abstract by Hricik et al). Since ∼half of the human red cell mass is constituted by lipids, this over-expression suggests that the erythroid stimulating activity of GR may include its metabolic effects.Synthetic glucocorticoids are the most potent anti-inflammatory agents known but metabolic side effects limit their clinical utility. The availability of the crystal structure of GR has fostered studies to design synthetic agonists more potent than DXM and/or lacking its undesired metabolic action. These studies have identified fluticasone (FP), a potent analogue currently used in asthma treatment. More recently non-steroidal agonists, such as 67A, have been developed which lack mineralocorticoid effects. Both FP and 67A bind GR with 100-fold increased affinity compared to DXM, and function as full agonists (Ray D, manuscript in preparation).The aim of this study was to clarify whether the metabolic activity of GR contributes to its erythroid stimulating effects in vitro by comparing the efficacy of 67A, FP and DXM under HEMA conditions. MNC from 2 blood donors were cultured under HEMA conditions stimulated with GFs plus DXM (1 mM) or 67A (1, 3 or 10 nM), or FP (1, 3 and 10 nM), either alone or in combination with ES (1 mM). The cells were cultured for 17 days and the number and morphology (FACS profiling for CD36 and CD235a) of the progeny evaluated at day 7, 10, 12, 14 and 17. At day 12, the frequency of progenitor cells generated under the various conditions was also evaluated while the maturation potential of cells obtained at day 14 was evaluated in differentiation cultures stimulated with EPO and insulin.MNC generated more erythroid cells [relative fold increase (FI) with respect to GFs alone: 7- or 20-fold for 67A or FP, 10 nM each] and for longer time (maximal cell numbers were observed after 17 days of culture instead of 12) if cultured with 67A and FP, instead of DXM (relative FI with respect to GFs alone: 3-fold) (Fig. A). The effects of 67A and FP were concentration dependent and maximal effects were observed with 10 nM in both cases (not shown). Addition of ES did not increase the number of erythroid cells generated in liquid cultures and increased the number of BFU-E persisting up to day 12 only in combination with DXM (Fig. B). BFU-E from 67A- and FP-stimulated cultures generated larger and better hemoglobinized colonies than BFU-E from DXM-stimulated cultures (Fig. C), suggesting that 67A and FP were equally potent at the progenitor cell level. However, day 14 erythroid precursors from FP-stimulated cultures matured better (greater frequency of CD36negCD235alow cells and darker red pellet) with EPO and insulin than those generated with 67A or DXM (Fig. D and not shown).These results indicate that as predicted by the relative affinity for GR of the different compounds, 10 nM of 67A and FP induce levels of in vitro erythroid expansion greater, in terms of extent and duration of expansion and persistence of erythroid progenitor cells, than 1 mM of DXM. However, the numbers and maturation ability of erythroid precursors generated with 67A, which lacks mineralocorticoid activity, are lower than those of erythroid precursors generated with FP, suggesting that the metabolic activity of GR is an essential component of its erythroid stimulating activity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells

Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

ASH 2010 meeting report—Top 10 clinically oriented abstracts in transfusion medicine

ASH 2010 meeting report—Top 10 clinically oriented abstracts in transfusion medicine

Large-scale production of embryonic red blood cells from human embryonic stem cells

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

A Comparison of Five Different Culture Protocols Shows That Growth of Human CD34+ Peripheral Blood Stem Cells in SCF, IL-3, Flt-3 Ligand and EPO Results in Exponential Erythroid Expansion and a Globin Gene Expression Pattern Which Is Similar to That Seen in Human Bone Marrow.

Pharmacologic reactivation of γ-globin globin gene expression offers a potential strategy for ameliorating the consequences of β-thalassemia and sickle cell disease. While previous clinical and laboratory studies have established the effectiveness of inhibitors of DNA methylation in stimulating the expression of the fetal globin genes, the molecular mechanisms by which this effect is achieved are not well understood. In order to study the mechanisms and pharmacologic properties of these agents in a clinically relevant laboratory model, we have compared five different in vitro human erythroid differentiation protocols. In performing these experiments we sought a system which would yield a large number of erythroid cells exhibiting a pattern of globin gene expression which closely matched the pattern seen in adult bone marrow. FACS purified CD34+ peripheral blood stem cells (PBSC) from healthy donors were obtained from the NHLBI Programs of Excellence in Gene Therapy Hematopoietic Cell Processing Core (PEGT-HCPC) at the Fred Hutchinson Cancer Research Center. PBSC were cultured in the following different combinations of recombinant human cytokines: A) EPO alone for 12d; B) EPO, SCF and IL-3 for 14d; C) EPO, SCF and IL-3 for 7d followed by EPO alone for 7d; D) EPO, GM-CSF and IL-3 for 12d; and E) SCF, IL-3 and Flt-3 ligand for 7d followed by EPO alone for 11d. Cells were counted every day and differentiation assessed by light microscopy and flow cytometry using CD34, glycophorin A (GPA) and transferrin antibodies. Globin gene expression was measured by real time RT-PCR. Cultures B, C and E underwent exponential expansion from d4, while A and D showed no appreciable expansion. By day 6, all cultures that had EPO from d0 (A–D) consisted mainly of CD34−, GPA+ proerythroblasts. Basophilic erythroblasts, followed by polychromatophilic forms were evident at 8–10 days. By the end of each experiment more than 90% of cells in these cultures were erythroid. In contrast, condition E showed persistent expression of CD34 until removal of IL-3, SCF and flt-3 ligand. Proerythroblasts appeared on day 10 followed by basophilic and polychromatophilic forms at 13–15 days. At the end of the culture period 63% erythroid cells (by flow) were seen in a background of maturing monocytes and granulocytes. RT-PCR showed that induction of globin mRNA occured in all cultures at or just before appearance of basophilic erythroblasts (day 7–9 for cultures A–D and day 11–13 for E). While conditions C and E showed the highest levels of globin gene expression, peak expression under condition C for γ- and β-globins were equivalent and their expression overlapped. Condition E showed a much higher level of β- than γ-globin expression (β/γ ratio of 8:1), the rise in β-globin mRNA (d9–14) was accompanied by a fall γ-globin mRNA and β-globin expression persisted at a high level until the end of the experiment (d14–18). Of the 5 differentiation protocols tested, condition E appears to be the best choice for future studies of pharmacological reactivation of γ-globin gene expression as it produced a large number of erythroid cells which exhibited a gene expression pattern similar to that seen in normal human bone marrow and had a period of stable high-level β-globin gene expression which persisted over several days.

Response of the murine hematopoietic system to chronic infection with Mycobacterium lepraemurium.

Mycobacterium lepraemurium infection of mice produces a chronic lethal disease that is characterized by massive accumulation of macrophages throughout the mononuclear-phagocyte system. We studied the influence of M. lepraemurium infection on the composition and function of the hematopoietic system. Medullary erythropoiesis was virtually abolished, as reflected by a small number of erythroid elements and a decrease in the number and frequency of erythroid progenitors in the bone marrow, together with reduced uptake of 59Fe into bone marrow hemin. On the other hand, erythropoiesis was observed in the spleen, as demonstrated by a large number of erythroid cells, a sixfold increase of 59Fe uptake, and a pronounced increase in the number of erythroid progenitors. A considerable increase of monocyte progenitors was observed in the spleen, and a more modest increase was observed in the bone marrow. This increase may be accounted for, at least in part, by greatly increased levels of macrophage-colony-stimulating factor in the serum of infected mice. Thus, M. lepraemurium infection produces important changes in the hematopoietic system, during the course of which the spleen becomes the major hematopoietic organ.

Histological changes associated with enlargement and regression of the thymus glands of the red-billed quelea Quelea quelea L. (Ploceidae: weaver-birds).

Thymic lobes form over 200 red-billed queleas, Quelea quelea L., were examined histologically. Samples were taken from embryos about to hatch, juveniles and adults. The lobes varied in size from very small to very enlarged (1- greater than 5 mm long). The constituent cell types are described in detail and the occurrence of these cells in different sized lobes is discussed. A cycle of events is proposed which accounts for the observations presented here. It is suggested that the large numbers of erythroid cells found in the cortex of some individuals were developing in situ. The significance of erythropoiesis within the thymus is discussed.

Fine structural observations on hemopoiesis in the chorioallantoic placenta of the marmoset.

Hemopoietic sinuses in the chorionic trabeculae of marmoset placenta were examined electron microscopically at several stages of pregnancy. Tissues were prepared both by standard technique and following injection of exogenous peroxidase into the maternal circulation prior to autopsy, and subsequent cytochemical localization of its reaction product in the fetal placenta. Aside from their dilated size and cellular content, the sinuses resembled the allantoic capillaries of the peripheral umbilical circulation in this species; and in early and midgestational placentas, sinusal lumina, differentiating elements in the erythroid line were present in placentas at all stages. Such cells appeared to arise both by a process of proliferation of undifferentiated progenitors from the sinusal lining and by mitosis of pre-existing erythroid cells. Only the latter process was observed in the mature placenta near term. Granulopoiesis was not observed at any stage; on the other hand, cells in the thromboid line were identified in placentas believed to be near midgestation; and large intrasinusal phagocytes were present in both midgestational and late placentas. In the latter, they were seen to have endocytosed peroxidase, and, by tenuous, dendriform processes, to be related anatomically to large numbers of erythroid cells. Early in gestation, the developing erythroid elements were closely related by areas of contact, both to each other and to the delimiting endothelium of the sinuses.