Proteome Expression Profile for Red Blood Cells Enables Diagnostics for Hepatocellular Carcinoma

Abstract BackgroudEarly diagnosis of hepatocellular carcinoma (HCC) has not been clinically resolved, which has been causing more death in patients with HCC. HCC is also a systemic disease related to disorders of blood homeostasis, and the association of red blood cells (RBCs) and HCC tumorigenesis is still elusive. This study explored the protein characteristics of RBCs at the progressive pathological stages comparing with healthy individuals, including liver cirrhosis (LC) and established HCC, to fully understand the tumorigenesis of HCC from a different view and identify potentially novel diagnostic biomarkers for HCC in RBCs.MethodsData independent acquisition (DIA) proteomic analyses were performed with 72 clinical RBCs samples from a cohort of subjects including HCC, LC and healthy controls. Bioinformatics analysis was conducted for significantly differentially expressed proteins (DEPs) through the whole process of tumorigenesis to characterize the clinical relevanve of RBCs and tumorigenesis in HCC. The highly potential tumorigenesis-associated molecular biomarkers were evaluated with clinical samples by parallel reaction monitoring (PRM) technology.ResultsWe observed that red blood cells number dynamically changes during the tumorigenesis of HCC, and LC is a developmental stage more closely approaching HCC based on the protein expresson profiles in RBCs. The expression of hemoglobin (HbA, HbF) in erythroid cells also dynamically alters during the whole process of HCC tumorigenesis, suggesting immature erythroid cells exist in peripheral blood of HCC patients and erythropoiesis in the patients starts to be influenced with the occurance of LC. We observed that the autophagy pathway is disturbed in RBCs with the onset of LC and maintained during the tumorigenesis of HCC. Oxytocin pathway and GnRH pathway are disturbed and first identified during the development of LC into HCC. SMIM1, ANXA7, HBA1 and HBE1 that are significantly altered during tumorigenesis were verified as promising biomarkers for HCC early diagnosis.ConclusionsThis study underlied the clinical relevance of the proteins in RBCs and the tumorigenesis of HCC, and provided the potential biomarkers for early diagnosis in HCC from a new perspective. Our results provided a novel strategy with RBCs for HCC early diagnosis, which will improve the translational research and application in diagnosis of HCC.

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Electrokinetic Focusing and Dispensing of Particles and Cells on Microfluidic Chips

Microelectromechanical Systems ◽

10.1115/imece2005-80223 ◽

2005 ◽

Author(s):

Xiangchun Xuan ◽

Edmond W. K. Young ◽

Dongqing Li

Keyword(s):

Red Blood Cells ◽

Analytical Model ◽

Blood Cells ◽

Lab On A Chip ◽

Microfluidic Chips ◽

Whole Process ◽

Sample Stream ◽

Custom Made ◽

Electrical Pulses

This work investigated the electrokinetic focusing and dispensing of polystyrene particles and red blood cells on microfluidic chips. Particles or cells were first electrokinetically focused using the merging of focusing streams on the sample stream, and subsequently separated as a result of the focusing. These particles or cells were then selectively dispensed from the focused sample stream using precise application of electrical pulses. The whole process of focusing, separation and dispensing of particles was visualized by a custom-made microscopy system. In particular, the width of the focused fluorescein stream and the accelerated electrophoretic motion of particles and cells were measured in a cross-channel and compared with a proposed analytical model. The electrokinetic manipulation of particles and cells demonstrated in this work can be used for developing integrated lab-on-a-chip devices for studies of cells.

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Production and fate of erythroid cells in anaemic Xenopus laevis

Journal of Cell Science ◽

10.1242/jcs.35.1.403 ◽

1979 ◽

Vol 35 (1) ◽

pp. 403-415

Author(s):

N. Chegini ◽

V. Aleporou ◽

G. Bell ◽

V.A. Hilder ◽

N. Maclean

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Xenopus Laevis ◽

Red Blood Cells ◽

Blood Cells ◽

Complete Recovery ◽

Cell Counting ◽

Erythroid Cells ◽

Spleen Cells ◽

Sudden Release

Adult Xenopus laevis, rendered anaemic by phenylhydrazine injection, have been studied during the recovery from such anaemia. Electron microscopy of liver and spleen sections indicates that both of these organs are active in the phagocytosis and destruction of the old damaged red blood cells. May-Grunwald and Giemsa staining of liver and spleen cells following anaemia has been used to show that erythropoiesis also occurs in both liver and spleen, and this has been confirmed by electron-microscope studies of these organs. Cell counting and radiolabelling of the new population of circulating erythroid cells in the period following phenylhydrazine injection suggests that a sudden release of basophilic erythroblasts from liver and spleen is followed by mitosis of this new cell population in circulation, and that no further release of erythroid cells from these organs is likely until complete recovery has occurred.

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Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

Nature Biotechnology ◽

10.1038/nbt0502-467 ◽

2002 ◽

Vol 20 (5) ◽

pp. 467-472 ◽

Cited By ~ 186

Author(s):

Thi My Anh Neildez-Nguyen ◽

Henri Wajcman ◽

Michael C. Marden ◽

Morad Bensidhoum ◽

Vincent Moncollin ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Large Scale ◽

Ex Vivo ◽

Erythroid Cells

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Differential Requirements for Survivin in Hematopoietic Cell Development.

Blood ◽

10.1182/blood.v104.11.1257.1257 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1257-1257

Author(s):

Yanfei Xu ◽

Sandeep Gurbuxani ◽

Ganesan Keerthivasan ◽

Amittha Wickrema ◽

John D. Crispino

Keyword(s):

Cell Cycle ◽

Red Blood Cells ◽

Blood Cells ◽

Caspase 3 ◽

Terminal Differentiation ◽

Dependent Manner ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Stage Of Development ◽

Primary Mouse

Abstract The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

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MicroRNAs 155 and 451 as Possible Key Molecules for Human Erythroid Differentiation.

Blood ◽

10.1182/blood.v110.11.4071.4071 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4071-4071

Author(s):

Tsukuru Umemura ◽

Shizuka Masaki ◽

Rie Ohtsuka ◽

Yasunobu Abe ◽

Koichiro Muta

Keyword(s):

Red Blood Cells ◽

Internal Control ◽

Blood Cells ◽

Human Peripheral Blood ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Expression Levels ◽

Final Maturation ◽

Normal Human ◽

U6 Rna

Abstract MicroRNAs (miRNAs) are 18–25-nucleotide noncoding RNAs which play important roles for cell death, proliferation, development and differentiation. MiRNA is an important molecule to regulate genes by suppressing the translation or inducing instability of miRNAs, and is consist of the network system to regulate gene functions in combination with transcription factors. Many recent works demonstrated that some of miRNAs are playing key roles for hematopoiesis and leukemogenesis. In this study, we analyzed the expression of miRNAs(miRNA-155, miRNA-221, miRNA-223, miRNA-451) during differentiation of purified normal human eryhroid progenitors in the liquid culture system. Cells increased almost 500-folds in a number, and differentiated to benzidine-positive mature erythroblasts after days 7 to 9 which were partly red blood cells on days 12 to 14. Since mature erythroid cells loose cellular nucleic acids at the final maturation stages, we measured changes in U6 RNA contents as the internal control for assays of miRNA. Each expression levels of miRNAs were normalized using U6 RNA contents. Analyses of miRNA expressions using quantitative real-time reversetranscriptase polymerase chain reaction have shown that the expression level of miRNA-155 decreased about 200-folds from day 3 to day 12 with almost 87.5% reduction between days 3 and 5. On the other hand, the expression levels of miRNA-451 increased about 270-folds by day 12 in parallel to an increase in benzidine-positive cell numbers. To extend our observation on the up-regulation of miRNA-451 in mature blood cells, we analyzed the miRNA-451 levels in each mature blood cells (red blood cells, granulocytes, lymphocytes and monocytes, platelets) purified from normal human peripheral blood by using a density centrifugation method. miRNA-451 was expressed in red blood cells about 104 folds more than in granulocytes, about 102 folds more than in platelets. Moderate down-regulations of miRNAs 221 and 223 were observed. In conclusion, our observations suggest that the down-regulation of miRNA-155 and the up-regulation of miRNA-451 are key events for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be useful tools for specifying the differentiation stage of each erythroid cells.

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The Path of Iron from Plasma Transferrin to Hemoglobin in Developing Red Blood Cells.

Blood ◽

10.1182/blood.v112.11.sci-26.sci-26 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-26-sci-26

Author(s):

Prem Ponka ◽

An-Shen Zhang ◽

Alex Sheftel ◽

Orian S. Shirihai

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Biosynthetic Pathway ◽

Protoporphyrin Ix ◽

Heme Iron ◽

Erythroid Cells ◽

Divalent Metal Transporter ◽

Receptor Complexes ◽

Endosomal Acidification ◽

In Erythroid Cells

Abstract An exquisite relationship between iron and heme in hemoglobin-synthesizing cells makes blood red. Erythroid cells are the most avid consumers of iron (Fe) in the organism and synthesize heme at a breakneck speed. Additionally, there is virtually no free Fe or heme detectable during hemoglobin (Hb) synthesis. Developing red blood cells (RBC) can take up Fe only from the plasma glycoprotein transferrin (Tf). Delivery of iron to these cells occurs following the binding of Tf to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification. Iron, following its reduction to Fe2+ by Steap3, is then transported across the endosomal membrane by the divalent metal transporter, DMT1. However, the post-endosomal path of Fe in the developing RBC remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones Fe in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron-binding intermediate has never been identified. In erythroid cells, more than 90% of iron must enter mitochondria since ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides in the inner part of the inner mitochondrial membrane. In fact, in erythroid cells, strong evidence does exist for specific targeting of Fe toward mitochondria. This targeting is demonstrated in Hb-synthesizing cells in which Fe acquired from Tf continues to flow into mitochondria, even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated a hypothesis that in erythroid cells a transient mitochondrion-endosome interaction is involved in iron translocation to its final destination. Recently, we have collected strong experimental evidence supporting this hypothesis: we have shown that Fe, delivered to mitochondria via the Tf pathway, is unavailable to cytoplasmic chelators. Moreover, we have demonstrated that Tf-containing endosomes move and contact mitochondria in erythroid cells, that vesicular movement is required for iron delivery to mitochondria, and that “free” cytoplasmic Fe is not efficiently used for heme biosynthesis. As mentioned above, the substrate for the endosomal transporter DMT1 is Fe2+, the redox form of iron that is also the substrate for ferrochelatase. These facts make the above hypothesis quite attractive, since the “chaperone”-like function of endosomes may be one of the mechanisms that keeps the concentrations of reactive Fe2+ at extremely low levels in oxygen-rich cytosol of erythroblasts, preventing ferrous ion’s participation in a dangerous Fenton reaction. In conclusion, the delivery of iron into Hb occurs extremely efficiently, since mature erythrocytes contain about 45,000-fold more heme iron (20 mM) than non-heme iron (440 nM). These facts, together with experimental data that will be discussed, indicate that the iron transport machinery in erythroid cells is an integral part of the heme biosynthetic pathway.

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Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽

10.1182/blood.v118.21.344.344 ◽

2011 ◽

Vol 118 (21) ◽

pp. 344-344

Author(s):

Daniel Garcia Santos ◽

Matthias Schranzhofer ◽

José Artur Bogo Chies ◽

Prem Ponka

Keyword(s):

Red Blood Cells ◽

Heme Oxygenase ◽

Blood Cells ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Wild Type ◽

Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

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In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

BioMed Research International ◽

10.1155/2013/807863 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

Jiafei Xi ◽

Yanhua Li ◽

Ruoyong Wang ◽

Yunfang Wang ◽

Xue Nan ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Red Blood Cells ◽

Stromal Cells ◽

Blood Cells ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

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Large Scale Culture and Differentiation of Erythroblasts from PBMC and iPSC

Blood ◽

10.1182/blood-2018-99-118882 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2319-2319

Author(s):

Maria Claessen ◽

Eszter Varga ◽

Steven Heshusius ◽

Esther Heideveld ◽

Martin Hansen ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Group ◽

Peripheral Blood ◽

Blood Cells ◽

Large Scale ◽

Mononuclear Cells ◽

Blood Group Antigens ◽

Erythroid Cells ◽

Expression Of Genes ◽

Blood Mononuclear Cells

Abstract Transfusion of donor-derived red blood cells to aleviate anemia is the most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs or enzymes. However, the source depends on donor availability and carries a potential risk of alloimmunization and blood borne diseases. More than 30 bloodgroup systems encode >300 bloodgroup antigens and bloodgroup matching becomes increasingly challenging in a multiethnic society. Particularly the chronically transfused patients are at risk for alloimmunisation. In vitro cultured, customizable red blood cells (cRBC) would negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications. We aim to produce human cRBC at large-scale and cost effective, for which we need to optimize culture conditions and reduce cost-drivers. We adapted our protocols to GMP culture requirements, which reproducibly provided pure human erythroid cultures within 25 days with a 3.4x107 times expansion from peripheral blood mononuclear cells without prior CD34+ isolation. This expansion depended on the serum free medium we produce, which is supplemented with erythropoietin (Epo, 1 U/ml), stem cell factor (SCF) and glucocorticoids. Expanded erythroblasts CD71 highCD235low/- were differentiated for 10 days in medium supplemented with 5% human plasma, heparin and a higher concentration of Epo (10U/ml) yielding CD71dimCD235a+CD44+CD117-DRAQ5- cRBC. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability was measured by an Automated Rheoscope and Cell Analyser (ARCA), and oxygen equilibrium curves were measured with a Hemox analyzer. Both parameters were similar in cRBC and freshly isolated reticulocytes. RNA sequencing was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, and erythroid specific metabolic enzymes, concommittant with loss of expression of genes involved in the formation of organelles, and cell proliferation. The culture process is compatible with upscaling using 5L G-Rex bioreactors., Currently we are preparing a clinical study using biotinylated cRBC. Ultimately, however, large scale production requires an immortal source, for which we aim to use human induced pluripotent stem cells (iPSC) established from rare donors that lack most blood group antigens. Using single cell passaging of iPSC and differentiation in colonies, we generate at average 2x105 cRBC per single iPSC. However, the cRBC cultured from iPSC were less stable following enucleation, and expressed embryonic type globins. Comparison of transcriptome data from iPSC-derived erythroid cells at distinct differentiation stages with erythroid cells at similar stages that were cultured from adult- or cord blood mononuclear cells, or from fetal liver confirmed that most iPSC-derived erythroid cells largely express an embryonic RNA profile. In conclusion, our current protocols enable us to test cRBC cultured from adult peripheral blood for their stability after transfusion. Concurrently, we develop novel bioreactors to upscale the production, and we optimise the protocol to generate cRBC from immortal iPSC lines with near 'universal donor' genotypes. Disclosures No relevant conflicts of interest to declare.

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Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v106.11.3631.3631 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3631-3631

Author(s):

Emmanuel N. Olivier ◽

Caihong Qiu ◽

Eric E. Bouhassira

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Red Blood Cells ◽

Human Embryonic Stem Cells ◽

Blood Cells ◽

Liquid Culture ◽

Embryonic Stem ◽

Erythroid Cells ◽

Early Human

Abstract Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.

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