Sequential Analysis of - and -Globin Gene Expression During Erythropoietic Diffe - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:58 |只看该作者 |倒序浏览 |打印

作者：Katsutsugu Umedaa, Toshio Heikea, Mami Nakata-Hizumea, Akira Niwaa, Masato Araia, Gen Shinodaa, Feng Maa, Hirofumi Suemorib, Hong Yuan Luoc, David H. K. Chuic, Ryuzo Toriid, Masabumi Shibuyae, Norio Nakatsujif, Tatsutoshi Nakahataa作者单位：aDepartment of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;bLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;cDepartment of Medicine, Boston University School of Medicine, Boston

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      【摘要】
   The temporal pattern of embryonic, fetal, and adult globin expression in the  (  ) and &szlig; ( and &szlig;) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2high CD34  cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic - and -globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of &szlig;-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans. 1 @! v- \" ?; }% U
      【关键词】 Embryonic stem cells Erythroid progenitors
   INTRODUCTION
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In all species, the shifting sites of erythropoiesis coincide with changes in the hemoglobin composition of erythrocytes. Primitive (embryonic) hematopoiesis initially occurs in the yolk sac, followed by definitive (fetal and adult) hematopoiesis, first in the aorta-gonad-mesonephros region and then in the fetal liver, spleen, and bone marrow .) k% D+ x0 }* y% X" }/ s: Q

Recently, established primate embryonic stem cell (ESC) lines ." t9 \) O! x- z, Z

Globin switching has been thoroughly investigated, both as a model of tissue- and temporal-specific transcriptional control and as a tool for drug discovery aimed at ameliorating the effects of fetal hemoglobin synthesis in patients with hemoglobinopathies . In the present study, we specifically analyze the temporal pattern of globin switching in the - and ß-cluster of erythrocytes induced from primate ESCs in the OP9 coculture system. For this purpose, we separated VEGFR-2high CD34  hemogenic progenitors and cultured them in the presence of appropriate cytokines. This system enables the sequential analysis of mature floating erythrocytes and immature erythroid clonogenic progenitors, both at the transcriptional and translational level, and may serve as a novel in vitro model for the globin switch in humans.6 h0 U" H5 H$ N5 {  Z8 R
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MATERIALS AND METHODS

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The ESC line CMK6, established from cynomolgus monkey blastocysts, was maintained according to the procedure of Suemori et al. .+ j9 {) o! C- F9 W& O- c

Cytokines and Growth Factors

Recombinant human granulocyte cell-stimulating factor (G-CSF), erythropoietin (EPO), interleukin-3 (IL-3), stem cell factor (SCF), and thrombopoietin (TPO) were kindly provided by Kirin Brewery Co. (Tokyo, http://www.kirin.co.jp/english). Recombinant human VEGF was purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com).# G3 t4 }% Z- U* J9 }& }+ \# u  E

Antibodies
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Primary antibodies used in this study included mouse anti-human hemoglobin (Hb) - and -ß mononuclear antibodies (mAbs) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com), mouse anti-human CD34 (clone 563) from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen), and rabbit anti-human Hb polyclonal antibodies from MP Biomedicals (Irvine, CA, http://www.mpbio.com). Mouse anti-human Hb-- and - mAbs and mouse anti-human VEGFR-2 mAb were used according to previous reports . The secondary antibodies used included cyanine 3 (Cy3)-conjugated donkey anti-mouse immunoglobulin G (IgG) and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, http://www.jacksonimmuno.com).8 g& t, N4 K* r0 d) ~2 \: \: R/ K
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In Vitro Hematopoietic Differentiation of Primate ESCs
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In vitro differentiation of ESCs and cell sorting were performed as reported previously . Nuclei were labeled with Hoechst 33342. Fluorescence was detected and photographed with an AxioCam photomicroscope (Carl Zeiss GmbH, Jena, Germany, http://www.zeiss.com). In sequential analyses, data are presented as means ¡À SDs of triplicate wells. Representative results from one of three independent experiments are shown.: K7 v, G5 J9 c& D) D% a; o. [% A

Colony-Forming Assays for Primitive and Definitive Cells% f3 e' D9 \. |( f
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Colony-forming assays were performed every 6 days in semisolid medium consisting of -MEM, 0.9% methylcellulose, 30% FCS, 10% bovine serum albumin, 50 µM 2-ME, and a mixture of 10 ng/ml G-CSF, 2 U/ml EPO, 20 ng/ml IL-3, 100 ng/ml SCF, and 10 ng/ml TPO. For colonies consisting of primitive cells, the medium was replaced with fresh semisolid medium . Data are presented as means ¡À SDs of triplicate wells. Representative results from one of three independent experiments are shown. After 7 days for primitive and 12 days for definitive cells, individual colonies were lifted with an Eppendorf micropipette under direct microscopic visualization, washed twice with PBS, and processed for May-Giemsa staining, immunostaining, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. At least 10 individual colonies were analyzed by immunostaining and RT-PCR analysis./ d7 ?) }% _) K# H$ i+ M0 s- l+ ]
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RT-PCR for Globin Gene Expression& v' I  @, D5 w. y

RNA isolation and RT-PCR were performed using the procedure of Umeda et al. ), forward 5'-TGC ATT TTT ACT GCT GAG GAG A-3', reverse 5'-TGC CAA AGT GAG TAG CCA GAA TAA-3'; -globin (221 bp), forward 5'-GGC AAC CTG TCC TCT GCC TC-3', reverse 5'-GAA ATA GAT TGC CAA AAC AG-3'; ß-globin (183 bp), forward 5'-CTC ATG GCA AGA AAG TGC TTG-3', reverse 5-AAT TCT TTG CCA AAG TGA TGG G-3'; -globin (327 bp), forward 5'-CCG CCA TGT CTC TGA CCA A-3', reverse 5'-GCT CGC TCA GCT TGG ACA GGG-3'; -globin (395 bp), forward 5'-CCG ACA AGA CCA ACG TCA AGG-3', reverse 5'-AGG TCG AAG TGC GGG AAG TA-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (360 bp), forward 5'-CAC CAG GGC TGC TTT TAA CTC TG-3', reverse 5'-ATG GTT CAC ACC CAT GAC GAA C-3'. The PCRs consisted of 35 cycles for floating HCs and 40 cycles for individual erythroid colonies. cDNAs from cynomolgus monkey bone marrow or human erythroleukemia K562 cells were used as positive controls. For semiquantitative comparisons, samples were normalized by dilution to produce equivalent signals for GAPDH.

The quantitative RT-PCR assay of globin transcripts was performed using gene-specific double-fluorescent-labeled probesin an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The florescent reporter and quencher were 6-carbocyfluorescein (FAM) and 6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA), respectively. The following primers and probes were used for real-time PCR: -globin, forward 5'-TGG CAA GGA GTT CAC CCC T-3', reverse 5'-AAT GGC GAC AGC AGA CAC C-3', probe 5'-FAM-TGC AGG CTG CCT GGC AGA AGC-TAMRA-3'; -globin, forward 5'-TGG CAA GAA GGT GAC TTC-3', reverse 5'-TCA CTC AGC TGG GCA AAG-3', probe 5'-FAM-TGG GAG ATG CCA TAA AGA ACC TGG-TAMRA-3'; ß-globin, forward 5'-CAA GAA AGT GCT TGG TGC CT-3', reverse 5'-GCA AAG GTG CCC TTG AGG T-3', probe 5-FAM-TAG TGA TGG CCT GGC TCA CCT GGA C-TAMRA-3'; -globin, forward 5'-GGA CCC TCA TTG TGT CCA TGT-3', reverse 5'-TGC GGG TAG CTG AGG AAG AG-3', probe 5'-FAM-TCC ACT CAG GCC GAC AC-TAMRA-3'; -globin, forward 5'-TCC CCA CCA CCA AGA CCT AC-3'; reverse 5'-CCT AAC CTG GGC AGA GCC-3', probe 5'-FAM-TCC CCA CTT CGA CCT GAG CCA-TAMRA-3'; 18S rRNA, forward 5'-AGT CCC TGC CCT TTG TAC ACA-3', reverse 5'-GAT CCG AGG GCC TCA CTA AAC-3', probe 5'-FAM-CGC CCG TCG CTA CTA CCG ATT GG-TAMRA3'. The - and ß-globin-specific primers and probes have been described previously . Data are presented as means ¡À SDs of triplicate wells. Representative results from one of three independent experiments are shown.+ l- j9 F  E% h* K
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RESULTS  i0 F4 o. d- D  X8 B
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Seeding VEGFR-2high CD34  Hemogenic Progenitors in the OP9 Coculture System Leads to Two-Wave Erythropoiesis
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In a previous report, our group showed that the numbers of HCs that develop from ESCs increase if, after the initial 6-day VEGF treatment, the whole cultures are replated onto a new confluent OP9 cell layer . Reanalysis of sorted cells confirmed purity of 96%¨C98%.7 b8 `5 c& u- F& D+ B0 j0 q9 r7 z
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Figure 1. Fluoresence-activated cell sorting analysis and cell sorting using antibodies against VEGFR-2 and CD34. The amounts of VEGFR-2high CD34¨C (R2, upper left quadrant) or VEGFR-2high CD34  cells (R3, upper right quadrant) are shown as a percentage of the total GFP  ES cells (R1). Abbreviations: APC, allophycocyanin; ES, embryonic stem; GFP, green fluorescent protein; PI, propidium iodide; VEGFR-2, vascular endothelial growth factor receptor-2.- i; ?7 g. s& L" Q0 [# d
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Sequential analyses demonstrated that floating HCs, mostly large nucleated erythrocytes, were generated on day 9 with maximum levels on day 12, 6 days after cell sorting, but gradually decreased thereafter (Fig. 2A, 2C). Subsequently, small nucleated and enucleated erythrocytes appeared on day 18 (Fig. 2D). As the floating cells increased in number, a second wave of hematopoiesis, approximately 90% of which involved small erythrocytes, developed and 20%¨C25% of the total cells were enucleated erythrocytes on day 21 onwards (Fig. 2A, 2B). In contrast, adherent HCs were generated beginning on day 8 and maximizing on day 12 (Fig. 2E). The adherent fraction contains more progenitors than the floating fraction in the OP9 coculture system . The clusters decreased on day 18 and regrew all over the stromal layer by day 20 and thereafter (Fig. 2F). Thus, with a two-step system using OP9 stromal cells, two-wave hematopoiesis with a relatively high proportion of floating erythrocytes was induced from primate ESCs, along with the development of immature adherent hematopoietic progenitors.

Figure 2. Two-wave erythropoiesis generated from VEGFR-2high CD34  cells. (A): Sequential analysis of the number of floating erythrocytes and total blood cells. (B): Sequential analysis of the proportion of each cell lineage. (C, D): May-Giemsa staining of floating erythrocytes (x400). (E, F): Micrographs of an adherent hematopoietic cell cluster (x100). Abbreviations: d, day; Ery, erythrocyte; TBC, total blood count; VEGFR-2, vascular endothelial growth factor receptor-2.: D& g8 M$ j! e! Q0 H
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Globin Switches Occur in Parallel with Transition from Primitive to Definitive Erythropoiesis3 v4 H; I+ Q) O0 Q% s9 |

Primitive erythrocytes are relatively large and nucleated, whereas in definitive erythropoiesis, erythroid progenitors mature sequentially and eventually lose their nuclei . To determine the mechanism of erythropoietic transition in this culture system, sequential immunostaining analyses of temporal expression patterns of embryonic, fetal, and adult Hbs in floating erythrocytes were performed. Cy3 detection of erythrocytes stained with -, -, ß-, -, and -globin mAbs and FITC detection of erythrocytes stained with Hb polyclonal Ab, which reacts with embryonic, fetal, and adult erythrocytes, were used to detect all erythrocytes in the total HCs. Until day 15, all floating erythrocytes were positive for -, -, - and -globins, but not ß-globin expression (Fig. 3A¨C3J). In contrast, the second wave of erythrocytes was positive for -, ß-, and -globin, but one- half and less than 10% of the total erythrocytes expressed - and -globin, respectively (Fig. 3K¨C3V). Notably, the proportion of total enucleated erythrocytes that were positive for - and -globin was equivalent to the proportion of total nucleated cells (data not shown). Thus, expression of ß-globin, but not embryonic - and -globins, defines the switch in parallel with the transition from primitive (EryP) to definitive (EryD) erythrocytes during culturing.# _, P* J- S: o: P0 s: L. Z+ x
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Figure 3. Immunostaining analysis of globin gene expression in floating erythrocytes. (A¨CT): Immunostaining of erythrocytes (x400). Red (cyanine 3) indicates globin-type stains, and green (fluorescein isothiocyanate) indicates hemoglobin. (U, V): Sequential analysis of the proportion of erythrocytes stained with -, -, and ß-globin monoclonal antibodies (mAbs) (U) and - and -globin mAbs (V). Abbreviation: Hb, hemoglobin.- T- b- S0 C0 G6 f: S$ e8 Y" T; ~
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Next, quantitative real-time PCR analysis of globin genes in floating erythrocytes was performed to confirm the globin switch at the mRNA level. cDNA obtained from adult bone marrow or floating erythrocytes on day 30 was used as a standard for estimating the linear ranges and amplification efficiencies of the globin and ribosomal RNA systems. The reaction efficiency (E) was calculated from the slope of the dilution curve . Collectively, analyses at the transcriptional and translational levels revealed that this culture system recapitulates transition from embryonic to fetal/adult globin expression during sequential development of erythrocytes in the yolk sac and early fetal liver.

Figure 4. Sequential real-time reverse transcription-polymerase chain reaction analysis of globin gene expression in floating erythrocytes. (A, B): Standard curves generated from the CT values from five dilution series of rRNA (A) and -globin cDNA (B). (C, D): Sequential analysis of mRNA expression in the ß-globin (C) and -globin (D) clusters. Abbreviations: CT, threshold cycle; E, polymerase chain reaction efficiency; r2, correlation coefficient.
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Sequential Development of Primitive and Definitive Erythroid Colonies
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To evaluate the mechanism of globin switching at the clonogenic progenitor level, we performed methylcellulose colony-forming assays for both primitive and definitive colonies, as depicted schematically in Figure 5A, 5B. The phenotypes of colony-forming cells included EryP (Fig. 5C, 5G), EryD (Fig. 5D, 5H), mixed colony-forming unit (CFU-Mix) (Fig. 5E, 5I), and colony-forming unit granulocyte-macrophage (CFU-GM) colonies (Fig. 5F, 5J). Sequential colony-forming assays revealed EryP colonies until day 18 and the initial development of EryD and CFU-Mix colonies on day 30 (Fig. 5K, 5L). Immunostaining experiments showed that all of the erythrocytes in individual EryP colonies were positive for -, -, -, and -globin, but not ß-globin (Fig. 6A¨C6F and data not shown), consistent with previous reports . In contrast, erythrocytes in individual EryD colonies were positive for -, -, and ß-globin, although ß-globin was expressed at low levels (Fig. 6I, 6L and data not shown). Analysis with embryonic globin mAbs revealed - and -globin expression in 83.7% ¡À 6.5% and 35.0% ¡À 6.5%, respectively, of the erythrocytes in individual EryD colonies (Fig. 6G, 6H, 6J, 6K, 6M). These results indicate that ß-globin is exclusively expressed in EryD, but not EryP, colonies and that the decline in embryonic globin expression occurs in individual definitive erythroid progenitors.+ j3 Y1 Y. c. ?+ R: y" L9 e

Figure 5. Development of primitive and definitive hematopoietic colonies. (A, B): Schematic representation of colony-forming assays. (C¨CF): Light micrographs of colonies (x100). (G¨CJ): May-Giemsa staining of colonies (x400). (K, L): Sequential analysis of colony-forming assays. Abbreviations: CFU-GM, colony-forming unit granulocyte/macrophage colonies; CFU-Mix, mixed colony-forming unit; EryD, definitive erythroid; EryP, primitive erythroid.
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Figure 6. Immunostaining analysis of erythrocytes from primitive and definitive erythroid colonies. (A¨CL): Immunostaining of erythrocytes obtained from EryP (A¨CF) and EryD (G¨CL) colonies (x400). Staining is as in Figure 3. (M): Proportion of - or -globin-positive erythrocytes in individual EryD colonies. Data are presented as the mean of 10 individual colonies. Abbreviations: EryD, definitive erythroid; EryP, primitive erythroid; Hb, hemoglobin.
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OP9 Stromal Cell-Derived Factors Enhance ß-Globin Gene Expression in Definitive Erythroid Colonies

To determine whether erythrocytes in EryD colonies expressed less ß-globin than floating definitive erythrocytes generated from the OP9 stromal layer, we examined the effect of OP9 stromal cells on definitive colony-forming assays. In semisolid medium with OP9 cells, EryD and CFU-Mix colonies developed earlier and increased with time (Fig. 7A, 7B). Immunostaining analyses revealed increased ß-globin in EryD colonies with OP9 stromal cells (Fig. 7C¨C7F). Single-colony RT-PCR data additionally showed that both EryP and EryD colonies expressed embryonic ( and ) as wells as fetal ( and ) or adult (ß) globin genes. These results are consistent with the globin expression patterns in erythroid colonies from human yolk sac or fetal liver . Low ß-globin expression was observed in EryD colonies in the absence of OP9 cells. In the presence of OP9 cells, however, ß-globin expression was upregulated in individual EryD colonies, whereas - and -globin levels were not affected (Fig. 7G). Thus, the microenvironment created by OP9 stromal cells enhanced not only the proliferation of definitive erythroid progenitors, but also ß-globin expression.

Figure 7. Effects of OP9 stromal cells on ß-globin expression. (A, B): Sequential analysis of definitive hematopoietic colonies. (C¨CF): Immunostaining of erythrocytes in EryD colonies (x400). (G): Single-colony reverse transcription-polymerase chain reaction analysis. Abbreviations: BFU-E, burst-forming unit-erythroid; BM, bone marrow; EryD, definitive erythroid colonies; EryP, primitive erythroid; ES, embryonic stem cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM, granulocyte/macrophage; Hb, hemoglobin; K, K562; M, size marker; Mix, mixed.! d3 g1 S1 m! w1 Y* w' o! _% z

DISCUSSION1 g  H5 T' g+ _

Hematopoiesis during embryogenesis is a dynamic process notable for the seque
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