Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online August 3, 2006
Stem Cells Vol. 24 No. 12 December 2006, pp. 2627 -2636
doi:10.1634/stemcells.2006-0199; www.StemCells.com
© 2006 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2006-0199v1
24/12/2627    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umeda, K.
Right arrow Articles by Nakahata, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umeda, K.
Right arrow Articles by Nakahata, T.

EMBRYONIC STEM CELLS

Sequential Analysis of {alpha}- and ß-Globin Gene Expression During Erythropoietic Differentiation from Primate Embryonic Stem Cells

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

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, Massachusetts, USA;
dResearch Center for Animal Life Science, Shiga University of Medical Science, Ohtsu, Japan;
eDivision of Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
fDepartment of Development and Differentiation, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan

Key Words. Embryonic stem cells • Erythroid progenitors

Correspondence: Tatsutoshi Nakahata, M.D., Ph.D., Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507 Japan. Telephone: 81-75-751-3290; Fax: 81-75-752-2361; e-mail: tnakaha{at}kuhp.kyoto-u.ac.jp

Received on April 7, 2006; accepted for publication on July 26, 2006.

First published online in STEM CELLS EXPRESS  August 3, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
The temporal pattern of embryonic, fetal, and adult globin expression in the {alpha} ({zeta} -> {alpha}) and ß ({varepsilon} -> {gamma} and {gamma} -> ß) 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 {zeta}- and {varepsilon}-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of ß-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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
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 [1, 2]. Concomitant switches in the {alpha} ({zeta} -> {alpha}) and ß ({varepsilon} -> {gamma} and {gamma} -> ß) clusters of erythrocytes occur during primate (human and monkey) development and coincide with a shift in the site of hematopoiesis. However, the regulatory mechanisms of globin switching in primates remain to be resolved, primarily due to the lack of available model systems that reproduce the process of hematopoiesis to reflect in vivo development accurately. Only limited in vitro studies of the switch from embryonic to fetal globin have been performed, due to restrictions on the use of primate embryos [3–5].

Recently, established primate embryonic stem cell (ESC) lines [6–9] have served as an experimental model for tissue growth and development, as an efficacy and toxicity screening system for new drugs, and as a cell source for regeneration therapy [10, 11]. Numerous reports also have demonstrated hematopoietic differentiation induced from primate ESCs in vitro [12–16]. We previously showed transition from primitive to definitive erythropoiesis in primate ESCs upon coculture with OP9 stromal cells [15]. Moreover, sequential fluorescence-activated cell sorting (FACS) analysis revealed that the vascular endothelial growth factor receptor-2 (VEGFR-2)high CD34+ cells emerging onto the OP9 stromal layer, after the initial 6-day differentiation period, contain hemogenic progenitors [16].

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 [17]. In the present study, we specifically analyze the temporal pattern of globin switching in the {alpha}- 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.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Cell Lines
The ESC line CMK6, established from cynomolgus monkey blastocysts, was maintained according to the procedure of Suemori et al. [9]. The green fluorescent protein (GFP)-transfected ESC subline [18] was used to exclude OP9 cells. The proportion of GFP+ ESC-derived cells within the culture was determined by FACS. OP9 stromal cells, kindly provided by Dr. Hiroaki Kodama, were maintained as reported previously [15].

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).

Antibodies
Primary antibodies used in this study included mouse anti-human hemoglobin (Hb) {gamma}- 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-{varepsilon}- and -{zeta} mAbs and mouse anti-human VEGFR-2 mAb were used according to previous reports [19–21]. Mouse anti-human Hb-{alpha} mAb was established in the laboratory of David H. K. Chui. All primary antibodies against human antigens used in this study cross-reacted with cynomolgus monkey compartments, as observed previously [15, 21, 22]. 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).

In Vitro Hematopoietic Differentiation of Primate ESCs
In vitro differentiation of ESCs and cell sorting were performed as reported previously [15, 16]. In brief, trypsin-treated undifferentiated ESCs were transferred to fresh confluent OP9 cells in six-well plates at a concentration of 1 x 104 cells per well and cultured in {alpha}-minimal essential medium ({alpha}-MEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 50 µM 2-mercaptoethanol (2-ME), and 20 ng/ml VEGF. On day 6, cells were harvested and sorted with phycoerythrin-conjugated CD34 and allophycocyanin-conjugated VEGFR-2 mAbs using a FACSVantage flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Nonviable cells were excluded from the analysis by propidium iodide costaining. The purity of isolated VEGFR-2high CD34+ cells was determined using FACS. Preparations that were 96%–98% pure were employed in the experiments. Sorted cells were transferred to fresh confluent OP9 cells in six-well plates at a concentration of 1 x 104 cells per well in {alpha}-MEM, 10% FCS, 50 µM 2-ME, and a mixture of 2 U/ml EPO, 20 ng/ml IL-3, 100 ng/ml SCF, and 10 ng/ml TPO. Floating hematopoietic cells (HCs) that emerged after cell sorting were processed every 3 days for May-Giemsa staining and immunostaining with anti-human Hb antibodies as described previously [15, 16]. 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 ± SDs of triplicate wells. Representative results from one of three independent experiments are shown.

Colony-Forming Assays for Primitive and Definitive Cells
Colony-forming assays were performed every 6 days in semisolid medium consisting of {alpha}-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 [15]. For colonies consisting of definitive cells, we took advantage of the nature of OP9 stromal cells that adhered faster to culture dishes than ESC-derived cells [23]. After floating HCs were gently washed three times with phosphate-buffered saline (PBS), and the remaining adherent cells, including OP9 cells, were treated with 0.25% trypsin/EDTA. Trypsinized cells were transferred to a new culture dish and incubated for 30 minutes to allow OP9 cells to adhere. Floating cells obtained from the resulting culture supernatant after incubation were transferred (3 x 104 cells per well) to a new 35-mm Petri dish or to a fresh OP9 cell layer in a 35-mm culture dish. Colonies (≥50 cells) were counted using an inverted microscope according to previously established criteria [15, 24, 25]. Data are presented as means ± 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.

RT-PCR for Globin Gene Expression
RNA isolation and RT-PCR were performed using the procedure of Umeda et al. [15]. Samples were initially denatured at 94°C for 5 minutes, followed by amplification cycles of 94°C for 1 minute (denaturing), 60°C for 1 minute (annealing), and 72°C for 1 minute (extension), and a final extension at 94°C for 7 minutes. The following primers were used for RT-PCR: {varepsilon}-globin (359 base pairs [bp]), forward 5'-TGC ATT TTT ACT GCT GAG GAG A-3', reverse 5'-TGC CAA AGT GAG TAG CCA GAA TAA-3'; {gamma}-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'; {zeta}-globin (327 bp), forward 5'-CCG CCA TGT CTC TGA CCA A-3', reverse 5'-GCT CGC TCA GCT TGG ACA GGG-3'; {alpha}-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: {varepsilon}-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'; {gamma}-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'; {zeta}-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'; {alpha}-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 {varepsilon}- and ß-globin-specific primers and probes have been described previously [26, 27]. For all samples, the globin expression levels normalized to the housekeeping gene, 18S rRNA, were determined using the comparative threshold cycle (CT) method [28, 29]. Briefly, cDNA was mixed with primers and the PCR master mix (Applied Biosystems) and amplified in an ABI PRISM 7900HT instrument (Applied Biosystems). The CT value (the cycle number at which the emitted fluorescence exceeded an automatically determined threshold) for each globin gene was normalized against the corresponding rRNA CT value and plotted against the log quantities of target. The efficiency of the reaction (E) was calculated from the slope of the dilution curve using the following equation: E = 101/–S – 1, in which E = PCR efficiency and S = slope [28]. Data are presented as means ± SDs of triplicate wells. Representative results from one of three independent experiments are shown.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Seeding VEGFR-2high CD34+ Hemogenic Progenitors in the OP9 Coculture System Leads to Two-Wave Erythropoiesis
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 [15]. However, this induced hematopoiesis is not sufficient for quantitative analysis of globin expression, primarily due to the concomitant development of other lineages. Therefore, we employed a two-step culture system to efficiently induce erythropoiesis by purifying and reseeding hemogenic progenitors. For FACS analysis, VEGFR-2 and CD34 were employed as key markers of early hematopoietic progenitors [30–33]. As shown in Figure 1, undifferentiated ESCs were all negative for CD34, whereas approximately 80% of the cells expressed VEGFR-2 at low levels. VEGFR-2low cells gradually decreased during culturing, whereas VEGFR-2high cells first detected on day 6 and half of these were CD34+. We purified the VEGFR-2high CD34+ cell fraction, which possessed greater hemogenic potential, consistent with previous data [16], and reseeded on a new OP9 feeder layer in the presence of EPO, IL-3, SCF, and TPO, all of which enhance erythroid lineage development [23, 34, 35]. Reanalysis of sorted cells confirmed purity of 96%–98%.


Figure 1
View larger version (39K):
[in this window]
[in a new window]

  		Figure 1. Fluoresence-activated cell sorting analysis and cell sorting using antibodies against VEGFR-2 and CD34. The amounts of VEGFR-2high CD34– (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.

 
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%–25% 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 [15, 23]. 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
View larger version (43K):
[in this window]
[in a new window]

  		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.

 
Globin Switches Occur in Parallel with Transition from Primitive to Definitive Erythropoiesis
Primitive erythrocytes are relatively large and nucleated, whereas in definitive erythropoiesis, erythroid progenitors mature sequentially and eventually lose their nuclei [36]. 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 {varepsilon}-, {gamma}-, ß-, {zeta}-, and {alpha}-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 {varepsilon}-, {gamma}-, {zeta}- and {alpha}-globins, but not ß-globin expression (Fig. 3A–3J). In contrast, the second wave of erythrocytes was positive for {gamma}-, ß-, and {alpha}-globin, but one- half and less than 10% of the total erythrocytes expressed {varepsilon}- and {zeta}-globin, respectively (Fig. 3K–3V). Notably, the proportion of total enucleated erythrocytes that were positive for {varepsilon}- and {zeta}-globin was equivalent to the proportion of total nucleated cells (data not shown). Thus, expression of ß-globin, but not embryonic {varepsilon}- and {zeta}-globins, defines the switch in parallel with the transition from primitive (EryP) to definitive (EryD) erythrocytes during culturing.


Figure 3
View larger version (44K):
[in this window]
[in a new window]

  		Figure 3. Immunostaining analysis of globin gene expression in floating erythrocytes. (A–T): 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 {varepsilon}-, {gamma}-, and ß-globin monoclonal antibodies (mAbs) (U) and {zeta}- and {alpha}-globin mAbs (V). Abbreviation: Hb, hemoglobin.

 
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 [27, 28], which was confirmed to be between 0.993 and 0.999 for each transcript. The linear regression equation, correlation (r2), and E are illustrated in Figure 4A and 4B. In the ß-globin cluster, embryonic {varepsilon}-globin gradually decreased on day 18 but was subsequently detected thereafter, whereas fetal {gamma}-globin was constantly expressed during culture. Notably, adult ß-globin had an approximately 30-fold increase in expression on day 36, compared with its expression on day 12 ({Delta}CT [globin – rRNA] was –14.86 ± 0.34 on day 12 and –9.52 ± 0.41 on day 36), although its expression was far below than that of {gamma}-globin (Fig. 4C). In the {alpha}-globin cluster, {zeta}- and {alpha}-globin genes were coexpressed on day 12 and {alpha}-globin was expressed constantly during subsequent culturing, whereas the {zeta}-globin level decreased from day 18 onwards (Fig. 4D). Early definitive erythrocytes in the fetal liver express large amounts of fetal hemoglobin and a small amount of adult hemoglobin [37]. 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
View larger version (19K):
[in this window]
[in a new window]

  		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 {varepsilon}-globin cDNA (B). (C, D): Sequential analysis of mRNA expression in the ß-globin (C) and {alpha}-globin (D) clusters. Abbreviations: CT, threshold cycle; E, polymerase chain reaction efficiency; r2, correlation coefficient.

 
Sequential Development of Primitive and Definitive Erythroid Colonies
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 {varepsilon}-, {gamma}-, {zeta}-, and {alpha}-globin, but not ß-globin (Fig. 6A–6F and data not shown), consistent with previous reports [15]. In contrast, erythrocytes in individual EryD colonies were positive for {gamma}-, {alpha}-, and ß-globin, although ß-globin was expressed at low levels (Fig. 6I, 6L and data not shown). Analysis with embryonic globin mAbs revealed {varepsilon}- and {zeta}-globin expression in 83.7% ± 6.5% and 35.0% ± 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.


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		Figure 5. Development of primitive and definitive hematopoietic colonies. (A, B): Schematic representation of colony-forming assays. (C–F): Light micrographs of colonies (x100). (G–J): 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.

 


Figure 6
View larger version (20K):
[in this window]
[in a new window]

  		Figure 6. Immunostaining analysis of erythrocytes from primitive and definitive erythroid colonies. (A–L): Immunostaining of erythrocytes obtained from EryP (A–F) and EryD (G–L) colonies (x400). Staining is as in Figure 3. (M): Proportion of {varepsilon}- or {zeta}-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.

 
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–7F). Single-colony RT-PCR data additionally showed that both EryP and EryD colonies expressed embryonic ({varepsilon} and {zeta}) as wells as fetal ({gamma} and {alpha}) or adult (ß) globin genes. These results are consistent with the globin expression patterns in erythroid colonies from human yolk sac or fetal liver [38]. 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 {varepsilon}- and {zeta}-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
View larger version (23K):
[in this window]
[in a new window]

  		Figure 7. Effects of OP9 stromal cells on ß-globin expression. (A, B): Sequential analysis of definitive hematopoietic colonies. (C–F): 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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Hematopoiesis during embryogenesis is a dynamic process notable for the sequential emergence of distinct populations of blood-cell progenitors [1, 2]. Because the hematopoietic development of primates is distinct from that of mice, including the pattern of globin switching during the shift of hematopoietic sites [39], in vitro and in vivo studies on primate hematopoietic development should be performed using primate-derived materials. However, experiments on human embryos or fetuses, particularly for genetic manipulation, have ethical limitations. Immortalized cell lines also have critical disadvantages, in that they always reflect restricted aspects of the in vivo differentiation processes and sometimes behave differently from their physiological counterparts [40]. In this regard, ESCs serve as an alternative tool for analysis of hematopoietic development and differentiation, because the developmental process induced during differentiation is reproduced in a similar manner as that in early embryos. Furthermore, genetic manipulation of ESCs provides a system that facilitates the evaluation of embryonically lethal genetic changes in vivo. Globin switches in murine ESCs have been extensively investigated using various culture systems [34, 41, 42], whereas there have been few reports about erythropoietic differentiation in primate ESCs. Qiu et al. [43] reported the mature primitive erythropoietic differentiation of human ESCs (hESCs) by coculture with immortalized human liver cells. Zambidis et al. [44] demonstrated the transition from primitive into definitive erythropoiesis in hESCs by a colony-forming assay, which did not allow for the investigation of erythroid proliferation and maturation into terminal differentiation.

The OP9 coculture system is preferred for the analysis of transition because mature HCs can be easily and repeatedly obtained as floating cells in culture medium in the presence of protease treatment [34]. Thus, this culture system is a powerful experimental tool for elucidating the regulation of murine hematopoietic development and differentiation [23, 35]. We previously adapted this culture system for hematopoietic differentiation of primate ESCs for analysis of sequential development of primitive and definitive erythropoiesis in vitro. However, the low efficiency of mesodermal differentiation hampered the quantitative analysis [15]. The existence of hemogenic progenitors during early mesodermal differentiation in this coculture system was additionally demonstrated by serial FACS analysis with VEGFR-2 and CD34 mAbs [16] because VEGFR-2 and CD34 are expressed at this stage [30–33]. To induce abundant erythropoietic production, we established a two-step culture system using a combination of previously reported methods [15, 16]: (a) an initial 6-day culture to generate VEGFR-2high CD34+ cells in the presence of VEGF (hemogenic progenitor differentiation), and (b) purification and further culturing of VEGFR-2high CD34+ cells in the presence of EPO, IL-3, SCF, and TPO (erythroid lineage differentiation). With this culture system, we have successfully achieved effective and sustained production of mature erythrocytes, which allows the quantitative analyses of erythropoiesis corresponding to that from yolk sac to the early fetal liver stage in vivo.

In this coculture system, two-wave erythropoiesis develops sequentially. Our data show that, morphologically, the appearance of EryP precedes that of EryD. Sequential RT-PCR analyses reveal low ß-globin in EryP, but increased expression, both at the RNA and protein level, in EryD. Thus, we propose that ß-globin is the most sensitive and distinctive marker of EryD, consistent with earlier data from human embryos [3]. In contrast, {varepsilon}- and {zeta}-globin expression decrease but are still detectable in EryD. It is unlikely that remnants of embryonic globin expression are solely due to coexisting EryP, because erythrocytes generated after day 18 are positive for ß-globin exclusively and a proportion of enucleated erythrocytes also expressed {varepsilon}- or {zeta}-globin. This finding is supported by a previous report that embryonic globin expression is still detected in erythrocytes, even after birth [45].

Recently, Chang et al. reported the development of definitive-like erythroid cells from hESCs by embryoid body (EB) formation [46]. Their observation that the erythroid cells coexpress high levels of embryonic and fetal globins is in agreement with our own study. In contrast, the erythroid cells in the report of Chang et al. are all nucleated and do not express adult globin even in the presence of OP9 stromal cells, whereas the definitive erythroid cells in our study contain enucleated ones and express adult globin. These differences may be partially due to differences in the culture conditions (the EB and OP9 coculture system) and/or the ESCs that were used for the induction of differentiation.

The sequential development of immature adherent hematopoietic progenitors can be induced in this coculture system, and the clonogenic potential of such progenitors alters during culture. EryP colonies and a few CFU-GM colonies develop initially, whereas EryD and CFU-Mix, as well as numerous CFU-GM colonies, appear subsequently. This finding is consistent with a previous report on human yolk sac and fetal liver hematopoiesis that demonstrated the rapid expansion of clonogenic cells, along with shifting hematopoietic sites [47]. EryP colonies develop only in the presence of OP9 stromal cells, whereas EryD and CFU-Mix colonies appear irrespective of OP9 stromal cells. Immunostaining analysis shows that all erythrocytes in individual EryP colonies express embryonic {varepsilon}- and {zeta}-globins, as well as fetal {gamma}- and {alpha}-globins. In contrast, individual EryD colonies express embryonic ({varepsilon} and {zeta}) as well as fetal ({gamma} and {alpha}) or adult (ß) globin genes. Single-colony RT-PCR analysis shows the same expression pattern, consistent with the data from hESCs [44]. Notably, {varepsilon}- or {zeta}-globin expression is restricted to a subset of the total cells within individual EryD colonies, strongly suggesting that the {varepsilon}-to-{gamma} (or {zeta}-to-{alpha}) switch occurs in a single definitive erythroid progenitor during the early stage. The results strongly suggest that the definitive erythroid progenitors encode the same regulatory programs of globin gene expression that control the {gamma}-to-ß switch in fetal and adult progenitor-derived erythroid colonies [17]. The decline of embryonic globin expression in floating EryD, compared with that in EryD colonies, may also correspond to the decreasing proportion of fetal globin synthesis during fetal, neonatal, and adult erythroid cell maturation [47, 48].

In colony-forming assays with an OP9 stromal layer, EryD and CFU-Mix colonies are more abundant. These results are consistent with the finding that the microenvironment created by stromal cells is essential for the rapid expansion of erythrocytes in murine fetal liver [49]. Additionally, ß-globin gene expression in each EryD is upregulated in the presence of OP9 stromal cells. It would be interesting to determine whether stromal regulation of erythropoiesis occurs through cell-cell contact, extracellular matrices, or factors secreted by stromal cells.

Summary
We have described a culture system for producing enriched mature erythrocytes and immature erythroid progenitors from primate ESCs, which reflects the transition from yolk sac to early fetal liver-stage erythropoiesis in vivo. This differentiation induction method should facilitate the analysis of the regulatory mechanisms of human globin switching and the establishment of a system for transfusion medicine and drug discovery for hemoglobinopathies.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Tanabe Seiyaku Co. Ltd. (Osaka, Japan) for help with primate ESC preparation and Prof. Ogawa for critical reading of the manuscript. This work was supported by grants from the Science Research on Priority Areas and the Creative Science Research programs. It was also supported by the Japan Society for the Promotion of Science, by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

  1. Dzierzak E, Medvinsky A. Mouse embryonic hematopoiesis. Trend Genet 1995;11:359–366.[CrossRef][Medline]

  2. Xu MJ, Matsuoka S, Yang FC et al. Evidence for the presence of murines primitive megakarycytopoiesis in the early yolk sac. Blood 2001;97:2016–2022.[Abstract/Free Full Text]

  3. Peschle C, Migliaccio AR, Migliaccio G et al. Embryonic—fetal Hb switch in humans: Studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver. Proc Natl Acad Sci U S A 1984;81:2416–2420.[Abstract/Free Full Text]

  4. Peschle C, Mavilio F, Care A et al. Haemoglobin switching in human embryos: Asynchrony of {zeta}->{alpha} and {varepsilon}->{gamma} globin switches in primitive and definitive erythropoietic lineage. Nature 1985;313:235–238.[CrossRef][Medline]

  5. Stamatoyannopoulos G, Constantoulakis P, Brice M et al. Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: Relevance to the cell-lineage models of globin switching. Dev Biol 1987;123:191–197.[CrossRef][Medline]

  6. Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.[Abstract/Free Full Text]

  7. Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchu) blastocysts. Biol Reprod 1996;55:254–259.[Abstract]

  8. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

  9. Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279.[CrossRef][Medline]

  10. Odorico JS, Kaufman DS, Thomson JA. Mutilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:192–204.

  11. Nakatsuji N, Suemori H. Embryonic stem cell lines of nonhuman primates. ScientificWorldJournal 2002;2:762–773.

  12. Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001;98:10716–10721.[Abstract/Free Full Text]

  13. Li F, Lu S, Vida L et al. Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. Blood 2001;98:335–342.[Abstract/Free Full Text]

  14. Chadwick K, Wang L, Li L et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 2003;102:906–915.[Abstract/Free Full Text]

  15. Umeda K, Heike T, Yoshimoto M et al. Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro. Development 2004;131:1869–1879.[Abstract/Free Full Text]

  16. Umeda K, Heike T, Yoshimoto M et al. Identification and characterization of hemoangiogenic progenitors during cynomolgus monkey embryonic stem cell differentiation. STEM CELLS 2006;24:1348–1358.[Abstract/Free Full Text]

  17. Stamatoyannopoulos G, Kurnit DM, Papayannopoulou T. Stochastic expression of fetal hemoglobin in adult erythroid cells. Proc Natl Acad Sci U S A 1981;78:7005–7009.[Abstract/Free Full Text]

  18. Furuya M, Yasuchika K, Mizutani K et al. Electroporation of cynomolgus monkey embryonic stem cells. Genesis 2003;37:180–187.[CrossRef][Medline]

  19. Chui DHK, Mentzer WC, Patterson M et al. Human embryonic {zeta}–globin chains in fetal and newborn blood. Blood 1989;74:1409–1414.[Abstract/Free Full Text]

  20. Luo HY, Liang XL, Frye C et al. Embryonic hemoglobins are expressed in definitive cells. Blood 1999;94:359–361.[Abstract/Free Full Text]

  21. Sawano A, Iwai S, Sakurai Y et al. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood 2001;97:785–791.[Abstract/Free Full Text]

  22. Sone M, Itoh H, Yamashita J et al. Different differentiation kinetics of vascular progenitor cells in primate and mouse embryonic stem cells. Circulation 2003;107:2085–2088.

  23. Suwabe N, Takahashi S, T., Nakano T et al. GATA-1 regulates growth and differentiation of definitive erythroid lineage cells during in vitro ES cell differentiation. Blood 1998;92:4108–4118.[Abstract/Free Full Text]

  24. Nakahata T, Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with extensive capacity to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 1982;70:1324–1328.[Medline]

  25. Tajima S, Tsuji K, Ebihara Y et al. Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 1996;184:1357–1364.[Abstract/Free Full Text]

  26. Jimenez DF, Tarantal AF. Quantitative analysis of male fetal DNA in maternal serum of gravid rhesus monkeys (Macaca mulatta). Pediatr Res 2003;53:18–23.[CrossRef][Medline]

  27. Fibach E, Bianchi N, Borgatti M et al. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood 2003;102:1276–1281.[Abstract/Free Full Text]

  28. Klein D, Janda P, Steinborn R et al. Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: Influence of mismatches on quantification. Electrophoresis 1999;20:291–299.[CrossRef][Medline]

  29. Mullen AC, Hutchins AS, High FA et al. Hix is induced by and genetically interacts with T-bet to promote heritable THI gene induction. Nat Immunol 2002;3:652–658.[Medline]

  30. Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995;376:62–66.[CrossRef][Medline]

  31. Shalaby F, Ho J, Stanford WL et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 1997;89:981–990.[CrossRef][Medline]

  32. Tavian M, Coulombel L, Luton D et al. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 1996;87:67–72.[Abstract/Free Full Text]

  33. Marshall CJ, Moore RL, Thorogood P et al. Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer. Dev Dyn 1999;215:139–147.[CrossRef][Medline]

  34. Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Science 1996;272:722–724.[Abstract]

  35. Era T, Takagi T, Takahashi T et al. Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system. Blood 2000;95:870–878.[Abstract/Free Full Text]

  36. Gregory CJ, Eaves AC. The stages of erythropoietic cell differentiation distinguished by a number of physical and biologic properties. Blood 1978;51:527–537.[Abstract/Free Full Text]

  37. Stamatoyannopoulos G, Grosvled F. Hemoglobin Switching: The Molecular Basis of Blood Disease. Philadelphia: W.B. Saunders,2001;135–182.

  38. Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol 2005;33:259–271.[CrossRef][Medline]

  39. Tomhon C, Zhu W, Millinoff D et al. Evolution of a fetal expression pattern via cis changes near the {gamma} globin gene. J Biol Chem 1997;272:14062–14066.[Abstract/Free Full Text]

  40. Rutherford T, Clegg JB, Higgs DR et al. Embryonic erythroid differentiation in the human leukemic cell line K562. Proc Natl Acad Sci U S A 1981;78:348–352.[Abstract/Free Full Text]

  41. Lindenbaum MH, Grosveld F. An in vitro globin gene switching model based on differentiated embryonic stem cells. Genes Dev 1990;4:2075–2085.[Abstract/Free Full Text]

  42. Trimborn T, Gribnau J, Gorsveld F et al. Mechanisms of developmental control of transcription in the murine {alpha}- and ß- globin loci. Genes Dev 1999;13:112–124.[Abstract/Free Full Text]

  43. Qiu C, Hanson E, Olivier E et al. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver recapitulates the globin switch that occurs early in development. Exp Hematol 2005;33:1450–1458.[CrossRef][Medline]

  44. Zambidis ET, Peault B, Park TS et al. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hemato-endothelial, primitive, and definitive stages resembling human yolk sac development. Blood 2005;106:860–870.[Abstract/Free Full Text]

  45. Chui DHK, Wong SC, Enkin MW et al. Proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation. Proc Natl Acad Sci U S A 1980;77:2757–2761.[Abstract/Free Full Text]

  46. Chang KH, Nelson AM, Cao H et al. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood 2006;108:1515–1523.[Abstract/Free Full Text]

  47. Migliaccio G, Migliaccio AR, Petti S et al. Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac -> liver transition. J Clin Invest 1986;78:51–60.[Medline]

  48. Papayannopoulou T, Kalmantis T, Stamatoyannopoulos G. Cellular regulation of hemoglobin switching: Evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells. Proc Natl Acad Sci U S A 1979;76:6420–6424.[Abstract/Free Full Text]

  49. Ohneda O, Yanai N, Obinata M. Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver. Development 1990;110:379–384.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Ma, Y. Ebihara, K. Umeda, H. Sakai, S. Hanada, H. Zhang, Y. Zaike, E. Tsuchida, T. Nakahata, H. Nakauchi, et al.
Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis
PNAS, September 2, 2008; 105(35): 13087 - 13092.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2006-0199v1
24/12/2627    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umeda, K.
Right arrow Articles by Nakahata, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umeda, K.
Right arrow Articles by Nakahata, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS