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作者：Ryo Kuritaa, Erika Sasakib, Tomoko Yokooa, Takashi Hiroyamac, Kashiya Takasugia, Hideyuki Imotoa, Kiyoko Izawad, Yan Dongd, Takao Hashiguchia, Yasushi Sodad, Toyoki Maedaa, Youko Suehiroa, Yoshikuni Taniokab, Yukoh Nakazakia, Kenzaburo Tania作者单位：aDepartment of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan;bDepartment of Biomedical Science, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan;cCell Bank, RIKEN BioResource Center, Tsukuba 1 h$ w! [( r- F) O% M" N6 W8 G9 h
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0 J2 }$ h, _5 x6 ^" w7 O 【摘要】6 |9 |3 h- o4 t) T" ~8 v3 B* w
The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.
, }# ^; W- y6 _0 q8 { 【关键词】 CD cells Transcription factor tal/scl Lentiviral vector Hematopoietic cells Gene transfer Embryonic stem cell Embryoid body
" i, L$ G }& ~* F2 _5 P INTRODUCTION
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Since the successful establishment of human embryonic stem cell (ESC) lines in 1998 ; however, in many of these cases, these results are not directly applicable to clinical medicine. Although human ESCs have been studied extensively in vitro, for ethical reasons their safety and efficacy in vivo cannot be confirmed. Preclinical studies using large animal models, including nonhuman primates, are essential.8 X3 b2 f9 f4 ?% @1 [
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The common marmoset (CM) (Callithrix jacchus) is a New World primate species with reproductive characteristics appropriate for ESC studies .
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In vitro hematopoietic differentiation has been achieved by coculturing ESCs from humans, nonhuman primates, or mice with S17 or OP9 stromal cells . These culture systems, however, are not appropriate for clinical application. Furthermore, these reports examined only a limited array of ESC lines; it remains unknown whether these results will be applicable to other cell lines.
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) [" V; n$ P; V7 Y8 m$ \9 e: H/ EIn this communication, we investigated the use of gene transfer with vesicular stomatitis virus (VSV)-pseudotyped lentiviral vectors to differentiate several CM ESC lines into hematopoietic cells stably and efficiently. In tal1/scl-overexpressing CM cells, the efficiency of hematopoietic induction from ESCs was dramatically increased in comparison with that seen in the general coculture method. These results suggest that our gene-transfer method may be a useful tool for future ESC therapies.
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! V, ~- a/ v8 a' Z# ]: b: W8 fMATERIALS AND METHODS
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ESC Lines and Cytokines: _# p6 }+ \7 Q$ D/ s3 k$ O. O: |% d
Y* Q4 ^: z1 UThe mouse (E14tg2a) and CM (cj11) ESC lines were purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org) and WiCell Research Institute, Inc. (Madison, WI, http://www.wicell.org), respectively. Additional CM ESC lines (CM ES 20, 30, and 40) were established previously . Human stem cell factor (SCF), interleukin (IL)-3 and IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) were the kind gifts of the Kirin Brewery (Tokyo, http://www.kirin.co.jp/english). Additional cytokines and growth factors, including flt-3 ligand, thrombopoietin (TPO), basic fibroblast growth factor (bFGF), activin-A, bone morphogenic protein-4 (BMP-4), and vascular endothelial growth factor (VEGF), were purchased from R&D Systems (Minneapolis, http://www.rndsystems.com)., F g' ]! L: o$ X* E8 t
4 i5 J4 @* N+ r- A6 t, dConstruction of the Pseudotyped Lentiviral Vectors
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/ ]& V9 p4 k* A bMarmoset cDNAs encoding tal1/scl, gata1, gata2, and lhx2 were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the following primers representing sequences with the highest homology to human and murine cDNAs: tal1/scl, 5'-atgacsgagcggccgccgag-3' (sense) and 5'-tcaccgagggccggcyccatc-3' (antisense); gata1, 5'-atggagttccctggcctggggt-3' (sense) and 5'-tcatgagctgagcggagcca-3' (antisense); gata2, 5'-atggaggtggcgccggagca-3' (sense) and 5'-ctagcccatggcggtcaccat-3' (antisense); and lhx2, 5'-atcgacgagatggaccgcagg-3' (sense) and 5'-tgcgagtcattagaaaaggttgg-3' (antisense). A vector encoding the human hoxB4 gene (pMSCV HoxB4-IRES-green fluorescent protein .
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# ^6 I3 a, ]) P+ ~Hematopoietic Cell Differentiation from ESCs by Either Embryoid Body Formation or Coculture with Stromal Cells
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The CM ESC lines cj11 . The mouse ESC line E14tg2a was cultured in the same manner as the CM ESCs with the addition of 10 ng/ml leukemia inhibitory factor. After dissociation in 0.25% trypsin/1 mM EDTA, undifferentiated ESCs cultured on irradiated (40 Gy) mouse embryonic fibroblast cell layer were seeded onto low cell-binding dishes (Nalge Nunc, Naperville, IL, http://www.nuncbrand.com) at 0.5¨C5 x 105 cells per 9-cm plate to induce embryoid body (EB) formation. Iscove¡¯s modified Dulbecco¡¯s medium (Invitrogen) supplemented with 15% fetal bovine serum (FBS), 200 µg/ml transferrin, 10 µg/ml insulin, 50 µg/ml ascorbic acid, and 0.45 mM monothioglycerol was used to induce hematopoietic cell differentiation. We seeded 1¨C5 x 105 dissociated ESCs onto irradiated (20 Gy) or nonirradiated stromal cells, which were grown to semiconfluence 1 day before coculturing. During differentiation, cultured cells were plated onto freshly prepared stromal cells every 4¨C7 days; the medium was changed every 2 days. The following stromal cells were used: OP9 (op/op mouse calvaria cells); S17 (mouse bone marrow cells); HS-5 (human bone marrow cells); mouse aorta-gonad-mesonephros (AGM) cells; CM AGM-1, -2, and -3 cells; and hFOB1.19 (human osteoblast cells). Lentivirus infections were performed twice on days 0 and 3 (>50% infection estimated by GFP, respectively) after the induction of hematopoiesis. The cj11 cell line was used in the following experiments unless otherwise mentioned.) Z5 s; f1 x' _7 ]$ h# u
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RT-PCR and Genomic PCR1 C% y* y8 l- T. `0 O
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Total RNA, prepared with spazol RNA I super (Nacalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp/en), was used as template for cDNA synthesis using superscript II reverse transcriptase (Invitrogen). Genomic DNA extraction used a GFX Genomic Blood DNA Purification Kit (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com) according to the manufacturer¡¯s instructions. Primer sequences and amplification cycle numbers were as follows: tal1/scl, sense described above and 5'-gaggtcatccgggggtgcact-3' (antisense), 35 cycles; gata1, 5'-gcaacagccactccactgtgg-3' (sense) and 5'-aggggtccagggaaaggcat-3' (antisense), 35 cycles; gata2, sense and antisense described above, 35 cycles; flk-1 (KDR), 5'-atgatcagctatgcNggcatgg-3' and 5'-ttggtRaggatgacMgtgtaRtttcc-3' (sense), 35 cycles; c-kit, 5'-gtcgaccaccatgagaggcgctcgtggcgc-3' (sense) and 5'-ggatccgtgtggggctggatttgctct-3' (antisense), 35 cycles; Runx1/AML-1, 5'-ggcgaccgcagcatggtgga-3' (sense) and 5'-tcagtagggcctccacacgg-3' (antisense), 35 cycles; AC133, 5'-gaagagtatgattcatactggtgg-3' (sense) and 5'-cctatgccaaaccaaaacaaattca-3' (antisense), 35 cycles; CD34, 5'-gtcgaccaccatgccgcggggctggaccgc-3' (sense) and 5'-gaatttctttcgggaataactctggtggct-3' (antisense), 35 cycles; CD45, 5'-gtccattccacccaaacagcta-3' (sense) and 5'-aatttactaactgggtgtccagaa-3' (antisense), 35 cycles; -globin, 5'-tggaggtgaagccttgggcaga-3' (sense) and 5'-ccagggcaatggccacagcaga-3' (antisense), 35 cycles; -globin, 5'-tgtggaagatgctgggggagaaa-3' (sense) and 5'-tcaggggtgaattctttgccataaa-3' (antisense), 35 cycles; ß-globin, 5'-gtgcatctgactggtgaagaaaaat-3' (sense) and 5'-cacaccagccaccactttctgat-3' (antisense), 35 cycles; glycophorin A, 5'-tgcagttgtccttggtgggttt-3' (sense) and 5'-ataaagaggatctttccaatgacaa-3' (antisense), 35 cycles; and ß-actin, 5'-aacggctccggcatgtgcaa-3' (sense) 5'-gccaggtccagacgcaggat-3' (antisense), 22¨C25 cycles. Amplification by PCR was performed using the Expand Long Template PCR system (Roche, Mannheim, Germany, http://www.roche.com) or KOD-plus taq polymerase (TOYOBO, Osaka, Japan, http://www.toyobo.co.jp/e).
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Colony-Forming Units Assay
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For hematopoietic colony-forming units (CFU) assays, dissociated EB cells were first grown in Methocult GF H4435 (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), which contained 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml G-CSF, and 3 units/ml Epo. For mouse ESC-derived cells, CFU assays were performed in Methocult GF M3434 (StemCell Technologies), which contained 1% methylcellulose, 15% FBS, 1% BSA, 10 µg/ml insulin, 200 µg/ml transferrin, 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6, and 3 units/ml Epo. Cells were plated in triplicate at 0.5¨C2.0 x 104 cells per 3-cm plate and cultured at 37¡ãC in a 5% CO2 incubator. After 10¨C14 days, hematopoietic colonies were counted microscopically according to standard criteria. Samples were then spun onto slides with cytospin 2 (Thermo Electron Corporation, Waltham, MA, http://www.thermo.com) to examine their cellular morphology. May-Giemsa staining of these samples was performed according to a standard protocol. To detect transgenes, PCR was performed on genomic DNA prepared from individual colonies.
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Anti-Hemoglobin and Double Esterase Staining
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Cells spun onto glass slides were washed briefly with phosphate-buffered saline (PBS)(-) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) and fixed in 2% paraformaldehyde/PBS for 10 minutes on ice. After permeabilization in 0.05% Triton X-100/PBS, cells were washed with PBS(-) three times. Nonspecific binding was blocked with 2% BSA/PBS for 1¨C3 hours. Samples were incubated with rabbit polyclonal anti-human hemoglobin antibody (Cappel, Aurora, OH, http://www.mpbio.com), diluted 1:1,000 in blocking solution, at 4¡ãC overnight. Alexa488-conjugated anti-rabbit immunoglobulin G was used as a secondary antibody. After extensive washing with PBS(-), cells were incubated with secondary antibody for 30 minutes to 1 hour. After washing, cells were observed using an inverted fluorescence microscope. Double esterase staining was performed using an esterase AS-D staining kit (Muto Total Products, Tokyo) according to the manufacturer¡¯s instructions.+ q/ M, U# w- j1 _. w5 _# p1 E1 V
3 Y0 z! o# l2 N& V: T- G$ |7 AIsolation of CD34 Cells from CM EB and Bone Marrow Cells
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Purification and biotinylation of the anti-CM CD34 (MA24) antibody from anti-CD34-producing hybridoma cells were performed as described . CM CD34 cells, which were bound by the biotin-labeled CD34 antibody, were isolated using magnetic-activated cell sorting streptavidin microbeads (Miltenyi Biotec, Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer¡¯s instructions.' l) Y. I" \( i3 `
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Flow Cytometry
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For flow cytometry, 1¨C5 x 106 cells were incubated with the CM CD34 antibody at 4¡ãC for 30 minutes in 100 µl of 2% FBS/PBS solution (fluorescence-activated cell sorting buffer). After washing twice with FACS buffer, phycoerythrin-labeled streptavidin was applied as a secondary antibody. Cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).$ k3 X! H0 b+ ?. M. ?0 ~1 O
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5 K T, ]" K7 j# FIn Vitro Hematopoietic Cell Differentiation from ESCs by Either EB Formation or Coculture with Stromal Cells
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) Y1 n* M) F' t2 l9 E, L, qWe induced hematopoietic differentiation of mouse and CM ESCs by coculture with OP9 stromal cells. As in previous reports of mouse ESCs, mesodermal induction was readily observed by day 2¨C3; cobblestone structures were seen after the first passage at day 4 (Fig. 1A, upper left). CFU assays of mouse ESC-derived cells cultured on OP9 cells for 8¨C10 days revealed numerous multilineage hematopoietic colonies, including erythroblasts, megakaryocytes, granulocytes, and macrophages (Fig. 1A, upper middle and right). The CM ESC lines (cj11 and CM ES 20, 30, and 40) did not develop in this way when cultured on OP9 cells; these cells did not form cobblestone structures when cultured under these conditions (Fig. 1A, lower left). No hematopoietic colonies could be observed by the CFU assay (Fig. 1B). Similar results were obtained for CM ESCs cultured in the presence of other stromal cell lines, such as S17, mouse AGM, CM AGM-1, -2, and -3, hFOB4.1, and HS-5 cells (data not shown). Next, we induced CM ESCs to undergo hematopoiesis by EB formation. Although EB formation was less effective in inducing hematopoiesis than coculture of mouse ESCs, this method successfully established hematopoietic precursors from CM ESCs by day 14 (Fig. 1A, lower middle and right). The induction, however, was inefficient; in addition, no blood cells were produced from the cj11 line (Fig. 1B). Only CFU-macrophage (CFU-M) colonies could be obtained from CM EB cells (Fig. 1A, lower right). Differentiation was unaffected by the presence of various cytokines (SCF, Epo, GM-CSF, IL-3, IL-6, TPO, and Flt-3L) or growth factors (VEGF, bFGF, BMP-4, and activin-A). To confirm these results at the molecular level, we examined the expression patterns of hematopoietic gene markers by RT-PCR in cj11 cells during in vitro differentiation (Fig. 1C). CD34, a stem and progenitor cell marker, was first detected on culture day 14. The markers c-kit, gata2, and AC133 were also expressed in the early phase. Expression of the flk-1, runx1 (AML1), and tal1/scl genes, all of which have important roles in early hematopoiesis, was observed on culture days 4 and 7. CD45 and gata1 expression, however, could not be detected under these conditions. These results corresponded well with the CFU assay at day 14. We also performed the CFU assay at days 16, 18, 21, 25, and 30 because the commitment of hematopoietic cells is thought to correlate with tal1/scl and CD34 expression in both mouse and human cells. The results at these time points were similar to those seen at day 14; thus, the induction did not complete at later stages (Fig. 1D). We occasionally obtained a few hematopoietic colonies from day-21 EB cells, but these numbers were not statistically significant. Taken together, our culture conditions were not appropriate to induce hematopoietic cell differentiation, although limited differentiation of ESCs into mesodermal cells did occur.
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7 A" K p3 ~7 d) i* X' E3 C7 Z0 UFigure 1. Hematopoietic cell differentiation from mouse and CM ES cells by EB formation or coculture with OP9 stromal cells. (A): In mouse ES cells, cobblestone structures were seen upon coculture with OP9 cells (upper left, day 4); multilineage hematopoietic colonies were observed by colony-forming unit (CFU) assay (upper middle and right, day 10). In CM ES cell cocultures, however, no cobblestone structures or hematopoietic colonies could be observed under the same conditions (lower left, day 4). When hematopoietic cell differentiation was induced by EB formation, CFU-macrophage colonies were occasionally obtained from CM EB cells (lower right, day 14). Scale bars = 100 µm (middle), 50 µm (right and left). (B): Hematopoietic cells could not be generated by coculture of any CM ES cell line with OP9 cells. EB formation also resulted in low induction activity. (C): Sequential analyses of hematopoietic gene expression during EB differentiation. We used reverse transcription-polymerase chain reaction to examine the expression patterns of various genetic markers for hematopoiesis in cj11 cells. A number of markers, including tal1/scl, flk-1, CD34, c-kit, runx1, gata2, and AC133, were expressed during EB formation; the expression of CD45 and gata1, however, could not be detected. (D): Kinetic CFU assays during EB differentiation of the cj11 line. The induction of hematopoietic cells from EB cells was poor at later stages. Abbreviations: CM, common marmoset; EB, embryoid body; ES, embryonic stem; F.L., fetal liver.) ?/ C% K: [! _! X8 F; a0 q
( B* z! l5 c( X4 g+ N, ]Gene Transduction into CM ESCs by Pseudotyped Lentiviral Vectors: t z% c7 p: O) o
. K3 }% E: z& Y/ ]8 ?- KPreviously reported methods were unable to induce CM ESCs to differentiate fully into hematopoietic lineages. Thus, we investigated the possibility of inducing hematopoietic progenitor cell (HPCs) and/or hematopoietic stem cell (HSC) differentiation by gene transduction. We used VSV-glycoprotein pseudotyped lentiviral vectors (VSV-G LVs) to introduce exogenous cDNAs into CM ESCs effectively. First, we constructed a VSV-G LV containing the GFP gene under the control of several different promoters (EF1-, cytomegalovirus = 60, light blue) and 85% (MOI = 180, red) of the differentiated cells (Fig. 2B). Because the highest levels of GFP expression were generated by the EF1- promoter-containing vector (VSV-G EF1- LV), we used the VSV-G EF1- LV for further experiments.- Z6 y) S& }# m$ k
& x7 U$ w0 P2 y# PFigure 2. Gene transduction into CM ES cells by pseudotyped lentiviral vectors. To introduce exogenous cDNAs into CM ES cells, we used vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vector (VSV-G LV)s. (A): Use of the VSV-G LV encoding the GFP gene under the control of the EF1- promoter produce high levels of GFP expression in both undifferentiated and differentiated CM ES cells. (B): During EB formation, GFP expression was detected in greater than 70% (multiplicity of infection = 60, light blue) and 85% (MOI = 180, red) of differentiated cells. The blue area indicates nontransduction control cultures. Scale bars = 100 µm. Abbreviations: CM, common marmoset; EB, embryoid body; ES, embryonic stem; GFP, green fluorescent protein.
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Hematopoietic Cell Differentiation by Enforced Gene Expression& s5 |( _; C k( l d$ B
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We constructed VSV-G LVs encoding the hematopoietic genes tal1/scl, gata1, gata2, hhoxB4, and lhx2 under the control of the EF1- promoter. These vectors were transduced into EBs on days 0 and 3. Subsequent CFU assays demonstrated that transgenic expression of tal1/scl, a basic helix-loop-helix (bHLH) transcription factor essential for both primitive and definitive hematopoiesis, produced large numbers of hematopoietic colonies from CM ESCs in the absence of additional cytokines and growth factors (Fig. 3A¨CC). In contrast, overexpression of gata1, gata2, hhoxB4, or lhx2 did not induce hematopoiesis. Hematopoietic colonies obtained by tal1/scl transduction were classified according to standard morphological criteria as CFU-erythroid (CFU-E), CFU-granulocyte/macrophage (CFU-GM), CFU-M, and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-Mix ), suggesting that multilineage hematopoiesis occurred (Fig. 3D). To confirm that these colonies arose from exogenous tal1/scl gene transduction, we randomly isolated a few hematopoietic colonies. We isolated genomic DNA from these cells and subjected it to PCR analysis. Genomic PCR revealed a band representative of the tal1/scl cDNA derived from the transduced lentiviral vector (Fig. 3E), indicating that the hematopoietic colonies contained the tal1/scl transgene. Next, we examined in detail the morphology of the induced cells by May-Giemsa staining, hemoglobin immunohistochemistry, and double-esterase staining. These results demonstrated that multiple lineages of hematopoietic cells, including erythroblasts, granulocytes, and macrophages, were produced by tal/scl gene transduction (Fig. 4A, B). Semiquantitative RT-PCR analyses revealed marked increases in expression of the erythroid markers gata1, glycophorin A, and - (embryonic), - (fetal), and ß- (adult) globins in EB-tal1/scl cells (Fig. 4C). Unexpectedly, these three globins were already expressed at early stages (day 4). Although we did not observe distinct globin switching during EB development, - and ß-globin exhibited the highest expression levels at days 7 and 10, respectively. The results might reflect globin switching in a subset of EB-tal1/scl cells. In contrast, -globin was maintained at similar expression levels throughout differentiation. Interestingly, ß-globin was expressed in both nontransduced and tal1/scl-transduced EB cells at day 4; expression, however, was downregulated immediately and completely in nontransduced EB cells for unknown reasons. Flow cytometric analysis revealed that the fraction expressing the megakaryocyte/platelet marker CD41 increased significantly in EB-tal1/scl cells; megakaryocyte-like cells were observed by immunohistological staining, indicating that megakaryocytes and/or platelets were induced by tal1/scl cDNA transduction (data not shown). Because similar results were obtained for all four CM ESC lines, lentiviral-mediated tal1/scl transduction can efficiently and reproducibly induce hematopoiesis of CM ESCs (Fig. 3B).
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Figure 3. Efficient hematopoietic cell differentiation induced by expression of the tal1/scl gene. (A): Several hematopoietic genes, including tal1/scl, gata1, gata2, lhx2, and hhoxb4, were individually transduced into common marmoset (CM) embryonic stem (ES) cells. Overexpression of tal1/scl, but not any of the other genes, dramatically increased hematopoietic colony number. (B): Similar results were obtained for all four CM ES lines. (C): Kinetic colony-forming unit (CFU) assay revealed that hematopoietic cell induction occurred in tal1/scl-transduced EB cells within 12¨C25 days; the induction activity was the highest at day 18. (D): Several types of hematopoietic colonies, including CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (left upper), CFU-erythroid (right upper), CFU-granulocyte/macrophage (left lower), and CFU-macrophage (right lower), were obtained after tal1/scl transduction. (E): Genomic polymerase chain reaction demonstrated that the tal1/scl cDNA band was derived from the transduced lentiviral vector. Scale bars = 100 µm. Abbreviations: EB, embryoid body; GFP, green fluorescent protein.8 t, w0 h) S3 z! U1 |, D+ }
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Figure 4. Production of multilineage hematopoietic cells by tal1/scl gene transduction. (A): Hematopoietic colonies obtained by colony-forming unit (CFU) assay analyzed at the cellular level using May-Giemsa staining. (B): Hematopoietic colonies obtained by CFU assay analyzed at the cellular level using hemoglobin immunohistochemistry (left and middle panels) and double-esterase staining (right panel). Multilineage hematopoietic cells, including erythroblasts, granulocytes, and macrophages, were observed. The arrow indicates hemoglobin-positive cells in the bright field. (C): Semiquantitative reverse transcription-polymerase chain reaction analyses revealed increases in the expression of the erythroid markers gata1, glycophorin A, and three globin genes; the - (embryonic), - (fetal), and ß- (adult) globins were remarkably increased. All three globins were already expressed in the early stages of differentiation (day 4); however, - and ß-globin exhibited their respective highest expressions on days 7 and 10. At day 4, ß-globin expression in EB cells was reproducible and downregulated immediately. Scale bars = 100 µm. Abbreviation: EB, embryoid body.( c+ N! ]1 E+ n* k. [
1 L) V o% u" o0 g4 m; N) JTo analyze the temporal pattern of colony formation during EB differentiation, we performed CFU assays at days 12 and 20. At day 12, the majority of colony-forming cells were erythroid, whereas myeloid cells were obtained more frequently than erythroid cells at day 20 (Fig. 5A, B). These results are compatible with the pattern of hematopoietic development that occurs during embryogenesis, suggesting that normal hematopoiesis was induced in tal1/scl-expressing ESCs.4 {# y$ I- x/ Z6 n7 g
2 }' B8 r/ h6 F0 N4 ?* gFigure 5. Sequential analyses of colony formation during EB differentiation. To analyze the changes in hematopoietic cell populations during the course of EB differentiation, we performed colony-forming unit assays at days 12 and 20. At day 12, the majority of colony-forming cells contained erythroid cells, whereas myeloid cells were more abundant than erythroid cells at day 20. (A): Colony number and types. (B): Percentage of each lineage colony. Abbreviations: E, erythroid; EB, embryoid body; GM, granulocyte/macrophage; M, macrophage; Mix, granulocyte, erythrocyte, macrophage, megakaryocyte.
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Examination of Colony-Forming Ability in the Presence of Various Growth Factors
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To examine the effect of growth factors on CM ESC hematopoiesis, growth factors reported to play important roles during hematopoiesis (bFGF, BMP-4, activin-A, or VEGF ) were added to cultures at day 5 after EB formation. CFU assays of tal1/scl-induced EBs at day 12 showed that the presence or absence of these growth factors did not significantly affect either colony number or the variety of differentiated cell types that developed (Fig. 6A and data not shown). Addition of bFGF, however, did increase cell proliferation and doubled the total cell numbers (Fig. 6B). This result suggests that, although the development of HPCs and HSCs is potentiated by bFGF, the induction of hematopoiesis itself is not affected., Q% F. U$ [ Q2 c
" O5 J' i/ @6 a+ O) a" a: f5 nFigure 6. Examination of colony-forming ability in the presence of various growth factors. We added the growth factors bFGF, BMP-4, activin-A, and VEGF to the culture medium after transduction of the tal1/scl gene. (A): Colony-forming unit assays demonstrated that, for equivalent numbers of cells, neither the colony numbers nor the number of different cell types was significantly different in the presence or absence of these growth factors. (B): The addition of bFGF, but not any of the other factors, increased cell proliferation and doubled the total cell numbers. Abbreviations: bFGF, basic fibroblast growth factor; BMP-4, bone morphogenic protein-4; VEGF, vascular endothelial growth factor.
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Characterization of CD34 Cells from CM ESCs
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To examine the suitability of ESC-derived HPCs as potential sources for transplantation, we focused on CD34 cells, which are enriched in the HPC/HSC population of human bone marrow. We first analyzed the time course of production of CD34 cells from tal1/scl-transduced EB cells (Fig. 7A, B). Flow cytometry demonstrated that CD34 cells are most abundant on day 9, whereas few or no such cells are present on day 5. From days 9 to 20, CD34 cells consistently comprised approximately 2% of the total cells (Fig. 7B). Our previous report using the same CM anti-CD34 antibody demonstrated that only 0.5% of CM bone marrow cells were CD34 . These findings demonstrate that larger proportions of CD34 cells could be isolated from tal1/scl-transduced EB cells than from CM bone marrow. To compare their respective hematopoietic capabilities, we performed CFU assays of 103 CD34 cells derived either from bone marrow or from tal1/scl-transduced EB cells. These assays revealed that ESC-derived CD34 cells produced similar numbers of hematopoietic colonies as bone marrow cells (Fig. 7C, upper graph). EB-derived CD34 cells, however, generated colonies primarily consisting of macrophages or erythroid cells (Fig. 7C, lower graph), which were smaller in size (data not shown). Few hematopoietic colonies could be obtained from CD34¨C cells from either bone marrow or ESC-derived cell cultures. These results suggest that, because of their quantity, ESC-derived CD34 cells are likely to be excellent sources of HPCs, despite different hematopoietic activities than BM cells.
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! s7 Z! z. s7 y( C, W. E6 y" iFigure 7. Characterization of CD34 cells derived from CM EB cells. We analyzed the temporal pattern of CD34 cell production from tal1/scl-transduced EB cells. (A): Flow cytometric analyses demonstrated that CD34 cells were most abundant at day 9. (B): Afterward, they were consistently present at almost 2%. (C): To compare the hematopoietic ability of CD34 cells derived from bone marrow and tal1/scl-transduced EB cells, we performed colony-forming unit assays. Embryonic stem (ES)-derived CD34 cells were able to produce a similar number of hematopoietic colonies as those isolated from bone marrow (upper graph). The majority of the colonies derived from EB-derived CD34 cells, however, consisted primarily of macrophages or erythroid cells (lower graph). In contrast, few hematopoietic colonies could be obtained from CD34¨C cells isolated from either bone marrow or ES-derived cells. Abbreviations: BM, bone marrow; CM, common marmoset; E, erythroid; EB, embryoid body; GM, granulocyte/macrophage; M, macrophage; Mix, granulocyte, erythrocyte, macrophage, megakaryocyte.
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DISCUSSION AND SUMMARY( Q! x2 f5 z: ^6 H- X+ i5 {
( z$ G6 y. }' G; @) fIn this study, we developed a highly efficient and stable methodology to induce hematopoietic differentiation of CM ESCs. Tal1/scl, one of the master regulators of hematopoiesis from fish to mammals, is an essential gene for the development of hemangioblasts .! V) I9 V( y- J2 }$ v
" D7 P' c* r3 c6 O. GForced expression of other genes implicated in hematopoiesis, including gata1, gata2 . Given that CM ESCs did not naturally express tal1/scl during the early stages of EB development, tal1/scl gene transduction may be essential for the in vitro differentiation of CM ESC/EB cells into hematopoietic cells; the transduction of other genes, however, was not essential for hematopoietic cell induction.+ L- B1 X3 @4 a8 S
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Although coculture with OP9 stromal cells has been shown to induce the effective hematopoiesis of primate ESCs .4 N' m2 _' o( G3 j% C$ n5 O
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One important unresolved issue raised by this study is whether tal1/scl-induced EB cells can support the long-term reconstitution of hematopoietic lineages in vivo. In this in vitro study, multilineage hematopoietic cells, including granulocytes and adult-type erythroid cells expressing ß-globin, were observed; however, most of the hematopoietic colonies were small and contained primarily CFU-M or CFU-E, rather than CFU-GM and CFU-Mix (Figs. 5A and 7C), suggesting that only minimal definitive hematopoiesis occurred in EB-tal1/scl cells. Precise in vivo analyses to examine these important issues are currently under way. In this study, we used VSV-G LVs to induce exogenous gene expression. This methodology, however, carries some risk of insertional mutagenesis by the vector. Gene therapy using a retroviral vector led to an uncontrolled clonal T lymphoproliferative syndrome, similar to acute lymphoblastic leukemia (ALL), in SCID (severe combined immunodeficient)-X1 patients due to exogenous gene integration into the LMO-2 gene of T lymphocyte lineage cells , transduction of the tal1/scl gene using a lentiviral vector may carry significant risk. Therefore, it may be necessary to develop a transient expression system using adenoviral vectors capable of transducing target genes without genomic integration. In summary, gene transduction is a very powerful tool to induce hematopoietic differentiation of ESCs, which may prove to be an effective source for future cellular therapies.
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DISCLOSURES
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The authors indicate no potential conflicts of interest.) [" O: |1 z/ R; i/ @
0 D0 \& i ^+ Q4 }/ @- sACKNOWLEDGMENTS
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We thank Dr. Sumiko Watanabe (Institute of Medical Science, University of Tokyo), Dr. Chieko Kai (Institute of Medical Science, University of Tokyo), Dr. Shigetaka Asano (Waseda University, Tokyo), Dr. Yukio Nakamura (RIKEN BioResource Center), and Dr. Ken-ichi Arai (Tokyo Metropolitan Institute of Medical Science) for their helpful advice and support. This work was supported by grants from the Japan Society for the Promotion of Science, the Research for the Future Program, the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labor, and Welfare of Japan.
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