Thrombopoietin Enhances Generation of CD34 Cells from Human Embryonic Stem Cell - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:53 |只看该作者 |倒序浏览 |打印

作者：Anand S. Srivastavaa, Elena Nedelcub, Babak Esmaeli-Azadc, Rangnath Mishrad, Ewa Carriera作者单位：aMoores UCSD Cancer Center, Department of Medicine, University of California San Diego, San Diego, California, USA;bDepartment of Pathology, University of California Los Angeles, Los Angeles, California, USA;


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      【摘要】
   The role of thrombopoietin (TPO) in adult hematopoiesis is well-established. A recent report suggests that TPO and vascular endothelial growth factor (VEGF) play a role in promoting formation of early erythropoietic progenitors in a nonhuman primate embryonic stem cell (ES) model. No such report exists for human ES cells as yet. Because TPO may become an important factor promoting human ES cell-derived hematopoiesis, we sought to investigate whether TPO in combination with VEGF can enhance human ES-derived hematopoiesis in an EB-derived culture system. The emphasis of this work was to demonstrate the molecular mechanisms involved in this process, specifically the role of c-mpl and its ligand TPO. Human ES cells were cultured to the EB state, and EB-derived secondary cultures supporting hematopoietic differentiation were established: condition 1, control (stem cell factor ); condition 2, SCF, Flt3L, and TPO; and condition 3, SCF, Flt3L, TPO, and VEGF. Cells were harvested daily, starting at day 2 and continuing until day 8, for reverse transcription-polymerase chain reaction and Western blot. There was no evidence of expression of c-mpl and VEGF receptor on the gene or protein level until day 8, when the formation of well-established hematopoietic colonies began. This correlated with the formation of CD34 /CD31¨C negative progenitors, mostly found in blast-forming units-erythroid-like colonies. We concluded that TPO and VEGF play an important synergistic role in the formation of early ES-derived hematopoietic progenitors that occurs through the c-mpl and VEGF receptors.- e: r9 |7 ?+ G' ^% U+ m5 Y

Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Human embryonic stem cells CD  progenitors Hematopoietic progenitors CD  progenitors
   INTRODUCTION. r3 f% b" z; P4 O5 ?) ^; n

Thrombopoietin (TPO), a primary regulator of megakaryocyte and platelet production in vitro and in vivo, acts as a lineage-specific hematopoietic growth factor and exerts effects on early hematopoietic stem cells .( G/ D; J) S' i4 }
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The second line of evidence comes from clinical studies of patients with amegakaryocytic thrombocytopenia. Recently, it was shown that this disease is caused by the mutations in the c-mpl gene, leading to the unresponsiveness of hematopoietic stem cells to the role of TPO. All these patients developed pancytopenia within the first 3¨C5 years of life, suggesting that TPO has a role in early hematopoiesis in humans . However, little is known about the role of TPO in the generation of hematopoietic stem cells from human embryonic stem (ES) cells.  W5 ]; t$ T7 k0 D# Y# v9 ^

For the study of recapitulation of early hematopoietic events, the EB differentiation model is frequently used .' C' x1 `( v' w# Z" L- @; E* `+ b
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Previous studies have shown that hematopoietic differentiation of human ES cells using the EB differentiation model requires bone morphogenic protein (BMP4) in combination with a few other cytokines, such as stem cell factor (SCF), Flt3 ligand (Flt3L), IL-3, IL-6, and granulocyte macrophage-colony stimulating factor . TPO alone was not able to stimulate formation of hematopoietic and endothelial progenitors, whereas the addition of VEGF enhanced their generation. In these conditions, the mRNA expression of c-mpl was also significantly upregulated, allowing for enhanced responsiveness of early hematopoietic and endothelial progenitors to TPO.  G5 O4 m4 ^6 e/ h- N4 O8 e  \6 r

It is known that addition of TPO increased formation of primitive hematopoietic progenitors in murine and primate EB differentiation models ; however, no such report currently exists for human ES cells. Since EB-derived hematopoiesis represents a good system model with which to study in vitro early hematopoietic events in humans, investigating the role of TPO and VEGF and their respective receptors on early hematopoietic events may enhance future protocols of blood formation from human ES cells.9 j. J: g8 K# ~4 ^: C, P
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MATERIALS AND METHODS& Z3 x# H, O! Z: s

Human ES Cell Culture Conditions3 I( `2 _# [0 p
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Human ES (hES) cell lines (H1 cell line, NIH code WA01) were obtained from WiCell Research Institute (Madison, WI, http://www.wicell.org) .
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EB Formation

On the day of passage, the ES cells were treated with 200 U/ml collagenase IV (Invitrogen), counted with a hemocytometer, and transferred at a density of 2 x 106 cells per well to a low-attachment plates to allow EB formation by incubation in knockout DMEM supplemented with 20% non-heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, http://www.hyclone.com), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM ¦Â-mercaptoethanol. EBs were cultured for 2 days at 37¡ãC and 5% CO2 in a humidified atmosphere for 48 hours. The number of hEBs was counted with a hemocytometer prior to transfer into a secondary medium conducive to hematopoietic differentiation. EB cells mechanically dispersed or obtained by dissociation with collagenase were used for the generation of CD34  cells, colony-forming units (CFU)-C assays, and molecular studies for mRNA expression of TPO-R (c-mpl) and VEGFR.0 [' s4 e/ Q( I& B# X' A, [) i3 [8 E

Generation of CD34  Cells from EB-Derived Cells8 ^" l. u9 N* F! d# J

After 3 days of culture, DMEM was replaced by Iscove's modified Dulbecco's medium (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) with the same supplements and two additional cytokines (100 ng/ml SCF and 100 ng/ml Flt3L). This culture condition represented the control group. To investigate the role of TPO and VEGF, additional treatment with 100 ng/ml TPO alone (TPO-treated group) or in combination with 100 ng/ml recombinant human VEGF (TPO/VEGF-treated group) was performed. All cytokines were from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Cytospin preparations were performed after 8 days of culture from these culture conditions, and the CD34  cells were detected by immunohistochemistry as described below. To detect the formation of double endothelio-hematopoietic and endothelial progenitors, CD31  staining was performed on all examined slides, which were read by primary and secondary (blinded) reviewers and compared against positive and negative controls.
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Histochemical Analysis and Quantification of hES-Derived CD34  Cells! B. T9 `/ v+ [, l) h
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At day 8 of secondary differentiation culture, cells were collected, washed, and used for m-RNA, CFU-C assay, and histochemical staining. Indirect immunohistochemistry was performed on cytospin preparations of cell cultures with TPO, TPO/VEGF, and control cultures (SCF and Flt3L). The antibodies used were rat anti-human CD34 and CD31 primary antibody and biotin-labeled anti-rat secondary antibody in a streptavidin horseradish peroxidase detection system. All antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). The slides were counterstained with hematoxylin and examined with an Olympus BH-2 microscope (Tokyo, http://www.olympus-global.com), photographs were taken with an Olympus DP11 camera, and membrane staining was correlated with cell morphology. Each group of cells was examined for the presence of cells with CD34  and CD31  membranous stain by a trained hematopathologist and a blinded second reader. The intensity of the staining was scored as follows: 1, weak; 2, moderate; 3, strong. The frequency of CD34  and CD31  cells was recorded for all staining intensities. The mean positivity index was calculated as a mean of all products between frequency of the CD34  and CD31  cells and corresponding staining intensity and correlated with morphology.
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Analysis of CFU-C Obtained from Plating EB Cells2 a. T% p/ \8 [; Q, M( l

Secondary differentiation cultures were set up with cells dispersed from 2-day-old EBs. Cells were washed, and 1 x 105 EB-dispersed cells were plated on 35-mm methylcellulose dishes. Human clonogenic progenitor assay was performed by plating 1¨C2 x 105 cells obtained from hES-derived EBs into methylcellulose as described by Chadwick et al. . Specific culture conditions were as follows: condition 1, SCF and Flt3L (control); condition 2, SCF, Flt3L, and TPO; and condition 3, SCF, Flt3L, TPO, and VEGF. The hematopoietic colonies were scored every second day up to 8 days and were considered positive when they were composed of 50 cells and exhibited typical CFU morphology. Quantitative comparisons were made between the control (e.g., SCF and Flt3L only) and each experimental condition (e.g., TPO with or without VEGF) with regard to the number of CFU derived per 100 cells from dissociated EB-derived cells (Fig. 1).

Figure 1. Generation of EB from human embryonic stem (ES) cells. To achieve EB formation, human ES cells were incubated in the Dulbecco's modified Eagle's medium without fibroblast growth factor 2 for 48 hours. Human ES cells formed the typical round EBs shown in photograph (phase contrast; magnification, x20).
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Analysis of mRNA Expression of c-mpl and VEGFR by Reverse Transcription-Polymerase Chain Reaction

To detect the kinetics of expression of TPO and VEGF receptors (c-mpl and VEGFR, respectively) on cells subjected to three different culture conditions, cells were isolated daily from day 2 to day 8 of secondary differentiation cultures, and total RNA was isolated from these cells et each time point using an RNeasy Mini Kit according to the manufacturer's protocol (Qiagen, Valencia, CA, http://www1.qiagen.com). Five hundred nanograms of total RNA was subjected to study c-mpl and VEGR gene expression using a single-step reverse transcription-polymerase chain reaction (RT-PCR) kit (Invitrogen). The reaction was set for cDNA synthesis and predenaturation for one cycle at 55¡ãC for 30 minutes and finally 94¡ãC for 2 minutes, which was followed by a DNA amplification cycle. The reaction was set for denaturation at 94¡ãC for 15 seconds, annealing at 58¡ãC for 30 seconds, and extension at 72¡ãC for 1 minute for 35 cycles followed by a final extension at 72¡ãC for 5 minutes. The ¦Â-actin gene was used as an internal control. The following nucleotide sequences were used for primers: VEGFR forward, 5'-AGCCCAGATTCTCCAGCCTGACTCGG-3'; VEGFR reverse, 5'-TGGGGCCATTGCTTGAAGCTCTTTGTTC-3'; c-mpl forward, 5'-TGCCCTGCTTCTGCAGAGGCCTCACT-3'; c-mpl reverse, 5'-CAGCACCGTGCCCTGCTGTGGTA-3'; ¦Â-actin forward, 5'-CTGTCTGGCGGCACCACCAT-3'; ¦Â-actin reverse, 5'-GCAACTAAGTCATAGTCCGC-3'.

Analysis of Protein Expression of c-mpl and VEGFR by Western Blot( E: F* I; h1 P$ w% S# M

Cells were isolated daily from day 2 to day 8 of secondary differentiation cultures for protein analysis by Western blot. Cell homogenates (15% wt/vol) were prepared in extraction buffer composed of 50 mM PIPES (1,4-piperazinediethanesulfonic acid)/HCl, pH 6.5, 2 mM EDTA, 0.1% 3-(dimethylammonio)-1-propanesulfonate, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 5 mM dithiothreitol, 2 mM sodium pyrophosphate, 1 mM Na3VO4, and 1 mM NaF, and centrifuged at 2,000g for 10 minutes at 4¡ãC. Protein content in the supernatant was assayed with a DC protein assay (Bio-Rad, Hercules, CA, http://www.bio-rad.com). An aliquot of the lysate (20 µg of protein) was boiled with SDS sample buffer, resolved on a 4%¨C12% SDS-polyacrylamide gel electrophoresis gradient gel, and transferred to a 0.2-µm nitrocellulose membrane. After blocking in 5% nonfat dry milk in Tris-buffered saline/Tween 20 (TBS-T) (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 hour, the membrane was washed three times for 5 minutes each time with TBS-T and incubated overnight at 4¡ãC with anti-human VEGF R2 monoclonal antibody (1:1,000; R&D Systems) or anti-human TPO-R antibody (1:1,000; R&D Systems) in 3% bovine serum albumin in TBS-T. After incubation with a suitable HRP-labeled secondary antibody (1:2,000) and extensive washing, the membrane was exposed to film with an average exposure duration ranging from 10 to 30 seconds.. d+ m) v; X2 ^! K- Z8 ]1 ]
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Statistical Analysis2 m& F9 \  v( k5 h4 H
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Statistical analysis was performed using the SPSS statistical package, version 9.0 (SPSS, Inc., Chicago, http://www.spss.com). To analyze differences in mean values between groups, a two-sided unpaired Student's t test was used.
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RESULTS! ]$ _" O9 K6 @/ n0 D8 t7 \
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Human ES Cell Cultures

We have established in vitro cultures of hES cells to study molecular mechanisms involved in the formation of early hematopoietic progenitors. These experiments were done using NIH-approved hES cell line H1 (code WA01). Human ES cells were maintained in the undifferentiated state by mitomycin-treated murine feeder layer and serum-free media (Invitrogen) as described in Materials and Methods. In this culture condition, we have obtained stable proliferating hES cells, which formed typical, well-defined hES cell colonies (data not shown).

Embryoid Body Formation
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To achieve EB formation, hES cells were incubated in DMEM without FGF2 for 48 hours. hES cells formed typical, round EBs (data not shown). The efficiency of plating was very high, and 122 ¡À 18 EBs per 1 x 10 5 hES-plated cells were counted. In culture condition 1, which contained SCF and Flt3L only, the average number of CFU-C per 105 hES cells plated was 5. In culture condition 2, in which TPO was added, the mean number of CFU-C was 116 (p , g( z/ p) C. P# c; f( U# D
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Figure 2. Number of CFU-C colonies in three different culture conditions. Number of CFUs obtained from 1 x 10 5 human embryonic stem (hES) cells cultured in secondary differentiation media: left column, control media (SCF Flt3L); middle column, TPO added; right column, TPO and VEGF added. Results are presented as mean ¡À SEM. Significant differences were determined by Student's t test. Data show that TPO with VEGF significantly enhanced differentiation of hES into hematopoietic progenitors (*, p / {& v$ c4 M& k2 O, B+ x

Formation of Early Hematopoietic Colonies
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Secondary differentiation cultures were set up with cells dispersed from 2-day-old EBs. EB-derived cells were washed, and 105 EB cells were plated on 35-mm methylcellulose-coated dishes. The condition media used in addition to standard media had SCF and Flt3L (control), TPO alone (medium 2), or TPO and VEGF (medium 3). Since we intended to study molecular mechanisms involved in the early hematopoietic events, CFU-C were scored daily up to day 8 using the method described by Chadwick et al. . The first evidence of well-formed CFU-C colonies was detected at day 8 of differentiation, which coincided with the expression of c-mpl (TPO receptor) and VEGFR, indicating their role in the formation of hematopoietic progenitors. The standard blast-forming units-erythroid (BFU-E) morphology (clustered, large colonies with brown pigment), colony-forming units-granulocyte-macrophage (CFU-GM) (disperse colonies with translucent cells), and CFU-E were detected (Fig. 1).' K, E( \/ ^5 A: m, L8 v  Y9 Z
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Figure 3. Generation of colony-forming unit (CFU)-C colonies from human embryonic stem (ES) cells. Secondary differentiation cultures were set up with cells dispersed from 2-day-old EBs (48 hours in culture). Cells were washed, and 1 x 105 EB-dispersed cells were plated on 35-mm methylcellulose dishes. Human clonogenic progenitor assay was performed by plating 1¨C2 x 105 cells obtained from human ES-derived EBs into methylcellulose as described by Chadwick et al. . The first evidence of well-formed CFU-C colonies was detected at day 8 of differentiation, which coincided with the expression of c-mpl (thrombopoietin receptor) and vascular endothelial growth factor receptor, indicating their role in the formation of hematopoietic progenitors. The standard blast-forming units-erythroid morphology (clustered, large colonies with brown pigment; data not shown), colony-forming units-granulocyte-macrophage (disperse colonies with translucent cells), and CFU-E were detected.& D& j( U  A' r# z  A1 d. n  `7 f7 q! O
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Eighty percent of the CFU-C colonies corresponded to BFU-E morphology (large colonies composed of blast-like cells with reddish-brown pigment), although CFU-GM and CFU-E colonies were observed as well. No distinct megakaryocyte colonies were noted in these cultures, indicating that TPO alone is not sufficient for their formation. In the recent report by Gaur et al. , additional cytokines, such as IL-6 and IL-11, were required in addition to TPO for the formation of definite megakaryocytes, and longer incubation time was required.7 U& B  {/ Q+ `3 H

Quantitation of EB-Derived CD34-Positive Cells by Immunohistochemistry

In this study, we wanted to correlate early hematopoietic differentiation with molecular events related to c-mpl and VEGR expression. The expression of the c-mpl receptor and VEGFR was tested daily by RT-PCR from day 2 to day 8 with no evidence of expression of their respective genes and proteins until day 8, suggesting that these receptors are involved in hematopoietic differentiation but not in early differentiation events. The CD31 staining in addition to CD34 staining was performed, with no evidence of CD31 staining, indicating that hematopoietic but not endothelial progenitors were formed in these culture conditions (TPO and VEGF). All slides were reviewed by primary and secondary (blinded) reviewers and discussed with a hematopathologist familiar with this staining. Microscopic examination revealed groups of cells positive for CD34 membranous staining but with different stain intensity. Weak CD34 staining (grade 1) was encountered in larger cells with more abundant cytoplasm and small nuclei, whereas strong staining (grade 3) was observed in smaller cells with a higher nuclear/cytoplasmic ratio (Fig. 4). There was no positive membrane staining for CD31, indicating that in these culture conditions (SCF, IL-3, TPO, and VEGF), endothelial progenitors were not formed. The percentage of cells positively staining for CD34 and the mean positivity index calculated as a mean of all products between frequency of the CD34  cells and corresponding staining intensity were both higher in cytospin preparation obtained from TPO and TPO/VEGF (15% vs. 5% and 10% vs. 3%, respectively), suggesting that TPO/VEGF culture conditions are inductive to the generation of ES-derived CD34  cells (Fig. 5). The two distinct morphologies observed may represent early erythropoietic progenitors (large cells with weak CD34  staining) and hematopoietic stem cells (small cells with strong CD34  staining), corresponding with the formation of CFU-C colonies. The percentage of staining cells for each culture condition is shown in Figure 5, and the intensity of staining is shown in Figure 6.9 K5 x. e0 D! f# G- w1 v

Figure 4. CD34 staining in cells obtained from hematopoietic colonies. CD34  cells derived from human embryonic stem cells showed staining of different intensities. Illustration of the intensity scoring system used for the CD34-positive cells: top left, negative control; top right, weak staining intensity (score 1); bottom left, moderate staining intensity (score 2); bottom right, strong staining intensity (score 3). Weak CD34 staining (score 1) was encountered in larger cells with more abundant cytoplasm and small nuclei, whereas strong staining (score 3) was observed in smaller cells with higher nuclear/cytoplasmic ratios. There was no positive membrane staining for CD31 indicating that in these culture conditions (SCF, interleukin 3, thrombopoietin, and vascular endothelial growth factor), endothelial progenitors were not formed.

Figure 5. Quantification of the human embryonic stem (hES) cell-derived CD34  cells by the mean positivity index. The hES-derived CD34  cells were cultured with SCF and Flt3L (column 1, control cultures), thrombopoietin (TPO) alone (column 2), and both vascular endothelial growth factor (VEGF) and TPO (column 3). The highest number of CD34-positive cells was observed in the presence of cultures with TPO and both VEGF and TPO (in addition to SCF and Flt3L).( N+ _0 s0 U: j3 ?, x" H

Figure 6. Staining intensity of CD34  cells in three different culture conditions. Percentage of cells displaying strong, moderate, and weak staining was determined. Dotted columns, weak staining; solid columns, moderate staining; striped columns, strong staining. Set 1, thrombopoietin (TPO); set 2, TPO and vascular endothelial growth factor (VEGF); set 3, control (SCF and Flt3L). As shown, culture condition 2 (TPO and VEGF) induced 10%¨C15% of cells with strong CD34 staining. These cells showed specific morphology, displaying small size and a high nuclear/cytoplasmic ratio, suggesting generation of hematopoietic progenitors. None of these cells displayed CD31 staining (data not shown).- v0 X/ W. D+ B1 ?' R1 K9 r
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TPO-Upregulated mRNA Expression of VEGFR and c-mpl During Formation of CD34  Cells  f' \4 ?: n' n0 z  I3 n2 @" n% x: @
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To investigate the mechanisms through which TPO alone or in combination with VEGF induced differentiation of hES cells to CD34  progenitors, we analyzed mRNA expression of c-mpl and VEGFR from RNA obtained on days 2¨C8 of secondary differentiation cultures. Three culture conditions were established: condition 1, SCF and Flt3L (control); condition 2, SCF, Flt3, and TPO; and condition 3, SCF, Flt3, TPO, and VEGF. RT-PCR analysis showed no expression of c-mpl and VEGFR in progenitors up to seven days in secondary differentiation culture (data not shown). This correlated with the poor formation of CFU-C colonies prior to day 8 and no evidence of megakaryocyte formation. In our culture conditions (TPO and VEGF), the first transition occurred at day 8, when we observed formation of typical CFU-C colonies with predominantly BFU-E morphology, suggesting early erythropoiesis, induced by the combination of TPO and VEGF through their respective receptors. This was correlated with the expression of c-mpl and VEGFR as demonstrated by the RT-PCR (Fig. 7A) and Western blot analysis (Fig. 7). Progenitors obtained from these colonies expressed CD34 but no CD31 staining, indicating that at day 8 of differentiation, only hematopoietic progenitors were formed. Thus, it appears that in human EB-derived cultures, signaling through TPO receptor (c-mpl) activates signaling pathways involved in early hematopoiesis and that VEGF enhances this mechanism. This process was independent of BMP4, which has been shown in other reports to effectively enhance human ES-derived hematopoiesis .

Figure 7. TPO-R and VEGFR gene and protein expression in EB-derived embryonic stem (ES) cell culture. (A): TPO-R and VEGFR gene expression during hematopoietic differentiation of ES cells exposed to different culture conditions by reverse transcription-polymerase chain reaction and exposed to different cytokines. mRNA was collected after 8 days of secondary differentiation culture of human embryonic stem (hES) cells. Each sample was subjected to TPO-R and VEGFR gene expression studies. ¦Â-Actin was used as internal control. Lanes 1¨C5 show expression of TPO-R; lanes 6¨C10 show expression of VEGFR. Lane 1, no cytokines; lane 2, Flt3 and SCF; lane 3, thrombopoietin (TPO), SCF and Flt3; lane 4, vascular endothelial growth factor (VEGF); lane 5, VEGF, TPO, SCF, and Flt3; lane 6, no cytokines; lane 7, Flt3 and SCF; lane 8, TPO; lane 9, VEGF, SCF, and Flt3; lane 10, VEGF and TPO. (B): Expression of TPO-R (c-mpl) protein in hES cells at day 8 in different differentiation media by Western blot. Upregulation of these receptors was observed in cultures with TPO, which was further enhanced when VEGF was added. Studies on progenitors prior to 8 days did not show expression of TPO-R and VEGFR (data not shown), which suggests that this signaling pathway operates later in the hematopoietic development. Lane 1, Flt3 and SCF; lane 2, TPO; lane 3, VEGF; lane 4, TPO and VEGF. (C): Expression of the VEGFR protein in hES cells at day 8 in different differentiation media. As in eight a, expression of VEGFR protein was noted on day 8, when the first colony-forming unit-C colonies were formed. No evidence of expression was noted on days 2¨C7 (data not shown). Lane 1, hES in Flt3 and SCF (control); lane 2, TPO; lane 3, VEGF; lane 4, TPO and VEGF. Abbreviations: TPO-R, thrombopoietin receptor; VEGFR, vascular endothelial growth factor receptor.
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Western Blot Analysis of c-mpl and VEGFR Proteins in Different ES Cell Culture Conditions

To study expression of c-mpl and VEGFR on a protein level, Western blot analysis was performed on the progenitors obtained from day 2 to day 8 of secondary differentiation culture in three different conditions as described in Materials and Methods. No expression of c-mpl (Fig. 7B) or VEGFR (Fig. 7C) protein was noted up to day 8, suggesting that signaling through these receptors is initiated at the onset of early hematopoiesis (day 8), whereas signaling through receptors other than c-mpl and VEGF receptors may take place in early differentiation events, such as the hematoendothelial progenitor, sometimes referred to as hemangioblast. Upregulation of these receptors was observed in cultures with TPO, which was further enhanced with VEGF. No c-mpl or VEGFR proteins were detected in culture without these cytokines, indicating that signaling through these receptors in human EB-derived secondary cultures requires both TPO and VEGF. We did not observe megakaryocyte formation in these cultures, which were devoid of IL-6 and IL-11. According to recent report by Gaur et al. , formation of megakaryocytes from human ES cells occurs later in secondary differentiation culture.0 R0 f7 Y9 O7 E  f
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DISCUSSION/ m) ~5 a2 o) u5 r5 Z$ q
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The role of TPO on hematopoietic differentiation is well-established in the in vitro and in vivo systems, but its role in the human ES-derived hematopoiesis is not known. It was previously shown that TPO promotes hematopoietic differentiation of rhesus monkey ES cells , suggesting that TPO not only acts as the main regulator of megakaryopoesis but also exerts its effect on early hematopoietic progenitors.6 ~/ W, y. h) B$ t6 y, m* s
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In previous studies, the generation of CD34  cells from human ES cells was sixfold enhanced by addition of BMP4 to a mixture of cytokines such as SCF, Flt3L, IL-3, IL-6, and granulocyte-colony-stimulating factor in differentiation media  but not on undifferentiated human ES cells.
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As the number of CFU-C colonies expanded with TPO and VEGF and most of the colonies found had BFU-E morphology, we hypothesized that the TPO and VEGF combination, in addition to standard hematopoietic cytokines, enhances production of early erythroid progenitors . These hypotheses are currently being tested in our laboratory. We demonstrate for the first time that TPO and VEGF induce early hematopoietic differentiation of EB-derived human ES cells and that c-mpl is involved in this process. Understanding of specific culture conditions and signaling pathways will allow optimization of the in vitro differentiation of hES cells toward hematopoietic progenitors and future ES-derived blood products.$ L# R: H/ h4 O7 R- n- }% b+ T
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST' t7 v/ {: b# Q$ g

The authors indicate no potential conflicts of interest.. A, e  \# ?/ X8 \7 a8 E
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