TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Thrombopoietin Enhances Generation of CD34 Cells from Human Embryonic Stem Cells ANAND S. SRIVASTAVA, a ELENA NEDELCU, b BABAK ESMAELI-AZAD, c RANGNATH MISHRA, d EWA CARRIER a a Moores UCSD Cancer Center, Department of Medicine, University of California San Diego, San Diego, California, USA; b Department of Pathology, University of California Los Angeles, Los Angeles, California, USA; c DNAmicroarray, Inc., San Diego, California, USA; d Division of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Key Words. Human embryonic stem cells CD34 progenitors Hematopoietic progenitors CD31 progenitors Disclosure of potential conflicts of interest is found at the end of this article. ABSTRACT 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 [SCF] and Flt3 ligand [Flt3L]); 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 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. STEM CELLS 2007;25: INTRODUCTION Thrombopoietin (TPO), a primary regulator of megakaryocyte and platelet production in vitro and in vivo, acts as a lineagespecific hematopoietic growth factor and exerts effects on early hematopoietic stem cells [1]. There are two lines of evidence supporting TPO in the survival and expansion of hematopoietic stem cells. The first line of evidence comes from in vitro and in vivo experimental studies. In vitro studies demonstrated that TPO supports survival of hematopoietic stem cells (HSCs) and stimulates their proliferation when added in the presence of interleukin 3 or steel factor to the cultures of purified populations of candidate stem cells [2]. TPO stimulates stem cells to generate colony-forming cells of all lineages. TPO not only supports the growth of megakaryocyte progenitors [3] but also enhances the proliferation of erythroid progenitors [4 6]. In vivo studies also point out the crucial role of TPO in the early development of hematopoietic stem cells; knockout mice in which genes coding for TPO or its receptor (c-mpl) were deleted had a profound reduction not only in the number of megakaryocytes and platelets but also in the number of committed myeloid progenitors of all types (primitive progenitors and hematopoietic stem cells). Therefore, it appears that TPO is not only a lineage-specific growth factor but also plays an important role in the proliferation and differentiation of hematopoietic stem cells [7]. Administration of recombinant TPO leads to a significant expansion of colony-forming units of all lineages in both normal and myelosuppressed mice [7, 8]. 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 5 years of life, suggesting that TPO has a role in early hematopoiesis in humans [9]. However, little is known about the role of TPO in the generation of hematopoietic stem cells from human embryonic stem (ES) cells. For the study of recapitulation of early hematopoietic events, the EB differentiation model is frequently used [10]. Previous studies have demonstrated that mouse-derived EB cells generated blast cell colonies and had hematopoietic and endothelial potential when cultured in endothelial cell-conditioned medium in the presence of vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) [11]. It is thought that the blast colony-forming cells, present in the mouse EBs for approximately 24 hours, represent the in vivo yolk sac progenitor of Correspondence: Ewa Carrier, M.D., Department of Medicine, Pediatrics and UCSD Moores Cancer Center, School of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. Telephone: ; Fax: ; ecarrier@ucsd.edu Received November 3, 2006; accepted for publication March 8, 2007; first published online in STEM CELLS EXPRESS March 22, AlphaMed Press /2007/$30.00/0 doi: /stemcells STEM CELLS 2007;25:

2 Srivastava, Nedelcu, Esmaeli-Azad et al dual hematopoietic and endothelial potential [12]. In another independent study, these vascular endothelial growth factor receptor 2 (VEGFR2)-positive progenitors generated blood islands composed of round clusters of hematopoietic cells surrounded by both endothelial and hematopoietic cells [13]. 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 [14, 15]. However, a recent report demonstrated that TPO regulates proliferation and differentiation of rhesus EBs to hematopoietic and endothelial progenitors without BMP4 but instead required VEGF, SCF, and Flt3L [16]. 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. It is known that addition of TPO increased formation of primitive hematopoietic progenitors in murine and primate EB differentiation models [16]; 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. MATERIALS AND METHODS Human ES Cell Culture Conditions Human ES (hes) cell lines (H1 cell line, NIH code WA01) were obtained from WiCell Research Institute (Madison, WI, wicell.org) [10]. hes cells were cultured on mitomycin-treated mouse embryonic fibroblast cells in Dulbecco s modified Eagle s medium (DMEM)/Ham s F-12 medium supplemented with 15% knockout serum replacement (Invitrogen, Carlsbad, CA, ng/ml of human recombinant basic fibroblast growth factor 2 (FGF2) (Invitrogen), 1% nonessential amino acids, and 1% L-glutamine. The cells were fed daily and passaged weekly with 0.05% trypsin, 0.53 mmol/l EDTA (Invitrogen) to maintain undifferentiated growth [17]. 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 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, 1% nonessential amino acids, 1 mm L-glutamine, and 0.1 mm -mercaptoethanol. EBs were cultured for 2 days at 37 C and 5% CO 2 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. Generation of CD34 Cells from EB-Derived Cells After 3 days of culture, DMEM was replaced by Iscove s modified Dulbecco s medium (Gibco-BRL, Gaithersburg, MD, 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 (TPOtreated 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, 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. Histochemical Analysis and Quantification of hes- Derived CD34 Cells 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, The slides were counterstained with hematoxylin and examined with an Olympus BH-2 microscope (Tokyo, 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. Analysis of CFU-C Obtained from Plating EB Cells Secondary differentiation cultures were set up with cells dispersed from 2-day-old EBs. Cells were washed, and EB-dispersed cells were plated on 35-mm methylcellulose dishes. Human clonogenic progenitor assay was performed by plating cells obtained from hes-derived EBs into methylcellulose as described by Chadwick et al. [17]. 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). 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, 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 C for 30 minutes and finally 94 C for 2 minutes, which was followed by a DNA amplification cycle. The reaction was set for denaturation at 94 C for 15 seconds, annealing at 58 C for 30 seconds, and extension at 72 C for 1 minute for 35 cycles followed by a final extension at 72 C for 5

3 1458 TPO Enhances Hematopoietic Differentiation 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, 20). minutes. The -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 ; -actin forward, 5 -CTGTCTGGCGGCACCACCAT-3 ; -actin reverse, 5 -GCAAC- TAAGTCATAGTCCGC-3. Analysis of Protein Expression of c-mpl and VEGFR by Western Blot 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-([3-cholamidopropyl]dimethylammonio)-1- propanesulfonate, 20 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml aprotinin, 5 mm dithiothreitol, 2 mm sodium pyrophosphate, 1mMNa 3 VO 4, and 1 mm NaF, and centrifuged at 2,000g for 10 minutes at 4 C. Protein content in the supernatant was assayed with a DC protein assay (Bio-Rad, Hercules, CA, com). An aliquot of the lysate (20 g of protein) was boiled with SDS sample buffer, resolved on a 4% 12% SDS-polyacrylamide gel electrophoresis gradient gel, and transferred to a 0.2- m 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 C 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. Statistical Analysis Statistical analysis was performed using the SPSS statistical package, version 9.0 (SPSS, Inc., Chicago, To analyze differences in mean values between groups, a two-sided unpaired Student s t test was used. RESULTS 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 Figure 2. Number of CFU-C colonies in three different culture conditions. Number of CFUs obtained from 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 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 05). Abbreviations: TPO, thrombopoietin; VEGF, vascular endothelial growth factor. NIH-approved hes cell line H1 (code WA01). Human ES cells were maintained in the undifferentiated state by mitomycintreated 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 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 EBs per hes-plated cells were counted. In culture condition 1, which contained SCF and Flt3L only, the average number of CFU-C per 10 5 hes cells plated was 5. In culture condition 2, in which TPO was added, the mean number of CFU-C was 116 (p.001). In culture condition 3, TPO and VEGF were added together with the SCF and Flt3L, and a mean of 153 CFU-C per 10 5 cells plated were obtained (p.001), suggesting that TPO and VEGF exerted a synergistic effect on the formation of hematopoietic progenitors (Fig. 2); differences between cultures 2 and 3 were statistically significant (p.05). The first evidence of well-formed CFU-C was detected at day 8 of differentiation, which correlated with the expression of c-mpl and VEGFR. Formation of Early Hematopoietic Colonies Secondary differentiation cultures were set up with cells dispersed from 2-day-old EBs. EB-derived cells were washed, and 10 5 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. [17]. 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), colonyforming units-granulocyte-macrophage (CFU-GM) (disperse

4 Srivastava, Nedelcu, Esmaeli-Azad et al 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 EB-dispersed cells were plated on 35-mm methylcellulose dishes. Human clonogenic progenitor assay was performed by plating cells obtained from human ESderived EBs into methylcellulose as described by Chadwick et al. [17]. 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 unitsgranulocyte-macrophage (disperse colonies with translucent cells), and CFU-E were detected. colonies with translucent cells), and CFU-E were detected (Fig. 1). 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. [18], 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. 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 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. 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. TPO-Upregulated mrna Expression of VEGFR and c-mpl During Formation of CD34 Cells 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 8 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

5 1460 TPO Enhances Hematopoietic Differentiation Figure 5. Quantification of the human embryonic stem (hes) cellderived 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). 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% 15% 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). mechanism. This process was independent of BMP4, which has been shown in other reports to effectively enhance human ES-derived hematopoiesis [17], but correlated with a recent report on nonhuman primate ES cells in which TPO and VEGF induced early erythroid differentiation [16]. 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 Figure 7. TPO-R and VEGFR gene and protein expression in EBderived 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. -Actin was used as internal control. Lanes 1 5 show expression of TPO-R; lanes 6 10 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 7 (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. 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. [18], formation of megakaryocytes from human ES cells occurs later in secondary differentiation culture. DISCUSSION 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 [16]; however, its effect on the generation of CD34 cells from human ES cells has not been established. In this report, we provide experimental evidence supporting hypothesis that TPO has a positive effect on the

6 Srivastava, Nedelcu, Esmaeli-Azad et al survival and expansion of early hematopoietic progenitors derived from human ES cells and that c-mpl receptor plays a role in this process [19, 20]. It is known that patients with congenital amegakaryocytic thrombocytopenia, who have TPO-R mutations, develop not only thrombocytopenia but also pancytopenia [21, 22], suggesting that TPO not only acts as the main regulator of megakaryopoesis but also exerts its effect on early hematopoietic progenitors. 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 [10]. Similarly, other researchers efficiently induced differentiation of hes cells into CD34 cells by coculturing them with the OP9 mouse bone marrow stromal cell line [23]. It is also well-established that the TPO-R is present on primitive hematopoietic progenitors [4, 24] but not on undifferentiated human ES cells. 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 [4]. However, the question remains why megakaryocytes were not formed in those cultures in which TPO was used as a main additive cytokine. In a recent report by Gaur et al., the formation of megakaryocytes from human ES cells was obtained using OP9 stroma-enhanced cultures [18]. TPO alone was not sufficient for the induction of megakaryocytes, and other cytokines, such as IL-6 and IL-11, were required in addition to TPO for megakaryocyte formation. The mrna expression of both c-mpl and VEGFR was significantly upregulated in TPO- and TPO/VEGF-stimulated cell cultures versus cultures containing SCF and Flt3L, suggesting synergy between these cytokines. No expression of c-mpl and VEGF was documented in the control cultures, indicating that TPO and VEGF were essential in promoting expression of their respective receptors, as well as formation of early hematopoietic colonies through these receptors. However, the mechanisms responsible for these effects are not known. TPO-R is one of the important members of the so-called homodimerizing cytokine receptor subfamily that is known to activate many of the same pathways as erythropoietin receptor, JAK2, STAT5, mitogenactivated protein kinase, PI3 kinase [4, 24]. Our findings suggest that TPO may initially act through a different, common receptor on hes cells in preparation for a more specific signaling through its own receptor, when definite hematopoietic progenitors are formed. It is also possible that the hes cells decode the signals transmitted generically through cytokine receptors in similar manner or that TPO is part of the cytokine network regulating the proliferation and differentiation of hes cell-derived uncommitted progenitors [25]. 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. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Sitnicka E, Lin N, Priestley GV et al. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood 1996;87: Kaushansky K, Lok S, Holly RD et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-mpl ligand thrombopoietin. Nature 1994;369: Lok S, Kaushansky K, Holly RD et al. Cloning and expression of murine thrombopoietin cdna and stimulation of platelet production in vivo. Nature 1994;369: Kobayashi M, Laver JH, Kato T et al. Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors. Blood 1995;86: Kaushansky K, Broudy VC, Grossmann A et al. Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest 1995; 96: Kaushansky K, Lin N, Grossmann A et al. Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice. Exp Hematol 1996;24: Alexander WS, Roberts AW, Nicola NA et al. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-mpl. Blood 1996; 87: Carver-Moore K, Broxmeyer HE, Luoh SM et al. Low levels of erythroid and myeloid progenitors in thrombopoietin-and c-mpl-deficient mice. Blood 1996;88: Germeshausen M, Schulze H, Gaudig A et al. Congenital amegakaryocytic thrombocytopenia (CAMT): A defect of the thrombopoietin receptor c-mpl]. Klin Padiatr 2001;213: Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colonyforming cells derived from human embryonic stem cells. Proc Natl Acad SciUSA2001;98: Jaffredo T, Bollerot K, Sugiyama D et al. Tracing the hemangioblast during embryogenesis: Developmental relationships between endothelial and hematopoietic cells. Int J Dev Biol 2005;49: Yamashita JK. Differentiation and diversification of vascular cells from embryonic stem cells. Int J Hematol 2004;80: Ema M, Faloon P, Zhang WJ et al. Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes Dev 2003;17: Cerdan C, Rouleau A, Bhatia M. VEGF-A165 augments erythropoietic development from human embryonic stem cells. Blood 2004;103: 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: Wang Z, Skokowa J, Pramono A et al. Thrombopoietin regulates differentiation of rhesus monkey embryonic stem cells to hematopoietic cells. Ann N Y Acad Sci 2005;1044: Chadwick K, Wang L, Li L et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 2003;102: Gaur M, Kamata T, Wang S et al. Megakaryocytes derived from human embryonic stem cells: A genetically tractable system to study megakaryocytopoiesis and integrin function. J Thromb Haemost 2006;4: Methia N, Louache F, Vainchenker W et al. Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993;82: Ku H, Yonemura Y, Kaushansky K et al. Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. Blood 1996;87: Ballmaier M, Germeshausen M, Schulze H et al. c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood 2001;97: Ballmaier M, Germeshausen M, Krukemeier S et al. Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: Development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia. Ann N Y Acad Sci 2003;996: Vodyanik MA, Bork JA, Thomson JA et al. Human embryonic stem cell-derived CD34 cells: Efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 2005;105: Borge OJ, Ramsfjell V, Cui L et al. Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34. Blood 1997;90: Watowich SS, Wu H, Socolovsky M et al. Cytokine receptor signal transduction and the control of hematopoietic cell development. Annu Rev Cell Dev Biol 1996;12: