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First published online January 12, 2006
Stem Cells Vol. 24 No. 5 May 2006, pp. 1348 -1358
doi:10.1634/stemcells.2005-0165; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS

Identification and Characterization of Hemoangiogenic Progenitors During Cynomolgus Monkey Embryonic Stem Cell Differentiation

Katsutsugu Umedaa, Toshio Heikea, Momoko Yoshimotoa, Gen Shinodaa, Mitsutaka Shiotaa, Hirofumi Suemorib, Hong Yuan Luoc, David H. K. Chuic, Ryuzo Toriid, Masabumi Shibuyae, Norio Nakatsujif, Tatsutoshi Nakahataa

a Department of Pediatrics, Graduate School of Medicine,
b Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences,
f Department of Development and Differentiation, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan;
c Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA;
d Research Center for Animal Life Science, Shiga University of Medical Science, Ohtsu, Japan;
e Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Key Words. Embryonic stem cell • Primate • Hemangioblast • Vascular endothelial growth factor

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

Received April 11, 2005; accepted for publication December 15, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2high CD34– and VEGFR-2high CD34+ cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2high cells, particularly that of VEGFR-2high CD34+ cells, in a dose-dependent manner. Although either population of VEGFR-2high cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2high CD34+ cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2high CD34–or VEGFR-2high CD34+ precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has been reported previously that development of hematopoietic cells (HCs) and endothelial cells (ECs) is closely associated [1, 2]. These observations suggest that both lineage cells share a common precursor, which has been called the hemangioblast. Further supporting the putative existence of the hemangioblast is an immunohistochemical study of murine embryos that revealed HC clusters adhering to the ventral floor of the dorsal aorta [3]. Additionally, both HCs and ECs share common surface markers such as vascular endothelial growth factor receptor (VEGFR)-2, CD34, CD31, tyrosine kinase with Ig and EGF homology domain (Tie)-1, and Tie-2 [3, 4]. Of these antigens, VEGFR-2 (also known as Flk-1 in the mouse) is a candidate marker for hemangioblasts, given that murine embryos or ES cells that do not express VEGFR-2 completely fail to produce cells from either lineage [5, 6]. Moreover, recent studies on the differentiation of murine ES cells have shown that both HCs and ECs can be generated from VEGFR-2+ cells under various culture conditions [7, 8].

Some immunohistochemical studies of human embryos have shown a spatially close association in the development of both HCs and ECs [9–11]. However, the developmental relationship between HCs and ECs in human embryogenesis has not been further elucidated because of various obstacles, including the ethical restrictions on experiments using human embryos.

To understand the mechanisms that regulate the differentiation of HCs and ECs in humans, it is currently promising to use primate (human and monkey) ES cells. We have recently established a culture system, which induces hematopoietic cell differentiation from cynomolgus monkey ES cells by coculture with OP9 stromal cells [12]. We investigated the close association of HC and EC development during ES cell differentiation in this culture system using fluorescence-activated cell sorting (FACS) and subsequent culture of the sorted fractions. Here, we show that both lineages developed after VEGFR-2high cells emerged on day 6, when neither lineage was observed. Both HCs and ECs were generated from single-cell cultures of VEGFR-2high cells, which strongly supports the existence of hemangioblasts in primates.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
The ES cell line CMK6, which was established from cynomolgus monkey blastocysts, was maintained as described previously [13]. The enhanced green fluorescent protein (GFP)-transfected ES cell subline, which we established previously [14], was used to distinguish ES cell-derived cells, except in the experiments that involved immunostaining with antibodies (Abs) to human hemoglobin. The OP9 stromal cell line, a kind gift from Dr. Hiroaki Kodama, was maintained as previously reported [12].

Cytokines and Growth Factors
Recombinant human granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), interleukin (IL)-3, and stem cell factor (SCF) were kindly provided by Kirin Brewery (Tokyo, http://www.kirin.co.jp/english). Recombinant human vascular endothelial growth factor (VEGF) was purchased from R&D Systems (Minneapolis, http://www.rndsystems.com).

Antibodies
The primary Abs used in this study were mouse anti-human CD34 (clone 563) and CD41a (clone HIP8) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen); mouse anti-human c-kit and rabbit anti-human vWF (Nichirei, Tokyo, http://www.nichirei.co.jp), mouse anti-human CD45 (clone 2B11+PD7/26), and CD41 (clone 5B12) (DAKO, Kyoto, Japan, http://www.dako.com), mouse anti-human vascular endothelial cadherin (VE-cadherin, clone TEA1/31) (Immunotech, Luminy, France, http://www.immunotech.com), mouse anti-human CD31 (clone WM59) (eBioscience, San Diego, CA, http://baybio.co.jp), mouse anti-human CD11b (clone Bear1) (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), rabbit anti-human hemoglobin (Hb) (Cappel, Aurora, OH), mouse anti-human {gamma}-globin (Hb{gamma}) (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), and mouse anti-TRA-1–60 (clone TRA-1–60) (Chemicon, Temecula, CA, http://www.chemicon.com). Mouse anti-stage-specific embryonic antigen (SSEA)-4 monoclonal antibody (mAb) developed by Kannagi et al. [15] was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). The mouse anti-human {varepsilon}-globin (Hb{varepsilon}) [16] and VEGFR-2 mAbs [17] were used as previously reported. All primary antibodies against human antigens that were used in this study cross-react to cynomolgus monkey proteins, as previously reported [12, 18, 19]. The secondary Abs used in this study were Cy3-conjugated donkey anti-mouse IgG, horseradish peroxidase-conjugated donkey anti-mouse IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG, and alkaline phosphatase (ALP)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc, West Grove, PA, http://www.jacksonimmuno.com), phycoerythrin (PE)-conjugated goat anti-mouse IgG (Dako), PE-conjugated goat anti-mouse IgM (eBioscience), and allophycocyanin (APC)-conjugated goat anti-mouse IgG (BD Pharmingen).

Staining and the Dil-Ac-LDL Incorporation Assay
May-Giemsa staining and immunostaining were performed as previously reported [12]. For the 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) incorporation assay, adherent cells were incubated with 10 µg/ml Dil-Ac-LDL (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) in culture medium for 4 hours at 37°C. These cells were then washed with {alpha}-minimum essential medium ({alpha}-MEM) (Gibco, Grand Island, NY, http://www.invitrogen.com) and observed by fluorescence microscopy (FLUOVIEW System; Olympus, Tokyo, http://www.olympus-global.com). After the DiI-Ac-LDL incorporation assay, the cells were then fixed and used for antibody staining.

FACS Analysis and Cell Sorting
Staining procedures, FACS analysis, and cell sorting were performed as reported previously [12]. Briefly, the cultured cells were harvested with cell dissociation buffer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and incubated with PE- or APC-conjugated Abs or unconjugated Abs for 30 minutes. Samples stained with unconjugated Abs were then incubated with PE- or APC-conjugated goat anti-mouse Abs. Nonviable cells were excluded from the analysis by propidium iodide costaining. FACS analysis was performed with a FACScaliber instrument with the CellQuest program (Becton Dickinson Labware, Bedford, MA, http://www.bd.com). Cell sorting with PE-conjugated CD34 and APC-conjugated VEGFR-2 mAbs was performed using a FACSVantage flow cytometer (Becton Dickinson).

Reverse Transcription-Polymerase Chain Reaction
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) were performed as described previously [12]. Samples were initially denatured at 94°C for 5 minutes, followed by 35–40 amplification rounds consisting of 94°C for 1 minute (denaturing), 60°C for 1 minute (annealing), and 72°C for 1 minute (extension), followed by a final extension at 94°C for 7 minutes. The primers used for RT-PCR were as follows: GATA-1 (498 bp), forward, 5'-CAC ATC CCC AAG GCG GCC GAA C-3', reverse, 5'-AGG TCT GGG CTC AGC CGC TCT-3'; MYB (307 bp), forward, 5'-CAC GCT GGG CCT GTC ATC AAC-3', reverse, 5-GCA TGG CTC TTC GTG TTA TAG C-3'; FLI-1 (412 bp), forward, 5'-ATG GAT CCA GGG AGT CTC CGG T-3', reverse, 5'-TTG GTC GGT GTG GGA GGT TGT-3'; Tie-1 (308 bp), forward, 5'-TGG TCG GAG AGA ACC TAG CC-3', reverse, 5'-GAC GCA TCA GCT CGT ACA CTT C-3'; eNOS (557 bp), forward, 5'-GAC ATT TTC GGG CTC ACG CTG-3', reverse, 5'-TGG GGT AGG CAC TTT AGT AGT TC-3'; GATA-2 (303 bp), forward, 5'-TGG CGC ACA ACT ACA TGG AAC-3', reverse, 5'-GAG GGG TGC AGT GGC GTC TT-3'; SCL (185 bp), forward, 5'-TCT CGG CAG CGG GTT CTT TG-3', reverse, 5'-AAG GCC CCG TTC ACA TTC TGC-3'; FLT-1 (508 bp), forward, 5'-GCT CAC CAT GGT CAG CTA CTG-3', reverse, 5'-CAG TGA TGT TAG GTG ACG TGA ACC-3'; Rex-1 (489 bp), forward, 5'-CGC GGT GTG GGC CTT ATG TG-3', reverse, 5'-TCT CAG GGC AGC TCT ATT CCT C-3'; Oct-4 (246 bp), forward, 5'-CGT GAA GCT GGA GAA GGA GAA GCT G-3', reverse, 5'-CAA GGG CCG CAG CTT ACA CAT GTT C-3'; GAPDH (360 bp), forward, 5'-CAC CAG GGC TGC TTT TAA CTC TG-3', reverse, 5'-ATG GTT CAC ACC CAT GAC GAA C-3'. cDNA from cynomolgus monkey bone marrow, human umbilical vein endothelial cells (HUVECs), and human erythroleukemia K562 cells served as positive controls. For semiquantitative comparisons, samples were normalized by dilution to give equivalent signals for GAPDH.

In Vitro Differentiation of ES Cells
For initial differentiation induction, trypsin-treated undifferentiated ES cells were transferred onto fresh confluent OP9 cells in six-well plates at a concentration of 4 x 103 cells per well and cultured with various concentrations of VEGF (0, 10, 20, and 40 ng/ml) in {alpha}-minimum essential medium supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and 50 µM 2-mercaptoethanol (2ME). The cultured cells were harvested in cell dissociation buffer (Invitrogen) and analyzed by FACS, as described above, on days 4, 6, 8, and 10.

To induce the differentiation of HCs and ECs, cells that had been cultured for 6 days in the presence of 20 ng/ml VEGF were harvested and sorted by FACS according to the expression of VEGFR-2 and CD34, as detailed in Results. Each sorted cell fraction was transferred onto fresh confluent OP9 cells in six-well plates at a concentration of 1 x 104 cells per well or in 12-well plates at a concentration of 1 x 103 cells per well. To analyze the development of HCs, the sorted cells were cultured in {alpha}-MEM supplemented with 10% FCS (Sigma-Aldrich), 50 µM 2ME, and a mixture of 10 ng/ml G-CSF, 2 U/ml EPO, 20 ng/ml IL-3, and 100 ng/ml SCF (hematopoietic cytokine mixture). Floating and adherent HCs were analyzed sequentially, as previously reported [12, 20]. To analyze EC development, the sorted cells were cultured in {alpha}-MEM supplemented with 10% FCS, 50 µM 2ME, and 20 ng/ml VEGF. Six days after cell sorting, the cells were stained with anti-VE-cadherin and ALP-conjugated anti-mouse IgG, and the EC clusters were scored by microscopy. At least three independent experiments were conducted.

Single-Cell Deposition Assay for Hematopoietic and Endothelial Differentiation
The deposition of single sorted cells into individual wells of 96-well plates was carried out by using the Clon-Cyt system of the FACSVantage flow cytometer (Becton Dickinson). Individual sorted cells from each fraction were seeded onto OP9 stromal cells in {alpha}-MEM supplemented with 10% FCS and a hematopoietic cytokine mixture. After 6 days in culture, HC development was evaluated by immunostaining with the anti-CD45, CD41, and HbF mAbs, whereas EC development was analyzed by immunostaining with the anti-VE-cadherin mAb or by the DiI-Ac-LDL incorporation assay. The concomitant development of both lineage of cells was confirmed by immunostaining with the anti-CD34 mAb.

Statistics
Differences in the number of HC or EC cluster between two groups were assessed using Student’s t test. Differences in the frequency of HC and/or EC cluster development in the single-cell deposition assay were assessed using the {chi}2 test. Statistical significance was defined as p values less than .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FACS Analysis of Hematopoietic and/or Endothelial Surface Markers During Early Primate ES Differentiation
The ES cell line CMK6 and the GFP-transfected ES cell subline that were used in this study both expressed the undifferentiated-state marker SSEA-4, even after being maintained in culture for more than a year (data not shown). We confirmed that both ES cell lines were equally capable of differentiating into HCs and ECs (data not shown).

By RT-PCR analysis of cultures in the OP9 coculture system, we have demonstrated previously that sequential expression of genes associated with both HC and EC lineage development was equivalent to that seen during primate ontogeny in vivo [12]. FACS analysis was used to determine the expression patterns of various surface markers involved in HC and EC development when GFP-transfected ES cells were induced to differentiate by coculture with OP9 stromal cells in the presence or absence of exogenous VEGF (Fig. 1Go). The numbers of cells expressing particular markers were quantified as a percent of the total live GFP+ cells in the culture (Fig. 1AGo). Although substantial fraction of the GFP+ cells were dead after being harvested with cell dissociation buffer, harvesting by other means, such as by using trypsin or collagenase, did not significantly alter the proportion of dead cells (data not shown). The markers could be classified into three groups depending on their expression kinetics. The first group includes CD34 and CD31, which are expressed by both early HCs and ECs (Fig. 1B, 1CGo). Their expression was not detected in undifferentiated ES cells, but became upregulated on day 6. Thereafter, CD34+ and CD31+ cells increased, especially in the presence of exogenous VEGF. The second group includes CD45, CD41a, and VE-cadherin (Fig. 1D–1FGo), whose expression is specific to either HCs (CD45 and CD41a) or ECs (VE-cadherin). Expression of these proteins was not detected in undifferentiated ES cells but became slightly upregulated by day 10 in the presence of VEGF. The third group includes c-kit and VEGFR-2 (Fig. 1G, 1HGo). Although expression of these proteins was detected in almost all undifferentiated ES cells, it became downregulated on day 4 and thereafter, with or without exogenous VEGF. These kinetics were similar to that of SSEA-4 (Fig. 1IGo), which is expressed by undifferentiated ES cells [13, 21]. These observations showed that during in vitro HC and EC differentiation, common surface markers such as CD34 and CD31 were expressed first, followed by the expression of lineage-specific markers.


Figure 1
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  		Figure 1. Fluorescence-activated cell sorting (FACS) analysis of surface markers expressed by hematopoietic cells and/or endothelial cells during embryonic stem (ES) cell differentiation. Undifferentiated green fluorescent protein (GFP)-transfected ES cells were seeded onto confluent OP9 stromal cells in six-well plates in the presence or absence of 20 ng/ml exogenous vascular endothelial growth factor (VEGF). The undifferentiated ES cells and the subsequent cultures on days 4, 6, 8, and 10 were stained with various monoclonal antibodies and quantified by FACS analysis. (A): The amounts of GFP+ ES-derived live cells (boxed region) are shown as a percentage of the total cells in cultures. (B–I): Cells that stained positive for each antigen, CD34 (B), CD31 (C), CD45 (D), CD41a (E), VE-cadherin (F), c-kit (G), VEGFR-2 (H), and SSEA-4 (I), are shown as a percentage of the total GFP+ cells. The white and black circles show the results in the absence and presence of VEGF, respectively. The data represent the mean ± SD of three independent experiments. Abbreviations: SSEA, stage-specific embryonic antigen; VE-cadherin, vascular endothelial cadherin; VEGFR, vascular endothelial growth factor receptor.

 
Generation of VEGFR-2highCD34– and VEGFR-2highCD34+ Cells Before HC or EC Development During Primate ES Cell Differentiation
Several studies have demonstrated that VEGFR-2 is a key marker of hemangioblasts during early murine development [5–8]. CD34 and CD31 are also expressed by early hematopoietic and endothelial progenitors [9, 11]. Therefore, to identify bipotential progenitor cells in primates, we analyzed the differentiating ES cell-derived cells by FACS using VEGFR-2, CD34, and CD31 mAbs. The undifferentiated ES cells did not express CD34 or CD31, whereas approximately 80% of them expressed VEGFR-2 at low levels (Fig. 2A, 2BGo). We examined the expression levels of VEGFR-2 more closely and found that the proportion of VEGFR-2low cells gradually decreased during coculture and that VEGFR-2high cells could be detected on day 6. More than half of the VEGFR-2high cells were CD34-negative on day 6, but VEGFR-2high CD34+ cells increased by day 8 and thereafter (Fig. 2AGo). The same temporal expression pattern of CD31 among the VEGFR-2high cells was observed (Fig. 2BGo). FACS and immunostaining analysis showed that the VEGFR-2high cells that emerged on day 6 were negative for CD41a, CD45, VE-cadherin, or any hemoglobins (data not shown), indicating that these cells did not yet express HC or EC lineage-specific markers.


Figure 2
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  		Figure 2. Fluorescence-activated cell sorting and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the VEGFR-2high cells that emerge during ES cell differentiation. The green fluorescent protein (GFP)-transfected ES cells and the subsequent cultures on days 4, 6, 8, and 10 in the absence of exogenous vascular endothelial growth factor were analyzed by FACS with antibodies against VEGFR-2 and CD34 (A), VEGFR-2 and CD31 (B), or VEGFR-2 and TRA-1–60 (C). (A, B): The amounts of VEGFR-2high CD34– or VEGFR-2high CD31– cells (upper left quadrant), or VEGFR-2high CD34+ or VEGFR-2high CD31+ cells (upper right quadrant), are shown as a percentage of the total GFP+ ES cells. (C): The numbers in each quadrant represent percentages of cells among the total GFP+ ES cells. (D): ES cells cocultured with OP9 cells for 6 days were subjected to RT-PCR analysis of genes associated with HC and/or EC development. The lanes contained mRNA from the following cells: adult cynomolgus monkey bone marrow cells (lane 1), the human erythroblastic cell line K562 (lane 2), human umbilical vein endothelial cells (lane 3), OP9 stromal cells (lane 4), undifferentiated ES cells (lane 5), the total GFP+ ES cell-derived cells (lane 6), the VEGFR-2high CD34– cells (lane 7), and the VEGFR-2high CD34+ cells (lane 8), harvested on day 6. Representative results from one of three independent experiments are shown. Abbreviations: EC, endothelial cells; ES, embryonic stem; HC, hematopoietic cells; M, size marker; VEGFR, vascular endothelial growth factor receptor.

 
We further examined the differentiation state of the VGEFR-2high cells by double staining with mAbs for VEGFR-2 and TRA-1–60, a surface marker indicative of undifferentiated ES cells. The expression of TRA-1–60 was detected in undifferentiated ES cells, as previously reported [13, 21], and was upregulated on day 4. However, although approximately half of the GFP+ ES cell-derived cells were positive for TRA-1–60 on day 6 and thereafter, the VEGFR-2high cells were always negative for TRA-1–60 at all time points (Fig. 2CGo).

To verify the potential of day 6 VEGFR-2high CD34– and VEGFR-2high CD34+ cells to differentiate into the HC and EC lineages, their gene expression profiles were investigated by RT-PCR. The genes analyzed were those representing HC (GATA-1, MYB, and FLI-1) [22], EC (Tie-1 and eNOS) [23, 24], or HC-EC potentials (SCL, FLT-1, and GATA-2) [25–27]. As shown in Figure 2DGo, GATA-1 and SCL were expressed by VEGFR-2high CD34+ cells but not by VEGFR-2high CD34–cells, whereas FLI-1 expression was up-regulated in both VEGFR-2high cell populations. In contrast, the expression profiles of the other HC and/or EC markers did not correlate with the development of VEGFR-2high cells.

We also analyzed the expression of Rex-1 and Oct-4, which are marker genes for undifferentiated ES cells [21]. Although their expression was still detected in the total GFP+ cell populations on day 6, they were not expressed by either VEGFR-2high cell population. These observations together indicate that the day 6 VEGFR-2high cell population differs from other ES cell-derived cells that emerge during the differentiation induction, as they express genes characteristic for the HC and/or EC lineages.

We then analyzed the effect of VEGF on VEGFR-2high cell development by adding various concentrations of VEGF to the culture (Table 1Go). FACS analysis demonstrated that the presence of VEGF increased both the proportion of VEGFR-2high cells in the culture and the percentage of VEGFR-2high CD34+ cells among the VEGFR-2high cell fraction. This effect was dose-dependent and saturated at 20 ng/ml VEGF. The same trends were observed by analysis with VEGFR-2 and CD31 mAbs.


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  		Table 1. Effects of VEGF on the generation of VEGFR-2high cells

 
HC Development from VEGFR-2high Fractions
Given the results described above, we hypothesized that the VEGFR-2high CD34– or the VEGFR-2high CD34+ cells on day 6 may contain hemoangiogenic progenitors. As shown in Figure 1D–1FGo, rather few HCs and ECs develop from ES cells over 10 days of culture. However, we have shown previously that the numbers of HCs that develop from ES cells markedly increased if the cells are replated onto a new confluent OP9 cell layer and that abundant hematopoiesis, in particular definitive hematopoiesis, cannot develop without additional VEGF [12]. Therefore, we sorted the cultures using anti-VEGFR-2 and CD34 mAbs after initial 6-day VEGF treatment, and each fraction was replated onto a new confluent OP9 cell layer. Fractions of VEGFR-2high CD34–, VEGFR-2high CD34+, VEGFR-2low CD34–, VEGFR-2– CD34–, and VEGFR-2low or – CD34+ cells were collected, replated, and analyzed for their capacity to generate HCs and/or ECs, as shown schematically in Figure 3AGo. FACS reanalysis of the sorted VEGFR-2high CD34+ cells showed their purity ranged from 93.0%–97.0%, whereas the purity of the other sorted fractions ranged from 99.0%–99.7% (Fig. 3BGo and data not shown).


Figure 3
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  		Figure 3. Schematic representation of the assays used to analyze the differentiation of HCs and ECs from ES cells. (A, B): The cells were cocultured with OP9 cells in the presence of 20 ng/ml vascular endothelial growth factor (VEGF) and sorted using VEGFR-2 and CD34 mAbs on day 6. The FACS plot (B) shows the regions (R3–R7) that were used to define the different populations of cells: VEGFR-2high CD34–, R3; VEGFR-2high CD34+, R4; VEGFR-2low CD34–, R5; VEGFR-2– CD34–, R6; VEGFR-2low or – CD34+, R7. The sorted cells from each population were then transferred onto fresh confluent OP9 cells. To analyze HC development, the sorted cells were cultured in {alpha}-minimum essential medium ({alpha}-MEM) supplemented with 10% fetal calf serum (FCS), 2-mercaptoethanol (2ME), and a mixture of granulocyte colony-stimulating factor, erythropoietin, interleukin-3, and stem cell factor (hematopoietic cytokine mixture). The number of adherent HC clusters were counted 4 days after cell sorting, and the floating cells were analyzed every 2 days. To analyze EC development, the sorted cells were cultured in {alpha}-minimum essential medium ({alpha}-MEM) supplemented with 10% FCS, 2ME, and VEGF. Cultures were analyzed by immunostaining with an antibody against vascular endothelial cadherin 6 days after cell sorting. For the single-cell deposition assay, individual sorted cells from the VEGFR-2high CD34– and VEGFR-2high CD34+ fractions were seeded onto OP9 stromal cells and cultured with a hematopoietic cytokine mixture. Six days later, HC and EC development was evaluated by immunostaining or by the 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein incorporation assay. FACS reanalysis showed that the purity of the sorted VEGFR-2high CD34– and VEGFR-2high CD34+ cell populations was 99.0%–99.7% and 93.0%–97.0%, respectively (see the right-hand dotplots for examples). Abbreviations: EC, endothelial cell; ES, embryonic stem; HC, hematopoietic cell; VEGFR, vascular endothelial growth factor receptor.

 
Adherent HCs first emerged from the VEGFR-2high CD34–and VEGFR-2high CD34+ fractions 2 days after cell sorting and replating (Fig. 4A, 4BGo). The adherent HCs, which formed clusters on or underneath the OP9 stromal layer, are known to contain immature hematopoietic progenitors [12, 20]. Almost all of the adherent HC clusters were positive for CD34 (Fig. 4HGo) and also contained cells that were positive for VEGFR-2- (Fig. 4IGo), CD45– (Fig. 4JGo), CD11b– (Fig. 4KGo), CD41– (Fig. 4LGo), and Hb{varepsilon} (Fig. 4MGo). The number of adherent HC clusters was maximal on day 10 but decreased thereafter. On day 10, the number of clusters generated from the VEGFR-2high CD34+ fraction was approximately four times the number generated from the VEGFR-2high CD34– fraction. Furthermore, larger clusters were detected from the VEGFR-2high CD34+ fractions (Fig. 4MGo), and adherent clusters from the VEGFR-2high CD34+ fraction covered the stromal layer by day 20 (Fig. 4EGo), whereas adherent clusters from the VEGFR-2high CD34– fraction were rarely observed (Fig. 4DGo) and disappeared over time in culture.


Figure 4
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  		Figure 4. HC development from the VEGFR-2high CD34–and VEGFR-2high CD34+ fractions. (A, B): Micrographs of adherent HC clusters generated on day 10 from the VEGFR-2high CD34– fraction (A) and the VEGFR-2high CD34+ fraction (B). (C, F): May-Giemsa staining of floating HCs on days 12 (C) and 27 (F). (D, E): Micrographs of adherent HC clusters generated on day 27 from the VEGFR-2high CD34–fraction (D) and the VEGFR-2high CD34+ fraction (E). (G–M): Alkaline phosphatase detection of adherent HCs stained with antibodies to CD34 (H), VEGFR-2 (I), CD45 (J), CD11b (K), CD41 (L), and Hb{varepsilon} (M). The staining with the isotype control IgG1 is shown in G. (N, R) Immunostaining of hemoglobin (Hb) (fluorescein isothiocyanate [FITC[) and Hb{gamma} (Cy3) in erythrocytes on days 12 (N) and 27 (R). (O–Q, S–U): Immunostaining of Hb (FITC) and Hb{varepsilon} (Cy3) in erythrocytes on days 12 (O–Q) and 27 (S–U). Merged images are shown in N, Q, R, and U. Nuclei were labeled with Hoechst 33342 (N–U). The anti-human Hb Ab, which reacts with embryonic, fetal, and adult erythrocytes, was used to detect all erythrocytes among the floating cells during culture. The same staining results were obtained from both the VEGFR-2high CD34– and VEGFR-2high CD34+ fractions. Original magnification, x100 (A, B, D, E, G–M) and x400 (C, F, N–U). Scale bars = 100 µm (A, B, D, E, G–M) and 10 µm (C, F, N–U). (V): The number of adherent HC clusters in the indicated fractions. Small clusters (white bars), which consisted of 20–49 round blast-like cells, and large clusters (black bars), which consisted of more than 50 cells, were counted on day 10. (W): Sequential analysis of the number of erythrocytes generated from the VEGFR-2high CD34– (white circles) and VEGFR-2high CD34+ (black circles) populations. (X): Sequential analysis of the proportion of definitive erythrocytes (EryD) among all the erythrocytes generated by the VEGFR-2high CD34– (white columns) and VEGFR-2high CD34+ (black columns) populations. EryD were defined as Hb{varepsilon}-negative, Hb-and Hb{gamma}-positive erythrocytes, whereas primitive erythrocytes were Hb-, Hb{gamma}-, and Hb{varepsilon}-positive. (V–X): Data represent the mean ± SD of triplicate wells, and representative results from one of three independent experiments are shown. Abbreviations: HC, hematopoietic cell; VEGFR, vascular endothelial growth factor receptor.

 
In this coculture system, HC development in the floating fractions first occurred on day 8 (2 days after seeding on day 6). The floating cells were found to consist exclusively of mature HCs, such as erythrocytes, myeloid lineage cells, and megakaryocytes (data not shown) [12]. We examined whether both VEGFR-2high fractions were capable of both primitive and definitive hematopoiesis by sequential May-Giemsa staining and immunostaining with hemoglobin Abs. Until day 15 (9 days after sorting), all of the floating HCs from both fractions were large nucleated erythrocytes positive for Hb{varepsilon}, Hb{gamma}, and Hb (Fig. 4C, 4N–4QGo). These cells correspond to primitive erythrocytes (EryP). On day 18 and thereafter, both VEGFR-2high CD34– and VEGFR-2high CD34+ cultures contained some small erythrocytes, including enucleated erythrocytes, that were positive for Hb{gamma} and Hb but negative for Hb{varepsilon} (Fig. 4F, 4R–4UGo). These cells correspond to definitive erythrocytes (EryD). The number of erythrocytes generated from either cell fraction was maximal on day 12 but gradually decreased thereafter. Subsequently, a second wave of erythrocytes appeared around day 21 (Fig. 4WGo). The proportion of EryD gradually increased around day 18 and thereafter, constituting up to 20% of erythrocytes, in parallel with the second wave of erythropoiesis (Fig. 4XGo). Thus, the VEGFR-2high CD34– and VEGFR-2high CD34+ fractions were capable of both primitive and definitive erythropoiesis, but the VEGFR-2high CD34– cells were less competent to differentiate than the VEGFR-2high CD34+ cells. In contrast, the VEGFR-2low CD34– fraction produced few floating HCs, whereas the VEGFR-2low or – CD34+ and VEGFR-2– CD34– fractions failed to produce any HCs (data not shown).

EC Development from VEGFR-2high Fractions
We also investigated the capacity of the various fractions sorted at day 6 to differentiate into ECs in the presence of exogenous VEGF. Some GFP-positive cells formed sheet-like or cord-like clusters that first appeared on the OP9 stromal layer on day 10 (Fig. 5AGo). These clusters took up DiI-Ac-LDL (Fig. 5BGo) and co-expressed VE-cadherin (Fig. 5CGo), CD34 (Fig. 5DGo), VEGFR-2 (Fig. 5EGo), vWF (Fig. 5FGo), and CD31 (data not shown), indicating that these cells are ECs. Immunostaining with a VE-cadherin mAb showed that 6 days after sorting, the VEGFR-2high CD34+ fraction generated significantly more VEGFR-2high VE-cadherin+ EC clusters than the VEGFR-2high CD34– fraction (Fig. 5HGo). Cluster formation was rare in the VEGFR-2low CD34– or VEGFR-2low or – CD34+ fraction, and no clusters were observed in the VEGFR-2– CD34– fraction. Thus, EC production was restricted to the VEGFR-2high cell fractions, and the VEGFR-2high CD34+ cells had more angiogenic potential than did the VEGFR-2high CD34– cells.


Figure 5
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  		Figure 5. EC development from the VEGFR-2high CD34– and VEGFR-2high CD34+ fractions. (A–G): Some green fluorescent protein-positive (GFP+) embryonic stem cell-derived adherent cells formed sheet-like or cord-like endothelial cell (EC) clusters (A) that took up 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (B). Alkaline phosphatase detection of EC clusters stained with antibodies vascular endothelial cadherin (C), CD34 (D), VEGFR-2 (E), and vWF (F). The staining with the isotype control IgG1 is shown in G. Original magnification, x100; scale bars = 100 µm (A–F). (H): The number of EC clusters in cultures of the indicated populations. The data represent the mean ± SD of triplicate wells, and representative results from one of three independent experiments are shown. Abbreviation: VEGFR, vascular endothelial growth factor receptor.

 
HC and EC Development from Single VEGFR-2high CD34– and VEGFR-2high CD34+ Cells
Finally, we performed a single-cell analysis by using a single-cell deposition system (the Clon-Cyt system) to analyze whether the VEGFR-2high CD34– or VEGFR-2high CD34+ fractions contain the common progenitor for both HC and EC lineages. Each well was observed by fluorescence microscopy 24 hours after cell deposition, and wells that contained more than one GFP-positive cell were excluded from further analysis. HC and EC clusters were produced by single cells from the VEGFR-2high CD34– and VEGFR-2high CD34+ fractions. Immunostaining with anti-CD45, CD41, Hb{gamma}, and VE-cadherin mAbs or by DiI-Ac-LDL incorporation assays confirmed the presence of HCs or ECs (Fig. 6A– 6C, 6E, 6FGo). When a mixture of anti-CD45, CD41, and Hb{gamma} mAbs was used for staining wells containing HC clusters, all round cells were positive (Fig. 6DGo). The concomitant development of both lineages of cells was also confirmed by immunostaining with a mixture of the three hematopoietic lineage mAbs and VE-cadherin mAb or with the anti-CD34 mAb (Fig. 6G, 6HGo). When 480 cells from each fraction were individually seeded, the potential for mono- or bipotential progenitor development was approximately 2-fold higher in the VEGFR-2high CD34+ cell population than the VEGFR-2high CD34+ cell population (the frequencies of HC development alone: 2.5% [12 wells] vs. 0.8% [four wells], p < .05; those of EC development alone: 15.4% [74 wells] vs. 7.9% [38 wells], p < .05; those of HC plus EC development: 2.3% [11 wells] vs. 1.0% [five wells], p = .13). Nevertheless, the data also strongly suggest that both VEGFR-2high fractions contain the common hemoangiogenic progenitors, the "hemangioblasts."


Figure 6
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  		Figure 6. Single-cell deposition assay showing the hemoangiogenic potential of VEGFR-2high CD34– and VEGFR-2high CD34+ cells. (A–D): VEGFR-2high CD34– and VEGFR-2high CD34+ cells were sorted, and single cells were seeded onto an OP9 stromal layer in 96-well plates. Six days later, hematopoietic cell (HC) development was evaluated by immunostaining with monoclonal antibodies CD45 (A), CD41 (B), or Hb{gamma} (C). (D): When a mixture of all these monoclonal antibodies was used for staining wells containing HC clusters, all of them were positive. (E, F): EC development was evaluated by immunostaining with vascular endothelial cadherin mAb (E) or by the 1,1'-dioctadecyl-1,3,3,3', 3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein incorporation assay (F). (G, H): Concomitant development was confirmed by a mixture of CD45, CD41, and Hb{gamma} (blue)/vascular endothelial cadherin (brown) double immunostaining (G) or immunostaining with CD34 mAb (H). Original magnification, x100; scale bars = 100 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ES cells are pluripotent and can differentiate into multiple cell types, including derivatives of all three germ layers. Although their high potential for differentiation has been intensively examined in many murine ES cell culture systems [28–32], it is inevitable that the development of particular cells will be contaminated with that of cells from other lineages. Isolating cells of interest using FACS is a particularly useful approach to enrich for tissue-specific cells during in vitro ES cell differentiation. HC and EC development is a good model for such an approach because both lineages of cells have many well known common surface markers, as well as lineage-specific antigens [3, 4]. This enhances our ability to select live cells of interest. Indeed, these features have been used to identify the progenitors of both HC and EC lineages during murine ES cell differentiation [8, 31, 32].

The selection of tissue-specific stem cells or progenitors and the tracing of their fates by further culture are essential for preclinical research using monkey ES cells that aims to examine the efficacy and safety of clinical applications using human ES cells [13, 33]. In this study, we have dissected the differentiation pathways by which primate ES cells generate HCs and ECs by analysis of HC and/or EC cell surface markers. Our results show that the surface markers associated with HC and EC development are expressed in a defined order during culture, consistent with earlier studies of murine ES cell differentiation [8, 31, 32]. Furthermore, we show here that it is possible to identify the progenitors of both lineages and to determine their fates by analyzing a combination of well known surface markers. These observations will facilitate further investigations on primate ES cells, including in vivo studies.

Disruption of the murine homologue of the VEGFR-2 (Flk-1) gene in murine embryos or ES cells resulted in a combined defect in HC and EC development, which may reflect the loss of a common progenitor, the hemangioblast [5, 6]. Furthermore, in vitro differentiation of murine ES cells shows that VEGFR-2+ cells indeed serve as hemangioblasts [7]. In primates, however, there is no direct evidence suggesting the presence of hemangioblasts during embryogenesis, although some studies have suggested that these cells are present in the fetus and during adulthood [34, 35]. Unlike murine ES cells, several studies have detected VEGFR-2 expression in undifferentiated primate ES cells [12, 19, 36–38]. We found that undifferentiated ES cells expressed low levels of VEGFR-2 and that this surface marker is down-regulated during culture of the ES cells in the OP9 coculture system. In addition, when VEGFR-2low cells were sorted from the cultures on day 6 or from undifferentiated ES cells, little HC and EC differentiation was observed (Figs. 4Go and 5Go). Moreover, markers of the undifferentiated state, such as TRA-1–60, Rex-1, and Oct-4, were expressed by the VEGFR-2low cells (Fig. 2C, 2DGo, and data not shown), which suggests that the former fraction still contained undifferentiated components. In contrast, VEGFR-2high cells emerged on day 6, immediately prior to HC and EC differentiation, and we showed here that these cells subsequently gave rise to both primitive and definitive HCs and ECs. It should be noted that the sorted VEGFR-2high cell populations were not completely pure (purity ranged from 93.0%–99.7%). However, it is unlikely that the hemoangiogenic cells came from contaminating VEGFR-2low or VEGFR-2– cells, since the sorted VEGFR-2low or VEGFR-2– cells were not able to differentiate into either HC or EC lineages. The results of the single-cell culture assays also strongly suggest that the VEGFR-2high fractions contain the common hemoangiogenic progenitors, the hemangioblasts. Recently, Wang et al. reported the identification of primitive endothelial-like cells derived from human ES cells by embryoid body (EB) formation [39]. Their observation that development of both lineages can be observed from a single ES cell-derived progenitor is in agreement with our own study. Furthermore, such progenitor cells expressed VEGFR-2, but not CD45, as has been observed in mesodermal differentiation of murine ES cells [7, 8]. In contrast, the VEGFR-2high hemoangiogenic progenitors in the report of Wang et al. expressed VE-cadherin, generally considered to be an EC marker [39], whereas the progenitor cells in our study did not. Our results demonstrate that the appearance of hemoangiogenic progenitors, without any HC or EC lineage-specific properties, clearly precedes differentiation into either of these cell lineages. Furthermore, this is the first report to demonstrate that both primitive and definitive HCs, as well as ECs, were generated in the VEGFR-2high fractions. These differences in hematopoietic and endothelial differentiation may be partially due to differences in the culture conditions (the EB and OP9 coculture system), and/or in the ES cells that were used for these studies.

Sequential FACS analysis with a combination of surface markers revealed that two distinct populations, VEGFR-2high CD34– and VEGFR-2high CD34+ cells, were present on day 6. The potential of the VEGFR-2high CD34+ cell to serve as a mono- or bipotential progenitor is approximately twice that of the VEGFR-2high CD34– cell, although both cell types produce equal proportions of HCs and ECs. Notably, when we analyzed the expression of HC and/or EC lineage marker genes by the VEGFR-2high CD34– and VEGFR-2high CD34+ cell populations on day 6, we found that only the VEGFR-2high CD34+ cells expressed SCL. Scl–/– murine embryos show lack of blood formation and a defect in yolk sac angiogenesis, indicating that this transcription factor is essential for HC and EC development [40–42]. Furthermore, recent reports on the developmental kinetics of VEGFR-2 and SCL suggest that VEGFR-2+ SCL+ cells may be hemangioblasts [43, 44]. These observations together suggest that CD34 is expressed by the VEGFR-2high cells during their differentiation into hemoangiogenic progenitors, concomitant with an upregulation of a set of factors that regulate the development of both lineages.

Recent studies, including our previous work, have reported that exogenous VEGF enhances early HC development [12, 45]. We also observed that ECs are generated more abundantly in the presence of VEGF (unpublished data). Unlike other reports with monkey or human ES cells [37, 39, 45], in our culture system, exogenous BMP-4 fails to induce hematopoietic differentiation, probably because it causes the OP9 stromal cells to differentiate and thereby impairs their interaction with ES cells [12]. We analyzed the effect of various concentrations of VEGF on the development of VEGFR-2high cells by FACS and found that it increases the proportion of CD34+ cells in the VEGFR-2high cell population in a dose-dependent manner. Taken together, the effect of VEGF on HC and EC development is mainly due to its ability to enhance the proliferation and/or differentiation of VEGFR-2high CD34+ cells with a higher hemoangiogenic potential during the initial 6-day differentiation induction.

In summary, we have been able to identify and characterize hemoangiogenic progenitors by sequential phenotypic analysis during primate ES differentiation. Our observations after cell sorting strongly suggest that the VEGFR-2high fraction of cells contains hemangioblasts. The approach we have taken in this study will contribute to investigations of early developmental steps in human biology and, in addition, will provide a cell source for regenerative medicine applications in the future.


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

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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