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Originally published In Press as doi:10.1074/jbc.M703919200 on September 7, 2007

J. Biol. Chem., Vol. 282, Issue 46, 33507-33514, November 16, 2007
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A New Role of Thrombopoietin Enhancing ex Vivo Expansion of Endothelial Precursor Cells Derived from AC133-positive Cells*

Toshie Kanayasu-Toyoda{ddagger}, Akiko Ishii-Watabe{ddagger}, Takayoshi Suzuki§, Tadashi Oshizawa§, and Teruhide Yamaguchi{ddagger}1

From the Divisions of {ddagger}Biological Chemistry and Biologicals, and §Cellular and Gene Therapy Products, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagayaku, Tokyo 158-8501, Japan

Received for publication, May 14, 2007 , and in revised form, September 5, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that CD31bright cells, which were sorted from cultured AC133+ cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31+ cells or CD31- cells, suggesting that CD31bright cells may be endothelial precursor cells. In this study, we found that CD31bright cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133+ cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31bright cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neovascularization is an important adaptation to rescue tissue from critical ischemia. Postnatal blood vessel formation was formerly thought to be primarily due to the migration and proliferation of preexisting, fully differentiated endothelial cells, a process referred to as angiogenesis. Recent studies provide increasing evidence that circulating bone marrow-derived endothelial progenitor cells (EPCs)2 contribute substantially to adult blood vessel formation (1–5). Cell therapy using EPCs is widely performed to rescue tissue damaged due to critical ischemia.

Although EPCs have been thought to be derived from many kinds of cells, cells characterized as CD34+ (6), AC133+ (7, 8), and CD14+ (9) are also thought to differentiate to EPCs. The main role of EPCs has been thought to be the release of angiogenic factors such as interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor, and vascular endothelial growth factor (VEGF) (9). To obtain a sufficient number of EPCs for the treatment may be very important in cell therapy for critical ischemia.

On the other hand, EPCs are mobilized from bone marrow by many substances such as G-CSF (10), granulocyte macrophage-colony stimulating factor (GM-CSF) (5), VEGF (3), erythropoietin (11–13), and statins (14, 15) in vivo. To get as many EPCs as possible without unduly burdening the patient, it is desirable to establish efficient expansion methods for EPCs in vitro.

Thrombopoietin (TPO), initially identified as the primary regulator of platelet production (16), plays an important and nonredundant role in the self-renewal of and expansion methods for hematopoietic stem cells (17–19). Recently, TPO has been found to exert a proangiogenic effect on cultured endothelial cells (20). The mechanism by which hematopoietic cytokines support revascularization in vivo, however, remains unknown. TPO has increased the number of colony-forming units-granulocyte-macrophage (21) and of burst-forming units-erythroid (22) in vivo and leads to a redistribution of colony-forming units-erythroid from marrow to spleen. Moreover, TPO acts in synergy with erythropoietin to increase the growth of burst-forming units-erythroid and the generation of colony-forming units-erythroid from marrow cells (21, 23, 24).

In our previous study (25), we isolated AC133+ cells and examined their endothelial differentiation in vitro. CD31(PECAM-1)+ and CD31bright cells appeared at an early stage of the in vitro differentiation of AC133+ cells, and CD31bright cells derived from AC133+ cells were identified as the precursors of endothelial cells because CD31bright cells had differentiated more efficiently to endothelial cells than others. Therefore, we conclude that CD31bright cells derived from AC133+ cells possess the typical character of EPCs. In this study, we analyzed the effects of TPO on the appearance of CD31bright cells from AC133+ cells, and we show that TPO plays an important role in in vitro EPC expansion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Recombinant TPO and recombinant stem cell factor (SCF) were kindly provided by Kirin-Amgen Inc. (Thousand Oaks, CA). Recombinant human VEGF was purchased from Strathmann Biotec AG (Hamburg, Germany). The AC133 magnetic cell sorting kit and phycoerythrin (PE)-conjugated anti-CD133/2 antibody were from Miltenyi Biotec (Gladbach, Germany). Allophycocyanin-conjugated anti-CD110 (TPO receptor) antibody, fluorescein isothiocyanate (FITC)-conjugated anti-CD31 monoclonal antibody, FITC-conjugated anti-CD34 monoclonal antibody, and anti-STAT3 monoclonal antibody were from Pharmingen. Phycoerythrin-conjugated vascular endothelial cadherin (VEcad/CD144) antibody was from Beckman Coulter (Marseilles, France). Anti-vascular endothelial growth factor receptor-2 (Flk-1/KDR) monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-human endothelial nitric oxide synthase (eNOS) rabbit polyclonal antibody (Cayman Chemical, Ann Arbor, MI) were obtained. Anti-phospho-Akt (Ser-473) antibody, anti-Akt antibody, and anti-phospho-STAT3 (Tyr-705) antibody were from Cell Signaling Technology (Beverly, MA). Fibronectin (FN)- and type IV collagen-coated dishes were purchased from Iwaki Co., Tokyo, Japan. Phycoerythrin-conjugated anti-CD14 antibody was from DakoCytomation (Glostrup, Denmark).

Preparation of Peripheral Blood Mononuclear Cells—Human cord blood was kindly supplied by the Metro Tokyo Red Cross Cord Blood Bank (Tokyo, Japan) with informed consent. The buffy coat fraction was prepared from voluntary donated human blood of Saitama Red Cross of Japan (Saitama, Japan). The blood sample was diluted with phosphate-buffered saline (PBS) containing 2 mM EDTA and was loaded on a LymphoprepTM tube (Axis-Shield PoC AS, Oslo Norway) (density = 1.077). After being centrifuged for 20 min 800 x g at 18 °C, mononuclear cells were collected and washed with sorting solution (PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin).

Flow Cytometric Analysis of AC133 and CD34 Expression in Mononuclear Cells—To eliminate the dead cells, dead cells were stained with 7-amino actinomycin D. Mononuclear cells were labeled with PE-conjugated anti-AC133 monoclonal antibody and FITC-conjugated anti-CD34 monoclonal antibody simultaneously at 4 °C for 30 min. After washing with the sorting solution, flow cytometric analysis was performed with a FACSCalibur (BD Biosciences).

Magnetic Cell Sorting of AC133+ Cells—Mononuclear cells were labeled with magnetic bead-conjugated anti-AC133 antibodies according to the protocol directed by the manufacturer. After the brief wash with the sorting solution, the cells were separated by a magnetic cell separator (autoMACS, Miltenyi Biotec, Gladbach, Germany), and the positive cells were then collected.

Culture of AC133+ Cells—Isolated AC133+ cells were cultured in EBM-2 (Cambrex Corp., East Rutherford, NJ) medium containing 20% heat-inactivated FBS and 30 mg/liter kanamycin sulfate at 37 °C under moisturized air containing 5% CO2 with 50 ng/ml VEGF as control medium. Control medium containing VEGF was added with TPO, SCF, or both. Cells were plated on FN- or type IV collagen-coated dishes at a cell density of ~106 cells/ml. We have previously shown that EPCs can tightly adhere to an FN-coated dish but weakly to type IV collagen-coated dish (25). Analysis of adherent EPCs was performed on FN-coated dish and that of suspended EPCs on type IV collagen-coated dish. Half of the medium was exchanged once every 3–4 days with fresh medium. Adherent cells on FN-coated dish were fixed with ethanol chilled to -20 °C and then subsequently subjected to an immunostaining procedure or other treatments. Cells on type IV collagen-coated dish were subsequently subjected to flow cytometric analysis.

Immunostaining of Adherent Cells—After fixation with chilled ethanol (-20 °C), the cell layer was washed three times with PBS. Cells were incubated with 1% bovine serum albumin in PBS (-) for 1 h at 4 °C for blocking and then with each first antibody in 1% bovine serum albumin in PBS (-) for 1 h at 4 °C. After washing with PBS, the cells were incubated with FITC-conjugated anti-mouse IgG antibody or rhodamine-conjugated anti-rabbit IgG antibody for 1 h at 4 °C. Cells were washed with PBS and then examined using a Zeiss LSM 510 microscope with an excitation wavelength of 488 nm and an emission of 530/30 nm for FITC or 570/30 nm for rhodamine.

In every experiment, we used nonspecific immunoglobulin corresponding to the first antibody species as a control and confirmed that the cells were not stained with control immunoglobulin. The fluorescence intensity of 20 randomly selected cells was calculated using the Scion Image program within the linear range for quantitation.

Analysis of Cytokines in the Supernatant of CD31bright and CD31+ Cells—The expression of CD31 on cultured AC133+ cells was determined with a flow cytometer. After AC133+ cells were cultured for several days on either FN-coated or collagen type IV-coated dishes, both adherent and nonadherent cells were collected. The collected cells were labeled with FITC-labeled anti-CD31 antibody for 15 min at 4 °C. After a brief wash with 0.5% bovine serum albumin in PBS, flow cytometric analysis was performed. CD31bright and CD31+ cells were sorted from cultured AC133+ cells with FACSAria (BD Biosciences). Sorted cells of both populations were subsequently cultured in EBM-2 supplemented with 20% FBS in the absence of any cytokines. After 5 days, the collected supernatant of cells was frozen at -20 °C. Cytokines were measured by a BDTM cytometric beads array Flex set system (BD Biosciences) according to the manufacturer's protocol.

Flow Cytometric Analysis of Various Cell Surface Markers in Cultured AC133+ Cells—After AC133+ cells were cultured for the indicated period, cells were co-stained with FITC-labeled anti-CD31 antibody and PE-labeled anti-CD14 antibody or PE-labeled VEcad antibody. Cells were also stained with FITC-labeled anti-CD31 antibody, allophycocyanin-labeled anti-CD110 antibody, and PE-labeled anti-AC133 antibody triply and then subjected to flow cytometry. Dead cells were eliminated by staining with 7-amino actinomycin D.

Calculation of the Absolute Number of CD31bright Cells—The absolute number of CD31bright cells was multiplied by the total cell number of each well, and the ratio of CD31bright cells was analyzed by fluorescence-activated cell sorter.

Preparation of Cell Lysates and Immunoblotting—After cell sorting, AC133+ cells were suspended in 20% FBS-EBM2 and cultured for 3 days in the presence of VEGF and TPO. Cells were collected and incubated in 2% FBS-EBM2 for 1 h. Cells were stimulated by 50 ng/ml TPO, 50 ng/ml VEGF, or both for 15 min. Cells (1 x 106) were collected and lysed in lysis buffer containing 1% Triton X-100, 10 mM K2HPO4/KH2PO4 (pH 7.5), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, and 50 mM beta-glycerophosphate, along with 1/100 (v/v) protease inhibitor mixture (Sigma) and 1/100 (v/v) phosphatase inhibitor mixture (Sigma). The cellular lysate of 5 x 105 cells/lane was subjected to Western blotting analysis.


Figure 1
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  		FIGURE 1.
In vitro differentiation of AC133+ cells of cord blood into endothelial cells. A, expression of AC133 and CD34 cells in human cord blood and peripheral blood mononuclear cells was analyzed by staining with AC133-PE (vertical axis) and CD34-FITC (horizontal axis). The numbers in the flow cytometric dot blots indicates the percentage of each population ± S.D. B, when AC133+ cells were cultured for 19 days in the presence of VEGF on FN-coated dishes, the appearance of CD31+ cells was analyzed. The upper panel shows the fluorescent photomicrograph of adhesion cells stained with FITC-conjugated anti-CD31 antibody after a 5-day culture. Quantitation of the fluorescence intensity of 20 CD31-positive cells was analyzed as described under "Experimental Procedures." Columns and bars represent the means ± S.D. from 20 cells (B, lower panel). C, the CD31-negative, positive, and bright cell populations prepared after 1-week cultivation of AC133+ cells are shown in a representative histogram stained with FITC-conjugated anti-CD31 antibody. The x axis represents the log fluorescence intensity of CD31-FITC, y axis relative cell number (panel a). Panels b and c show phase-contrast microscopic photographs of cultured CD31-positive and bright cells, respectively, subsequently cultured for 1 week after cell sorting. The bottom panels d and e show the fluorescent photomicrographs of adhesion cells from the CD31bright fraction stained with anti-KDR antibody and anti-eNOS antibody, respectively. Scale bar, 100 µm.

 
Statistical Analysis—Statistical analysis was performed using the unpaired Student's t test, and the dose response of TPO was compared between the four groups by one-way analysis of variance and the Tukey test using Prism 4 software. Values of p < 0.05 were considered to indicate statistical significance. Each experiment was repeated three times, and the representative data are indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We previously reported that during the in vitro differentiation of peripheral blood AC133+ cells into the endothelial cells, the expression of CD31 was the earliest marker among all of the tested markers (25). Moreover, by analyzing the ability of differentiation into endothelial cells, CD31bright cells were shown to exhibit EPC character when compared with the CD31+ fraction. Since cord blood is a rich source of blood stem cells such as CD34+ and AC133+ cells, it is expected to be a useful source for CD31bright cells. At first, we attempted to determine whether the CD31bright fraction derived from cord blood AC133+ cells contained EPCs. As shown in Fig. 1A, the populations of AC133+ CD34- cells, AC133- CD34+ cells, and AC133+ CD34+ cells in cord blood were approximately four times greater than those in peripheral blood (Fig. 1A). After 5 days of cultivation of AC133+ cells on an FN-coated dish, adherent CD31-positive cells were observed (Fig. 1B, upper panel). Analysis of the fluorescence intensity of CD31-positive cells revealed that the average fluorescence intensity in CD31+ cells was highest on day 5 (Fig. 1B, lower panel), corresponding to the results of peripheral blood cells.

After 1 week of cultivation of AC133+ cells on a collagen type IV-coated dish, on which cells adhered more loosely when compared with the FN-coated dish, cells were collected and sorted into CD31+ and CD31bright fractions, as shown in Fig. 1C, panel a, and both cell types were cultured on an FN-coated dish for 1 week after the sorting. The number of cells adhering and spreading was higher in the CD31bright fraction (Fig. 1C, panel c) than in the CD31+ fraction (Fig. 1C, panel b), and these adhering cells are apparently KDR- (Fig. 1C, panel d) and eNOS-positive (Fig. 1C, panel e). The large areas of intense green fluorescence represent the colonies of CD31bright cells. These data indicate that CD31bright cells derived from AC133+ cells of both peripheral blood and cord blood are EPCs.


Figure 2
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  		FIGURE 2.
Various cytokines released from CD31+ cells and CD31bright cells. Production of various cytokines from CD31+ cells and CD31bright cells derived from AC133+ cells cultivated for 5 days was measured. Gray columns indicate the cytokine production by cells from peripheral blood and open columns from cord blood. Columns and bars represent the means ± S.D. from three separate experiments. TNF, tumor necrosis factor; Pos, positive; MCP-1, monocyte chemoattractant protein-1.

 
Several reports have shown that EPCs produce cytokines (9, 26, 27), but the ability of CD31+ or CD31bright cells derived from AC133+ cells to produce cytokines is not known. After cell sorting, quantitative analysis of cytokines released by CD31+ cells and CD31bright cells was carried out at 5 days after the cultivation. As shown in Fig. 2, IL-8 was markedly produced by CD31bright cells from both peripheral blood and cord blood when compared with CD31+ cells. The production of monocyte chemoattractant protein-1 (MCP-1) by CD31bright cells was also higher than that of CD31+ cells. The production of VEGF was higher by CD31bright cells than by CD31+ cells but not significantly. The production of all cytokines by CD31bright cells from peripheral blood was higher than that from cord blood. Tumor necrosis factor-{alpha}, GM-CSF, and G-CSF were hardly produced by CD31bright and CD31+ cells. These data indicate that CD31bright cells derived from AC133+ cells have a strong ability to produce chemokines.

It has been reported that TPO and SCF are potent stimulators of multipotent cell proliferation (17, 19). Next, the effects of both growth factors on EPC growth and differentiation in our culture system were determined. After the addition of both TPO and SCF for 2 weeks, the expression of eNOS and KDR in adhered cells was analyzed (Fig. 3A). Fig. 3A clearly indicates that AC133+ cells from both peripheral blood and cord blood differentiate into eNOS+ and KDR+ cells more efficiently in the presence of the mixture of TPO, SCF, and VEGF than of VEGF alone. Flow cytometric analysis revealed that the ratio of CD31bright CD14- cells increased in the presence of the mixture of TPO, SCF, and VEGF when AC133+ cells were cultured on collagen type IV-coated dish for 1 week (Fig. 3B).

We next examined which growth factor is dominant in the induction and proliferation of CD31bright cells. The total cell number of cultured AC133+ cells from both peripheral blood (Fig. 4A, upper panel) and cord blood (Fig. 4A, lower panel) significantly increased in the presence of TPO, SCF, or both growth factors when compared with that of VEGF alone during a 1-week period. As shown in Fig. 4B, however, the increment in the ratio of the CD31bright cell population was observed only in the presence of TPO. The absolute number of CD31bright cells, calculated by the total cell number and the ratio of the CD31bright cell population, was markedly increased by TPO (Fig. 4C). In contrast, SCF induced the increase in total cell number to the same level as TPO (Fig. 4A), but it did not induce the increase in either the ratio of the CD31bright cell population (Fig. 4B) or the number of CD31bright cells (Fig. 4C). Next, we examined whether TPO and VEGF can synergistically affect the induction of CD31bright cells during a 1-week cultivation. As shown in Fig. 4D, although VEGF had no effects on the total cell number (Fig. 4D, panel a), it increased the ratio of the CD31bright cell population to 1.4-fold higher than that of the control (Fig. 4D, panel b), resulting in a slight increase in the number of CD31bright cells (Fig. 4D, panel c). Thrombopoietin alone induced an increase in not only the total cell number (Fig. 4D, panel a) but also the ratio of the CD31bright cell population (Fig. 4D, panel b), resulting in an ~24-fold increment of the absolute number of CD31bright cells when compared with the control (Fig. 4D, panel c). The concomitant treatment with both VEGF and TPO showed a synergic increase in the number of CD31bright cells (Fig. 4D, panel c).


Figure 3
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  		FIGURE 3.
Increment of EPCs from AC133+ cells in the presence of TPO and SCF. A, AC133+ cells were differentiated for 2 weeks in the presence of either VEGF alone or the combination of TPO, SCF, and VEGF on an FN-coated dish. The upper and middle panels indicate the fluorescent photomicrographs of cells stained with anti-eNOS antibody and anti-KDR antibody, respectively. The bottom panels indicate the merged images of both antibodies. From the left side, control (Cont) and the mixture of peripheral blood, control, and the mixture of cord blood. Scale bar, 100 µm. B, CD14 and CD31 expression in cultured AC133+ cells for 1 week was stained with CD14-PE (vertical axis) and CD31-FITC (horizontal axis). The upper panel indicates cells treated with VEGF alone, and the lower panel indicates cells treated with the mixture of VEGF, SCF, and TPO. The number on the right side of the flow cytometric dot blot indicates the percentage of the CD14- CD31bright population.

 


Figure 4
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  		FIGURE 4.
Stimulative effects of TPO on induction of CD31bright cells. A, alteration of the cell number of cultivated AC133+ cells for 1 week in the combination of growth factors. Mix, VEGF + SCF + TPO. B, the flow cytometric histogram of AC133+-derived cells stained with FITC-labeled anti-CD31 antibody after a 1-week culture. The representing number in the flow cytometric histogram indicates the percentage of the CD31bright cell population. The left panels are peripheral blood, and the right panels are cord blood. C, CD31bright cells numbers were calculated by both the total cell number and the ratio the of CD31bright population. D, the effects of TPO alone on EPC differentiation derived from AC133+ cells of cord blood. The upper left panel (a) shows the total cell number after a 1-week culture, the right panels (b) show the flow cytometric histogram of AC133+-derived cells stained with FITC-labeled anti-CD31 antibody, and the lower left panel (c) shows the calculated CD31bright cell number. Columns and bars represent the means ± S.D. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). NS, not significant; Cont, control.

 


Figure 5
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  		FIGURE 5.
Dose-dependent effects of TPO on the induction of CD31bright cells from AC133+ cells. AC133+ cells were treated with various concentrations of TPO for 1 week. The left panels (A) are the total cell number of cultured AC133+ cells from peripheral blood (upper panel) and cord blood (lower panel). The right panels (B) are the calculated CD31bright cell number from peripheral blood (upper panel) and cord blood (lower panel). Columns and bars represent the means ± S.D. (**, p < 0.01; ***, p < 0.001). Striped and dotted columns represent CD31brightVEcad+ cells and CD31brightVEcad- cells, respectively.

 
When AC133+ cells were cultured with various concentrations of TPO in the presence of constant concentrations of VEGF (50 ng/ml), the total cell number from both peripheral blood (Fig. 5A, upper panel) and cord blood (Fig. 5A, lower panel) significantly increased at 5 ng/ml of TPO when compared with the control, and there was no significant difference in the total cell number from 5 to 50 ng/ml of TPO. However, TPO increased the ratio of CD31bright cells of flow cytometry dose-dependently as follows: control, 0.50%; 5 ng/ml, 1.36%; 10 ng, 1.42%; 50 ng/ml 1.90% in peripheral blood and control, 1.16%; 5 ng/ml, 1.99%; 10 ng, 2.51%; 50 ng/ml 2.96% in cord blood. TPO markedly induced the differentiation of AC133+ cells into CD31brightVEcad+ cells in the case of both peripheral blood (Fig. 5B, upper panel) and cord blood (Fig. 5B, lower panel) in a dose-dependent manner. In the case of cord blood cells, differentiation into CD31brightVEcad- cells was also induced by TPO.

The effects of TPO on total cell number during 6-day culture of AC133+ cells were determined. Although the total cell number from AC133+ cells slightly and constantly increased from day 0 to day 6 in the absence of TPO, total cells markedly increased after the third day in the presence of TPO (Fig. 6A). Next, the alternation of TPO receptor (CD110) expression was analyzed during the cultivation of AC133+ cells. Although the percentages of both AC133+ CD110+ cells and CD31+ CD110+ cells were 0% just after magnetic cell sorting, 3 days after the cultivation, ~2% of CD31+ CD110+ cells (Fig. 6B, left panel) and 1% of AC133+ CD110+ cells (Fig. 6B, right panel) appeared from AC133+ cells in the peripheral blood and cord blood, respectively. These data indicate the possibility that sorted AC133+ cells may differentiate into AC133+ CD110+ cells and may subsequently proliferate and differentiate into EPCs in response to TPO.


Figure 6
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  		FIGURE 6.
Time-course analysis of TPO-treated AC133+ cells and expression of TPO receptor (CD110). A, alteration of cell number was counted at 2, 3, and 6 days. Solid and dotted lines indicate TPO-treated cells and control ((Cont) VEGF alone) cells, respectively. The results represent mean ± S.E. of triplicate wells. B, flow cytometric analysis of CD110 expression on AC133+ cells cultured for 3 days was carried out. The y axis represents the log fluorescence intensity of CD110-allophycocyanin (APC), and the x axis represents that of CD31-FITC (left panels) and AC133-PE (right panels). The number in the flow cytometric dot blot indicates the percentage of CD110+ CD31+ and CD110+ AC133+ populations, respectively. The upper panels are peripheral blood, and the lower panels are cord blood.

 
It has been reported that TPO activates the PI3K/Akt pathway (28) or JAK/STAT pathway (20, 29, 30) in target cells. In addition, in the present study, TPO induced a marked proliferation of AC133+ cells after 3-day culture, and CD110 expression in cells cultured for 3 days from both cord blood and peripheral blood was also observed (Fig. 6, A and B). We then attempted to determine whether TPO activates Akt or STAT in AC133+ cells cultured for 3 days by analyzing the phosphorylation at Ser-473 of Akt or the phosphorylation at Tyr-705 of STAT3, which are the active forms of Akt or STAT3, respectively. As shown in Fig. 7A, phosphorylation at Ser-473 of Akt was stimulated by both VEGF and TPO at 15 min and was more markedly stimulated by concomitant treatment with VEGF and TPO than by a single treatment (Fig. 7A, top panel). Phosphorylation at Tyr-705 of STAT3 was observed only in the presence of TPO, and unlike in the phosphorylation at Ser-473 of Akt, an increased amount of phosphorylation was not observed in the concomitant presence of VEGF and TPO (Fig. 7A, third panel). On the other hand, there was no difference in the expression of Akt and STAT3 protein levels (Fig. 7A, second panel and bottom panel, respectively). The induction of CD31bright cells was not perfectly but significantly inhibited by wortmannin, an inhibitor of PI3K, suggesting that the PI3K/Akt pathway plays an important role in TPO-induced EPC differentiation (Fig. 7B).


Figure 7
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  		FIGURE 7.
Analysis of TPO-induced signal transduction on AC133+ cells of cord blood. A, activation of Akt or STAT3 was analyzed by Western blotting with anti-phospho-specific Ser-473-Akt antibody (top panel) and reprobed with anti-Akt antibody (second panel), or with anti-phospho-specific Tyr-705-STAT3 antibody (third panel) and reprobed with anti-STAT3 antibody (lower panel) after stimulation by VEGF, TPO, or both VEGF and TPO for 15 min using 3-day-cultured AC133+ cells. C, control; V, VEGF; T, TPO. B, the effects of wortmannin on CD31bright cell induction were investigated. The right panel shows peripheral blood, and the left panel shows cord blood. The y axis represents the CD31bright cell number. Wort, 100 nM wortmannin. Columns and bars represent the means ± S.E. (*, p < 0.05; ***, p < 0.001). Cont, control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously reported that CD31bright cells derived from AC133+ cells in human peripheral blood are EPCs (25). In the present study, CD31bright cells also appeared from AC133+ cells prepared from cord blood, which are a rich source of stem cells during the early period of cultivation (Fig. 1, A and B). When cells were separated in terms of CD31 expression (Fig. 1C), CD31bright cells differentiated into KDR-positive and eNOS-positive adherent cells. These data indicate that CD31bright cells derived from AC133+ cells in cord blood have some characteristics similar to those of EPCs in peripheral blood. Although these EPCs in both cord blood and peripheral blood could not form tube-like structure by themselves on Matrigel (data not shown), they secreted angiogenic growth factors (Fig. 2) such as VEGF, IL-8 (31, 32), and monocyte chemoattractant protein-1 (MCP-1) (33). It has been reported that there are at least two types of EPCs: early EPCs and late EPCs. Early EPCs are unable to form tube-like structures and secrete VEGF and IL-8 showing peak growth at 2–3 weeks (9, 26, 27). Late EPCs with the ability to proliferate and having a cobblestone shape appear late at 2–3 weeks, show exponential growth at 4–8 weeks, and have the ability to form tube-like structures (26, 27, 34). Rehman et al. (9) have reported that EPCs derived from monocytes/macrophages do not proliferate but instead release potent proangiogenic growth factors. In many studies (9, 26, 27, 35–37), because the origin of early EPCs was CD14+ cells or was not precluded by monocytic cells, CD14 expression was still observed in the EPCs after cultivation. In our study, in which AC133+ cells were used as the origin of the EPCs, CD14 expression was not observed in CD31bright cells induced by TPO (Fig. 3B). Although the CD31bright cells identified as EPCs in this report and in a previous report did not correspond to their cells in terms of the origin of the cells or cell surface markers, these cells may be early EPCs that can release potent proangiogenic growth factors (Fig. 2). In any event, EPCs are thought to be a heterogeneous population, unlike late EPCs, which have a high ability to proliferate.

Circulating EPCs are up-regulated under physiological or pathological conditions and also by 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (14, 15) and cytokines such as erythropoietin (11–13) and G-CSF (10). In this report, we have revealed the possibility of marked expansion of EPCs in vitro by TPO. Brizzi et al. (20) have reported that TPO directly stimulates endothelial cell motility and neoangiogenesis. In the present study, TPO may have played a stimulatory role in the differentiation of EPCs from circulating stem cells.

Although both TPO and SCF have the same potency with regard to proliferation of AC133+ cells (Fig. 4A), TPO specifically induces an increase in the ratio of the CD31bright cell population when compared with SCF (Fig. 4, B and C). To develop useful cell therapy products for severe ischemia, it has been considered desirable to establish the efficient expansion of EPCs in vitro. Thrombopoietin could increase CD31bright cells (EPCs) even in the absence of VEGF. Kirito et al. (38) have reported that TPO enhances expression of VEGF in hematopoietic cells through induction of hypoxia-inducible factor 1{alpha}. These observations suggest the possibility that the production of EPCs by TPO may be supported by VEGF produced by AC133+ cells. However, from the perspective that TPO and VEGF have synergistic effects on the induction of EPCs, TPO seems to induce EPCs through another signaling cascade.

Thrombopoietin is a major regulator of the proliferation, differentiation, and maturation of megakaryocytes (39, 40). The results from recent studies suggest that TPO can act not only as a lineage-specific hematopoietic growth factor but also can affect other hematopoietic cell types. For example, TPO alone does not induce proliferation of long term repopulating hematopoietic stem cells. However, in combination with SCF or IL-3, TPO has several synergistic effects on cell proliferation (19). Our results have revealed a new role of TPO in the production of EPCs.

In the process of differentiation of AC133+ cells into CD31bright cells, both peripheral blood and cord blood appear to be very similar. AC133+ cells of cord blood, however, have a stronger ability to proliferate than those of peripheral blood (Fig. 6A). Moreover, TPO stimulates the induction of CD31brightVEcad- cells only from cord blood (Fig. 5B) at high concentrations. Hur et al. (26) have reported that VEcad- EPCs are thought to be an early EPC. It is therefore thought that AC133+ cells of cord blood are more immature than those of peripheral blood.

Although the total cell number treated with TPO slightly increased in a dose-dependent manner (Fig. 5A), the CD31bright cell number markedly increased as the TPO concentration increased (Fig. 5B). These data suggest the possibility that a higher concentration of TPO may be needed for CD31bright cell induction from AC133+ cells.

When AC133+ cells were stimulated by TPO or VEGF, an increase in the phosphorylation of Akt at Ser-473 was observed. This increase was strongly enhanced by concomitant treatment with VEGF and TPO (Fig. 7A). The induction of CD31bright cells by these growth factors (Fig. 4D) was consistent with the increase in the phosphorylation of Akt at Ser-473. TPO but not VEGF could also stimulate the phosphorylation of STAT3 at Tyr-705. We previously reported that the PI3K/p70 S6 kinase pathway and the JAK/STAT3 pathway were important for proliferation and differentiation, respectively, in neutrophilic differentiation (41, 42). Owing to the stimulation of both the PI3K/Akt and the JAK/STAT pathways, we postulated that TPO may be a stronger stimulator of EPC production than VEGF. As shown in Fig. 7B, however, wortmannin could not completely inhibit the induction of CD31bright cells. Therefore, a pathway other than the PI3K/Akt pathway may also work for the proliferation and differentiation of EPCs.

The observation of unfavorable angiogenesis has recently been reported after transplantation of bone marrow mononuclear cells in patients with thromboangiitis obliterans (43). Moreover, transfer of both spleen cell-derived EPCs and bone marrow mononuclear cells accelerate atherosclerosis in apoE knockout mice, whereas EPC transfer reduces markers associated with plaque stability (44). These observations suggest that transplantation of differentiated cells from EPCs may be useful therapy as regenerative medicine.

In conclusion, we have demonstrated a new role of TPO in enhancing the differentiation of AC133+ cells into CD31bright cells (EPCs) in vitro. These findings may contribute to further development of cell therapy for critical ischemia.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid for health and labor science research from the Japanese Ministry of Health, Labor, and Welfare, and in part by a grant-in-aid for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-3-3700-9064; Fax: 81-3-3707-6950; E-mail: yamaguch{at}nihs.go.jp.

2 The abbreviations used are: EPCs, endothelial precursor cells; VEGF, vascular endothelial growth factor; FN, fibronectin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PE, phycoerythrin; TPO, thrombopoietin; SCF, stem cell factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; VEcad, vascular endothelial cadherin; eNOS, endothelial nitric oxide synthase; FBS, fetal bovine serum; STAT, signal transducers and activators of transcription; JAK, Janus kinase; KDR, kinase insert domain-containing receptor. Back


   ACKNOWLEDGMENTS
 
We thank Saitama Red Cross of Japan (Saitama, Japan) and Metro Tokyo Red Cross Cord Blood Bank (Tokyo, Japan) for their kind cooperation. We also thank Kirin-Amgen Inc. for their kind gift of recombinant TPO and recombinant SCF.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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