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a Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan;
b Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Yonago, Tottori, Japan;
c The Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga, Japan;
d Department of Pathology, Stanford University School of Medicine, Beckman Center, Stanford, California, USA
Key Words. Clonal assays • Development • Embryonic stem cell • Experimental models • Hematopoiesis • In vitro culture • Mice • Progenitor • Osteoclast
Shin-Ichi Hayashi, M.D., Ph.D., Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Machi, Yonago, Tottori, 683-8503, Japan. Telephone: 81-859-34-8269; Fax: 81-859-34-8272; e-mail: shayashi{at}grape.med.tottori-u.ac.jp
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand (RANKL) have been reported to be critical for osteoclastogenesis [10–12], the mechanisms of osteoclast development in the early phase have not been clearly elucidated.
To investigate osteoclast development from the earliest stage, we have developed an in vitro culture system that supports the differentiation of embryonic stem (ES) cells to mature osteoclasts. In this system, undifferentiated ES cells are cocultured with a stromal cell line (ST2) in the presence of 1
,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dexamethasone (Dex) for 10 days without any passage or manipulation (hereafter called the "one-step culture") [13, 14]. This is an appropriate system for examining the potential of ES cells to generate osteoclasts, as these cultures are stable and enable us to assess the whole process of osteoclastogenesis. We also employed a step-wise culture system, which experimentally divides the formation of osteoclasts from ES cells into three phases. In the first phase (phase I), ES cells are cultured on another stromal cell line (OP9) for 5 days. ES cells generate colonies containing hematopoietic progenitors [15], including OCPs, which was demonstrated by the transfer of these cells onto ST2 cells and further cultivation for 6 days in the presence of 1,25-(OH)2D3 and Dex (two-step culture) [13, 14]. This system allows the separation of the maturation phase on ST2 cells from the phase for the generation of OCPs on OP9 cells. When the cells are transferred on day 5 onto fresh OP9 cells and cultured for an additional 5 days, hematopoietic cells increase. From the cells from wild-type ES cells recovered on day 10, osteoclasts can be generated more efficiently on ST2 cells after 6 days of culture in the presence of 1,25-(OH)2D3 and Dex (three-step culture) than in the two-step culture [13, 14]. In addition to the generation phase (phase I) and the maturation phase of OCPs, we can also observe an amplification and/or another generation phase (phase II) of OCPs between these two phases using the three-step culture system. This culture system enables us to observe OCPs at the time of transfer onto ST2 stromal cells [13, 14].
Using mutant ES cells in this culture system, molecules required for early osteoclast development were assessed [14]. Moreover, distinct dependency on signaling via Kit receptor tyrosine kinase and on the GATA-2 transcription factor accounted for the difference between OCPs and CFUs-M [14]. The majority of osteoclasts are generated in a Kit-independent manner [5, 9, 13]. OCP but not CFU-M generation from GATA2-null ES cells is severely reduced [14]. These results suggest that a part of the osteoclast lineage might diverge from other myeloid lineages at a relatively early stage of hematopoiesis.
Hematopoietic cells share common progenitor cells, named hemangioblasts, with endothelial cells. In Vegfa-null embryos, both vasculogenesis and hematopoiesis may be impaired, resulting in death at midgestation [16, 17]. This is why the effect of the mutation on the osteoclast lineage has not been explored. In this study, we attempted to examine osteoclast development from the earliest stage by adding an Flt-1 (vascular endothelial growth factor receptor [VEGFR]-1-human [h] IgG1-Fc portion chimeric protein [VEGFR-1/Fc]) to our one-step and step-wise ES cell cultures. The addition of VEGFR-1/Fc, which binds to VEGFs and neutralizes their function, should mimic the effect of knocking out the Vegfa gene.
We report here the characteristics of OCPs developed in the first 5 days of culture (phase I) and OCPs after culturing for a further 5 days (phase II) as indicated by the effects of VEGFR-1/Fc and antagonistic anti-Kit antibody (Ab) on the number of osteoclasts derived from single OCPs and the period of differentiation into mature osteoclasts. The majority of OCPs appeared to be derived from ES cells via a mechanism dependent on the binding of factor(s) to VEGFR-1 in phase I.
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MATERIALS AND METHODS |
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-MEM; GIBCO/BRL) containing 20% FBS, streptomycin, and penicillin [13, 21, 22].
Induction of Differentiation of ES Cells
Induction of the differentiation of ES cells was described previously [13, 21, 22]. For one-step cultures, undifferentiated ES cells (75 cells/well) were added onto confluent ST2 monolayers in 24-well culture plates (Corning Inc.; Acton, MA; http://www.corning.com) and cultured in -MEM supplemented with 10% FBS, streptomycin, penicillin, 10-7 M Dex (Sigma Chemical Co. Ltd.; St. Louis, MO; http://www.sigmaaldrich.com), and 10-8 M 1,25-(OH)2D3 (Biomol Research Laboratories, Inc.; Plymouth Meeting, PA; http://www.biomol.com). Cultures were fed every 3 days by replacing spent medium with fresh medium. In one-step cultures, formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining on day 10.
For multistep cultures, undifferentiated ES cells (104 cells) were inoculated in 6-well culture plates (Corning), seeded with OP9 stromal cells, and cultured in -MEM supplemented with 20% FBS (Intergen Company; Burlington, MA; http://www.intergen.com), streptomycin, and penicillin. On day 5, a cell suspension in phase I was prepared by treatment with 0.25% trypsin/0.5 mM EDTA (GIBCO/BRL). Harvested cells (103) were induced to differentiate into osteoclasts on ST2 cells for 6 days in the same medium as used for the one-step culture (two-step culture). For the three-step culture, the harvested cells (105) were further cultured for 5 days on a fresh OP9 stromal layer under the same conditions used for phase I. Hematopoietic cells generated on the OP9 cells were harvested by pipetting culture medium over the cell layers on day 10. The cells recovered were induced to differentiate into osteoclasts, as described previously, and TRAP staining was performed 6 days later. All cultures were incubated at 37°C with 5% CO2 in a humidified incubator [13, 14, 21].
In these experiments, 100 ng/ml of a recombinant mouse VEGFR-1 (Flt-1)/Fc chimera (R&D Systems, Inc.; Abington, UK; http://www.rndsystems.com), 100 ng/ml of human IgG1 (Chemicon International, Inc.; Temecula, CA; http://www.chemicon.com), or 0.01 mg/ml of a rat anti-mouse monoclonal antagonistic Ab against Kit (ACK2) [23] was added to the cultures.
OCP Frequency Analysis
The frequency of OCPs was determined by limiting dilution assay [9]. Various numbers of cells harvested on day 5 or day 10 from phase I or II, respectively, were inoculated into wells of 96-well plates (Corning), preseeded with ST2 cells, and cultured for 6 days in the presence of 1,25-(OH)2D3 and Dex. The presence of osteoclasts was determined by TRAP staining. After calculating the frequency of OCPs, the numbers of TRAP-positive cells in the wells containing TRAP-positive cells were counted.
TRAP Staining
Cultured cells were fixed with 10% formalin (3.7% formaldehyde; Wako) in phosphate-buffered saline for 10 minutes with ethanol-acetone (50:50; v/v; Wako) for 1 minute at room temperature and incubated in acetate buffer (pH 5.0; Sigma) containing naphthol AS-MX phosphate (Sigma) as a substrate and fast red violet LB salt (Sigma) as a stain in the presence of 50 mM sodium tartrate (Wako) [24].
Antibodies
Monoclonal rat anti-mouse Abs directed against Kit (ACK2) [23] and CD31 (Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen) were used for staining.
Reverse Transcription-Polymerase Chain Reaction
Hot-lid polymerase chain reaction (PCR) amplification of cDNA equivalent to 20 ng of total RNA was carried out in 1 x PCR buffer (1.5 mM MgCl2) containing 0.2 mM deoxynucleoside triphosphate (Takara; Shiga, Japan; http://www.takara-bio.co.jp/english), 0.75 U of rTaq DNA polymerase (Toyobo; Osaka, Japan; http://www.toyobo.co.jp/e), and 500 nM primers. Amplifications were carried out on a DNA thermal cycler (PTC DNA Engine; MJ Research Inc.; Watertown, MA; http://www.mjr.com). Following an initial 1-minute denaturation step (94°C), each PCR cycle consisted of 30 seconds of denaturation (94°C), 1 minute of annealing (55°C), and 1 minute of elongation (72°C). After the final cycle, the reaction was held for 3 minutes at 72°C. The PCR products were then separated on 1.5% agarose gel, stained with ethidium bromide, and photographed. The primers used were as follows: ß-globin (Hbb): 5'-CAC AAC CCC AGA AAC AGA CA-3' and 5'-CTG ACA GAT GCT CTC TTG GG-3'; 
-globin (Hbb-bh1): 5'-GCT CAG GCC GAG CCC ATT GG-3' and 5'-TAG CGG TAC TTC TCA GTC AG-3'; and hypoxanthine phosphoribosyl transferase (Hprt): 5'-AAT GAT CAG TCA ACG GGG GAC A-3' and 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'.
Statistical Analysis
The numbers of OCPs derived from single ES cells on day 5 (phase I) and on day 10 (phase II) were calculated as follows: phase I = [(the frequency of OCPs on day 5) x (the number of recovered cells per well on day 5)] / 104 and phase II = [(the frequency of OCPs on day 10) x (the number of recovered cells per well on day 10)] / 105 x [(the number of recovered cells per well on day 5) / 104]. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed by the Student’s t-test.
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RESULTS |
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,25-(OH)2D3 and Dex. Usually, TRAP-positive osteoclasts were located at the periphery of the colonies (Fig. 1A
), while endothelial cells were arrayed radially (Fig. 1B) [26, 27]. Under these conditions, we continuously supplemented the culture medium with 100 ng/ml VEGFR-1/Fc and found that, on day 10, the generation of endothelial cells expressing CD31 and of TRAP-positive osteoclasts were markedly reduced. The majority of ES cell colonies lacked both CD31-positive cells and TRAP-positive cells (Fig. 1C), but some of the colonies contained small numbers of CD31-positive cells and/or TRAP-positive cells (Fig. 1D and 1E). We used the same dose of hIgG1 as an Fc control and ascertained that it did not influence the generation of CD31-positive endothelial cells or TRAP-positive osteoclasts.
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). The plating efficiency of ES cells in the one-step culture was estimated based on the numbers and sizes of the colonies formed, and no difference was observed between cultures with versus without VEGFR-1/Fc for 10 days (data not shown). These results indicate that VEGFR-1/Fc affects osteoclastogenesis negatively during days 0–5 in this culture system.
VEGFR-1/Fc Inhibits OCP Generation in the Early Phase of ES Cell Culture
To assess the stage of osteoclast development affected by the addition of VEGFR-1/Fc, we performed the two-step culture with VEGFR-1/Fc (Table 1). ES cells (104/well) were cultured on OP9 stromal cell layers with or without VEGFR-1/Fc, anti-Kit Ab, or hIgG1 for 5 days (phase I), and growing cells were harvested. These cultures were repeated four times independently using two different ES cell lines (D3 and CCE) (upper section of Table 1). On day 5, the numbers of cells recovered from cultures with and without VEGFR-1/Fc, anti-Kit Ab, or hIgG were comparable (Table 1). Flow cytometric analysis demonstrated that CD45-positive hematopoietic cells comprised less than 0.3% of the recovered cells with or without VEGFR-1/Fc (data not shown). The harvested cells (103/well) were transferred onto ST2 cell layers in the presence of 1,25-(OH)2D3 and Dex for 6 days. In three of five trials in which VEGFR-1/Fc was added during phase I of the cultures, the generation of TRAP-positive MNCs was totally abolished (Experiments #1, #3 with CCE cells, and #4 of the middle section of Table 1), and only a few TRAP-positive MNCs were detected in the other two experiments (Experiments #2 and #3 with D3 cells in Table 1).
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,25-(OH)2D3 and Dex for 6 days. In three of four trials, no osteoclasts were obtained in the presence of VEGFR-1/Fc and, therefore, the frequency of OCP-I was estimated as less than 1 in 1,920 or 3,840 cells. In Experiment #2 (the bottom section of Table 1), OCP-I were obtained at a frequency of 1 in 940 cells in the presence of VEGFR-1/Fc, which is only 3.7% of the frequency of OCP-I obtained without VEGFR-1/Fc (1 OCP-I in 34 cells). Using the results from Experiment #2, we calculated that a single ES cell gave rise to 9.9 OCPs in the cultures with medium alone, but only 0.2 OCPs in those with VEGFR-1/Fc (the upper section of Table 2). Similar results from Experiment #3 are also shown in Table 2.
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) [13].
OCPs in Phase II Are Inhibited by VEGFR-1Fc and/or Anti-Kit Abs
After ES cells are cultured on OP9 for 5 days, if the harvested cells are recultured on a fresh OP9 cell layer for an additional 5 days (phase II), hematopoietic progenitors expand efficiently [15]. To determine the effect of VEGFR-1/Fc on OCP generation in this phase, we performed three-step cultures under various conditions (Table 2 and Fig. 2). We reported previously that the increase in the OCP number in phase II was 10- to 20-fold [14].
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; day 5 in Table 2). On day 10 of two-step culturing with or without VEGFR-1/Fc in phase I and without VEGFR-1/Fc in phase II, the developing cells were transferred into the osteoclast culture conditions for 6 days. The OCPs present on day 10 of phase II are referred to as OCP-II. The number of TRAP-positive MNCs obtained from cells treated with VEGFR-1/Fc in phase I was approximately one-third the number obtained by culturing the cells in medium alone (Fig. 2). The number of TRAP-positive MNCs was further decreased by the addition of VEGFR-1/Fc or the anti-Kit Ab in phase II; however, neither reagent inhibited the generation of these cells completely. The combination of both VEGFR-1/Fc and anti-Kit Ab further reduced the number of TRAP-positive MNCs compared with the addition of either one alone (Fig. 2).
We also assessed the frequency of OCP-I and OCP-II using the limiting dilution assay. D3 ES cells cultured in medium alone in both phases I and II contained OCP-II at a frequency of one OCP-II in 5.7 or 4.8 cells, and single ES cells gave rise to 107 or 13 OCP-II (Experiments #2 or #3, respectively, in Table 2). In the cultures treated with VEGFR-1/Fc in phase I, the frequency of OCP-II was significantly greater than that of OCP-I; however, since the number of cells recovered on day 10 was lower in this experiment (#2), the number of OCPs obtained from a single ES cell was still low (Table 2). In Experiment #3 using CCE ES cells, the number of cells recovered in phase II after culturing with VEGFR-1/Fc in phase I was approximately half the number of cells recovered after culturing with medium alone, but the frequency of OCP-II and the number of OCP-II obtained from a single ES cell in the cultures with VEGFR-1/Fc in phase I were still significantly lower than those with medium alone (Table 2). Therefore, the reduction of OCP-II generation was related to the treatment with VEGFR-1/Fc in phase I, even if the number of cells recovered was different in phase II.
Moreover, the presence of both VEGFR-1/Fc and anti-Kit Ab during phase II following the addition of VEGFR-1/Fc during phase I further decreased the frequency of OCP-II (Table 2). In contrast, the addition of anti-Kit Ab during phase II reduced the number of TRAP-positive MNCs from ES cells cultured with VEGFR-1/Fc in phase I, but the frequency of OCP-II was comparable with that obtained in medium only (Table 2). Continuous addition of either hIgG1 or anti-Kit Ab for 10 days throughout phases I and II did not decrease the number of TRAP-positive MNCs (Fig. 2). This result suggests that OCPs may require different signaling to develop in phases I and II.
Early Steps in Erythropoiesis also Require VEGF Family Cytokines
Signaling via VEGFRs is critical for the early development of hematopoietic cell lineages [16, 17]. Therefore, the addition of VEGFR-1/Fc to ES cell cultures may widely affect the development of hematopoietic cell lineages. To examine this effect, we assessed erythropoiesis in our culture system. Semiquantitative reverse transcription (RT)-PCR for - (Hbb-bh1) and ß-hemoglobin (Hbb) gene expression was carried out to detect the presence of erythropoiesis. ES cell cultures treated with VEGFR-1/Fc in phase I contained approximately 10 times less Hbb-bh1 transcript than those cultured with medium alone or with hIgG1 (Fig. 3A and 3B). We could not detect any message from the Hbb gene in the VEGFR-1/Fc-treated cultures (Fig. 3A). These results indicate that the addition of VEGFR-1/Fc during phase I inhibits the development of both - and ß-hemoglobin-producing erythrocytes.
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. The number of CFUs-M present in the VEGFR-1/Fc-treated culture (1 CFUs-M per 27,027 recovered cells) was only 1.2% of the number obtained with medium alone (1 per 322 cells) (Table 3). The number of CFUs-M generated from a single ES cell was 1.06 with medium alone and 0.008 with VEGFR-1/Fc (Table 3).
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). The number of CFUs-M in phase II derived from a single ES cell was almost identical to the number of CFUs-M cultured with (0.97 CFUs-M/ES cell) or without (0.95) VEGFR-1/Fc in phase I. Surprisingly, the number of CFUs-M from a single ES cell in phase I (1.06 CFUs-M/ES cell) was also similar to that in phase II (0.95) (Table 3). This indicates that the CFUs-M in the control culture did not increase during phase II, whereas those in the cultures treated with VEGFR-1/Fc (0.008 CFUs-M/ES cell) in phase I increased to the level of those in cultures treated with medium only (0.97).
The Number of TRAP-Positive Cells Derived from Single OCPs
To compare the characteristics of the OCPs generated in phases I and II, we investigated the number of TRAP-positive cells derived from single OCP-I and OCP-II (Table 4). After calculating the frequency of OCPs, the numbers of TRAP-positive cells in the wells containing TRAP-positive cells were assessed. Since no TRAP-positive cells were detected in the VEGFR-1/Fc-treated cultures in three of five trials when VEGFR-1/Fc was added in phase I of the two-step culture, we could not make comparisons using these experiments. In Experiment #2 (Table 1), a single VEGFR-1/Fc-treated OCP-I gave rise to approximately one sixth the number of TRAP-positive cells (23) derived from an OCP-I cultured in medium only (136) (Table 4).
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). Comparable numbers of TRAP-positive cells were generated from a single OCP-II in cultures with (57) and without (67) VEGFR-1/Fc during phase I, although the frequency of the OCP-II cultured with VEGFR-1/Fc was extremely low (Table 2). Interestingly, the OCP-I remained in the culture with VEGFR-1/Fc in phase I and gave rise to low numbers of TRAP-positive cells similar to the numbers derived from the OCP-II resistant to the VEGFR-1/Fc-treatment in both phases I and II (23 cells/OCP-I and 26 cells/OCP-II, respectively) (Table 4).
These results indicate that a single OCP-I produces a higher number of osteoclasts than a single OCP-II, and that treatment with VEGFR-1/Fc in phase I does not affect the number of osteoclasts generated from a single OCP-II. Similar results were obtained by using CCE ES cells (Experiment #3 in Table 4), although OCP-I were not present.
The Timing of TRAP-Positive Cell Appearance in the Osteoclast Culture
The period of TRAP-positive cell appearance after the induction of ES cells into osteoclasts was examined. Cells transferred to osteoclast culture conditions on day 5 of culturing with or without VEGFR-1/Fc differentiated into TRAP-positive cells 4 or 3 days after the transfer, respectively. Cells from phase II on day 10 gave rise to TRAP-positive cells for 2 days, regardless of whether or not they had been treated with VEGFR-1/Fc in phase I (Fig. 4A). It is thus likely that OCP-II are more mature than OCP-I.
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). In this respect, OCP-I resembled embryonic OCPs rather than OCPs in adult BM. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Supplementation with VEGFR-1/Fc in phase I of ES cell step-wise cultures severely inhibited the generation of both OCP-I and OCP-II. In some of the repeated experiments, a small number of OCP-I were detected in the VEGFR-1/Fc-treated cultures, and the lower number of OCPs compared with the control (medium only) was maintained after the passage of these OCP-I through phase II without VEGFR-1/Fc. Maturation of OCP-I to TRAP-positive cells was delayed for 1–2 days compared with that of OCP-II. Moreover, we found that OCP-I produced higher numbers of osteoclasts than OCP-II (Table 4). These results support the notion that OCP-I may be more immature than OCP-II and suggest that OCP-II may be the progeny of OCP-I.
However, the signal requirements for OCP generation in phases I and II are different. The generation of OCP-I was not affected by the presence of the anti-Kit Ab. In contrast, that of OCP-II was partially inhibited by either the anti-Kit Ab or VEGFR-1/Fc, and severely inhibited in the presence of both reagents (Fig. 2). Although treatment with VEGFR-1/Fc in phase I completely abolished the generation of OCP-I in three of five trials, OCP-II were detected in all experiments. OCP-II might have been generated from very few OCP-I we could not detect; alternatively, OCP-II may newly form in phase II. If so, OCP-I and OCP-II may be derived from different origins.
The reduction of CFUs-M by VEGFR-1/Fc in phase I did not influence the number of CFUs-M that arose in phase II (Table 3). The number of CFUs-M in control cultures was consistent in both phase I and phase II, indicating that CFU-M might newly form in each phase. It is unlikely that treatment with VEGFR-1/Fc in phase I totally eliminates cells with the potential to differentiate into hematopoietic progenitors. Using the GATA2-/- ES cell line, we previously demonstrated that the frequency of GATA2-/- OCP-I was significantly reduced, while that of CFUs-M was only slightly diminished [14]. Lee et al. have reported that OCPs might be more immature than macrophage progenitors that respond to M-CSF but more mature than multilineage progenitors that respond to stem cell factor [8]. These findings further support the idea that the majority of OCPs are derived from cells other than CFUs-M.
Adult hematopoiesis depends on signaling via the Kit receptor because Kit-deficient KitW/KitW mice and anti-Kit Ab-treated mice harbor severe anemia [23, 29], whereas the development of osteoclasts from ES cells is totally Kit independent [13], and KitW/KitW mice also have normal numbers of osteoclasts [3]. Continuous addition of anti-Kit Ab for 10 days throughout phases I and II did not decrease the number of TRAP-positive MNCs (Fig. 2). The addition of anti-Kit Ab in phase II reduced the number of TRAP-positive MNCs from ES cells cultured with VEGFR-1/Fc in phase I (Fig. 2). However, the frequency of these OCP-II was comparable with that obtained in medium only (Table 2). This result suggests that Kit signaling may be associated with the growth of part of the OCP-II fraction resistant to treatment with VEGFR-1/Fc in phase I. Since they would constitute only a small fraction of the total OCPs, we may not have detected these OCPs previously [5, 9, 13].
Definitive hematopoiesis has been reported to depend on Kit signaling, but primitive hematopoiesis in the yolk sac and early fetal liver does not [23, 30]. These facts imply that the majority of osteoclasts may develop through a primitive hematopoietic pathway, which the erythroid and monocytic lineages follow only at the earliest stage in yolk sacs. The OCP-I in control cultures were present at frequencies of 1/300 (0.3%) to 1/34 (3%) in phase I (Table 1), although less than 0.3% of cells expressed CD45, which is a hematopoietic cell-specific marker. This may mean that the OCP-I are very immature cells that have not yet committed to the hematopoietic cell lineage.
Endothelial cells were normally induced from a bloodless ES cell colony. The addition of M-CSF and RANKL increased the numbers of TRAP-positive osteoclasts and changed their locations from the peripheries to the centers of colonies [26, 27]. However, endothelial cells were normally generated in this culture condition. Subsequently, the addition of ligands for VEGFR, VEGF164 but not PlGF accelerated endothelial cell growth, and reciprocally, VEGF164 but not PlGF reduced the generation and frequency of OCPs and CFUs-M in ES cell cultures (Okuyama, unpublished observation). These results may suggest that osteoclasts do not regulate vasculogenesis, but exogenous VEGF leads to the acceleration of endothelial cell growth, resulting in a decline in the differentiation toward hematopoietic cells, including the osteoclast lineage, in cultures. VEGF (VEGF-A) binds to not only VEGFR-1 but also to VEGFR-2 (Flk-1) and neuropilin-1 [31–39]. VEGF-A, PlGF, and VEGF-B bind to VEGFR-1 [40]. PlGF does not affect osteoclastogenesis or vasculogenesis in vivo [40]. The function of VEGF-B is not obvious [41]. In the future, we should identify the VEGFR-1/Fc-neutralized signaling that is critical for OCP development.
Finally, the OCP-I that remained in the cultures treated with VEGFR-1/Fc in phase I and the OCP-II resistant to VEGFR-1/Fc treatment in phases I and II gave rise to significantly fewer numbers of TRAP-positive cells (Experiment #2 in Table 4). The lack of signaling via VEGFR(s) may reduce the growth ability of the few OCPs that develop under these conditions. Alternatively, this result may suggest the presence of another OCP subpopulation that is independent of signaling via VEGFR(s). Further studies are needed to examine these possibilities.
In this study, we showed that the majority of OCPs may be derived from ES cells in a manner dependent on factor(s) binding to VEGFR-1 in phase I, which is a very early developmental stage.
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ACKNOWLEDGMENT |
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REFERENCES |
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