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First published online January 12, 2006
Stem Cells Vol. 24 No. 5 May 2006, pp. 1381 -1388
doi:10.1634/stemcells.2005-0464; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS

Ciliated Cells Differentiated from Mouse Embryonic Stem Cells

Yusuke Nishimuraa, Tatsuo S. Hamazakib,c, Shinji Komazakid, Shinji Kamimurac, Hitoshi Okochib, Makoto Asashimaa,c,e

a Department of Biological Science, Graduate School of Science, The University of Tokyo, Tokyo, Japan;
b Department of Tissue Regeneration, Research Institute, International Medical Center of Japan, Tokyo, Japan;
c Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan;
d Department of Anatomy, Saitama Medical School, Saitama, Japan;
e International Cooperative Research Project, Japan Science and Technology Agency, Tokyo, Japan

Key Words. Mouse embryonic stem cells • Bone morphogenetic protein • Knockout serum replacement • Hepatocyte nuclear factor-3/forkhead homolog 4 • Ciliated cells

Correspondence: Tatsuo S. Hamazaki, Ph.D., Department of Tissue Regeneration, Research Institute, International Medical Center of Japan, Toyama 1-21-2, Shinjuku, Tokyo, Japan. Telephone: +81-3-3202-7181 (ext. 2866); Fax: +81-3-3202-7192; e-mail: hamazaki{at}ri.imcj.go.jp

Received September 23, 2005; accepted for publication December 28, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as ß-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17–20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mouse embryonic stem (ES) cells are derived from the inner cell mass of day-3.5 blastocysts. ES cells can be maintained in an undifferentiated state in vitro and can differentiate into all cell lineages of the developing mouse, including the germ line, when transplanted into a mouse blastocyst [1–3]. Thus, ES cells are valuable tools for analyzing cell differentiation in vitro and for regenerative medicine. Embryoid bodies (EBs) are widely used to study the differentiation of ES cells. EBs are aggregates of ES cells that differentiate into ectoderm, mesoderm, and endoderm [1, 2, 4, 5]. EBs can also be differentiated into various cell types, including neurons [6–8], keratinocytes [9], cardiomyocytes [10–13], skeletal muscle cells [14, 15], ß cells of the pancreas [16, 17], and hepatocytes [18].

Fetal bovine serum (FBS) is commonly used in the differentiation of ES cells. However, FBS contains undefined growth factors, and the constituents of FBS can vary between batches, making it unsuitable for analyses of differentiation mechanisms. Recently, Knockout Serum Replacement (KSR; Gibco, Grand Island, NY, http://www.invitrogen.com) was introduced for the culturing of ES cells. Since the contents of KSR are defined chemically and do not differ among lots, it provides a viable alternative to FBS in mechanistic studies of ES cell differentiation. However, differences have been reported between ES cells cultured in the presence of KSR and those cultured in the presence of FBS [19, 20]. In the present study, we show that ES cells could be differentiated into ciliated cells by culturing EBs in medium containing KSR. The only other report of ciliated cells being induced in vitro from ES cells involved culturing ES cells at an air-liquid interface (ALI) [21]. The ALI method mimics the condition of an adult trachea, which is different from the conditions of ciliated cell differentiation during normal development. Therefore, we thought to establish an inductive system for ciliated cells that would more closely mimic in vivo development.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ES Cell Culture and Differentiation of EBs
ES cells of line E14 were maintained on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 15% FBS (Gibco), 0.1 mM nonessential amino acids (Gibco), 0.1 mM ß-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 100 U/ml penicillin (Sigma-Aldrich), 100 U/ml streptomycin (Sigma-Aldrich), and 1,000 U/ml leukemia inhibitory factor (ESGRO; Chemicon, Temecula, CA, http://www.chemicon.com). The medium was changed daily, and a passage was performed every 3 days.

Dissociated ES cells were seeded on a culture dish and incubated for 30 minutes at 37°C to remove the feeder cells. To make aggregates of ES cells, dissociated ES cells were seeded on a low-attachment 96-well plate (1,000 cells per well) and incubated in DMEM containing 10% FBS (10% FBS medium) on a declined 96-well plate for 1 day. The aggregates were cultured for 2 more days on the 96-well plate to form EBs. These EBs were transferred to gelatin-coated dishes (10 EBs per 60-mm dish) and cultured in DMEM containing either 10% KSR (Gibco; 10% KSR medium), 10% FBS (10% FBS medium), insulin-transferrin-selenium A supplement (ITS-A; Gibco), or B-27 supplement without vitamin A (Gibco). To study the effect of the concentration of KSR or FBS, EBs were cultured in DMEM containing KSR or FBS at 1%–20%. To investigate the effect of FBS on differentiation of ciliated cells, EBs were cultured in 10% KSR medium containing FBS concentrations of 0.1%–10%. To study the precise culture conditions for optimal differentiation of ciliated cells, EBs were cultured in KSR or FBS medium for 1–5 days of attachment culture and then cultured in FBS or KSR medium, respectively, for up to 15 days of total attachment culturing time. The medium was changed every 3 days.

To examine the effect of growth factors on the differentiation of ES cells into ciliated cells, EBs were cultured in 10% KSR medium supplemented with various growth factors at different concentrations during the initial 5 days of attachment culture. Several growth factors were used in these experiments: bone morphogenetic protein-2 (BMP-2; 0.5–50 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), BMP-4 (0.5–50 ng/ml; R&D Systems), BMP-7 (0.5–50 ng/ml; R&D Systems), transforming growth factor-ß1 (TGF-ß1; 0.5–50 ng/ml; R&D Systems), human activin A (1–100 ng/ml; a gift from Dr. Y. Eto, Central Research Laboratories of Ajinomoto Co. Inc.), nodal (4–400 ng/ml; R&D Systems), fibroblast growth factor-2 (FGF-2; 1–50 ng/ml; Upstate, Charlottesville, VA, http://www.upstate.com), FGF-10 (10–1,000 ng/ml; Chemicon), epidermal growth factor (EGF; 2–200 ng/ml; R&D Systems), noggin (3–300 ng/ml; R&D Systems), and sonic hedgehog (Shh; 5–100 ng/ml; R&D Systems). After the 5th day of attachment culture, the medium containing growth factors was replaced with 10% KSR medium for 10 days (total attachment culture time was 15 days).

Light Microscopy and Immunohistochemistry
EBs were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 minutes at room temperature. After being rinsed three times with phosphate-buffered saline (PBS), the samples were dehydrated through a graded ethanol series, embedded in LR Gold resin (Electron Microscopy Sciences, Hatfield, PA, http://www.emsdiasum.com), and sectioned serially at 500 nm. The sections were stained with 1% toluidine blue and observed under a light microscope. For immunohistochemistry, the sections were treated with PBS containing 3% bovine serum albumin (BSA) for 30 minutes to block nonspecific binding. The sections were then immunostained with anti-ß-tubulin IV antibody (1:200; BioGenex) as a primary antibody for 1 hours. After being washed three times with PBS, they were incubated with anti-mouse IgG antibody-Alexa Fluor 594 (1:200; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) as a secondary antibody for 1 hour at room temperature. After being washed twice PBS, the sections were treated with PBS containing 1 mg/ml of 4',6-diamidine-2'-phenylindole dihydrochloride for 15 minutes, the sections were then washed finally and mounted for observation by epifluorescence microscopy (Olympus IX71 with ORCA-3CCD camera; Olympus, Tokyo, http://www.olympus-global.com).

To examine whole EB outgrowth, sections were also fixed in 4% paraformaldehyde in 0.1 M phosphate buffer. The sections were permeabilized with PBS containing 0.5% Triton X-100 for 30 minutes and then blocked in PBS containing 3% BSA for 30 minutes. Primary antibody incubations were then carried out for 1 hour at room temperature with anti-ß-tubulin IV antibody (1:200), anti-thyroid transcription factor-1 (TTF-1; 1:500; Santa Cruz Biotechnology Inc., Delaware, CA, http://www.scbt.com) or anti-hepatocyte nuclear factor-3/forkhead homolog-4 (HFH-4 antibody; 1:500; Lab Vision, Fremont, CA, http://www.labvision.com). After being washed in PBS, the sections were incubated in secondary antibodies for 1 hour: fluorescein isothiocyanate-conjugated anti-mouse IgG (1:500; Sigma-Aldrich) or tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG (1:500; Sigma-Aldrich). To estimate the efficiency of induction of ciliated cells, we randomly selected three or four areas from each of the expanded outgrowth areas of EBs stained by anti-HFH-4 antibody and counted the ciliated cells in these areas using NIH Image 1.61 software.

Electron Microscopy
EBs were fixed with 0.1 M sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde for 30 minutes and washed with 0.2 M sodium cacodylate buffer three times. EBs were postfixed with 1% osmium tetroxide for 30 minutes, dehydrated through an ethanol and acetone series, and then embedded in epoxy resin. Ultrathin sections cut at 80–90 nm thickness were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (JEM-200CX; JEOL, Tokyo, http://www.jeol.com).

Measurement of Ciliary Movement with a High-Speed Camera
To examine ciliary movement of the induced ciliated cells, we observed these cells with a high-speed camera at 200 frames per second (5 ms per frame), and ciliary beat frequency was measured from six different areas. To inhibit ciliary movement by vanadate treatment, EBs were exposed to solution A, comprising 102 mM potassium acetate, 10 mM HEPES, 1 mM dithio-threitol, 2 mM MgCl, 0.5 mM EGTA, 0.1 mM EDTA, and Triton X-100. After cessation of ciliary movement, solution A was replaced with solution B (solution A without Triton X-100). The ciliary beating resumed after the addition of 1 mM ATP, and then we added vanadate (final concentration, 10 mM) and ATP (final concentration, 1 mM) to make sure the beating stopped again.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted using Isogen (Nippon Gene, Tokyo, http://www.nippongene.com) according to the manufacturer’s protocol. DNase-treated total RNA (500 ng) was used for first-strand cDNA. Reverse transcription (RT) reaction was performed using Super Script II (Invitrogen) and oligo(dT) primer. The cycling parameters of polymerase chain reaction (PCR) were as follows: denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 40 seconds. The PCR primers, the length of the amplified products, and the number of cycles were as follows: HFH-4 (5'-GAG CTG GAA CCA CTC AAA GC-3', 5'-GGA ACA TGG GTG GAT GAA AC-3'; 606 bp, 30 cycles), TTF-1 (5'-TCC ACG CGC TTC TAC TTT TT-3', 5'-TAA GCT TGG GAA CCC ATT TG-3'; 453 bp, 30 cycles), surfactant protein C (SP-C; 5'-TGG AGA GTC CAC CGG ATT AC-3', 5'-TTT TCC AAT CAG GCT GCT TTA-3'; 736 bp, 22 cycles), Clara cell 10-kDa protein (CC10; 5'-CGC CAT CAC AAT CAC TGT GGT CA-3', 5'-GAG GGT ATC CAC CAG TCT CTT CA-3'; 201 bp, 30 cycles), MUC5AC (5'-GTG CAG GGC TCA GTT CTT TC-3', 5'-TGA CCC AGA TCC TCC ATC TC-3'; 539 bp, 30 cycles), oviductal glycoprotein 1 (Ovgp1: 5'-TGG ACC CCT TTC TTT GTA CG-3', 5'-AGC CAC TCC TTC CCC TTA AA-3'; 852 bp, 22 cycles), '-actin (5'-AGC CAT GTA CGT AGC CAT CC-3', 5'-TAG AAG CAC TTG CGG TGC AC-3'; 740 bp, 25 cycles).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differentiation of ES Cells into Ciliated Cells In Vitro
In the present study, cells with cilia-like motility were induced by culturing EBs in 10% KSR medium (Fig. 1AGo; supplemental online movie). These ciliated-like cells localized in the area of expanded outgrowth from almost all of the EBs cultured under these conditions. Light microscopy analysis revealed the cilia-like structure (Fig. 1BGo). The cells were confirmed as containing true cilia by immunostaining for the ciliary marker, ß-tubulin IV, shown in a section of an outgrowth region of a differentiated EB (Fig. 1CGo). Electron microscopic observation revealed that these cells showed the 9 + 2 arrangement of microtubes typical of a cilia structure, with axial filaments in the center of each cilium (Fig. 1D–1FGo). Double immunostaining for ß-tubulin IV and HFH-4 (a transcription factor essential to ciliogenesis) revealed that all nuclei of ß-tubulin IV-positive cells expressed HFH-4 in EBs cultured in 10% KSR medium (Fig. 2A–2DGo). Although some cells that expressed HFH-4 were not positive for ß-tubulin IV, these results confirm the true nature of the ciliated cells. In contrast, EBs cultured in 10% FBS medium contained very few ciliated cells, and HFH-4-positive cells were rarely found (Fig. 2E, 2FGo). To estimate the efficiency of induction, we counted the number of ciliated cells from randomly selected areas of the EBs (after 20 days of attachment culture). For EBs cultured in 10% KSR medium, 11.85% ± 2.25% cells were positive for HFH-4 (n = 22; mean cell number, 3,000). In the case of EBs cultured in 10% FBS medium, the approximate efficiency was less than 1%.


Figure 1
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  		Figure 1. Differentiation of embryonic stem cells into ciliated cells with knockout serum replacement medium. (A): Phase contrast image of ciliated-like cells from embryoid bodies (EBs) after 27 days of attachment culture. Arrows indicate the cells with ciliated-like motility. (B): Toluidine blue-stained 500-nm cross-section of induced ciliated cells. Multiple cilia were observed by light microscopy. Arrows indicate cilia structure. (C): Immunostaining of induced EBs for ß-tubulin IV, a ciliary marker protein. Arrows indicate cilia-like structure, which is positive for ß-tubulin IV. Arrowheads indicate nuclei stained with 4',6-iamidine-2'-phenylindole dihydrochloride. (D–F): Observation of induced ciliated cells by electron microscopy. (D): Low-magnification image of induced ciliated cells. (E): Cross-section of cilia in the induced ciliated cells showing the typical 9 + 2 arrangement of microtubules. (F): Longitudinal section of cilia showing axial filaments along the centers of the cilia. Scale bars = 50 µm (A), 20 µm (B), 50 µm (C), 5 µm (D), 200 nm (E), and 300 nm (F).

 

Figure 2
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  		Figure 2. Immunostaining of embryoid bodies (EBs) outgrowth for ß-tubulin IV and HFH-4. (A–D): EBs cultured in 10% KSR medium. (E, F): EBs cultured in 10% FBS medium. (A): Anti-ß-tubulin IV antibody staining. (B, E): Anti-HFH-4 antibody staining. (C): Merged fluorescent image of ß-tubulin IV and HFH-4. Arrow indicates a cell double-positive for ß-tubulin IV and HFH-4. Arrowhead indicates a cell showing staining for HFH-4 but not ß-tubulin IV. All ß-tubulin IV-positive cells showed nuclear staining for HFH-4, but not all HFH-4-positive cells were immunopositive for ß-tubulin IV. (D, F): DAPI staining. Scale bar = 100 µm (A–D). Abbreviations: DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; FBS, fetal bovine serum; HFH-4, hepatocyte nuclear factor-3/forkhead homolog-4; KSR, knockout serum replacement.

 
We next measured the movement of the induced ciliated cells with a high-speed camera to reveal a ciliary beat frequency of 17–20 Hz (Fig. 3Go). Furthermore, the ciliary movement was completely stopped by treatment with vanadate, which is a known potent inhibitor of ciliary movement (data not shown). Taken together, these findings indicate that EBs can differentiate into ciliated cells by culturing in KSR medium but not under when cultured in FBS medium.


Figure 3
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  		Figure 3. Ciliary movement of induced ciliated cells. High-speed camera images of ciliated cells taken at 5-ms intervals. Initial location of the center is indicated by the white dotted line. The location of the apical point at each time is shown by arrowheads. Note that the cilia is beating to both the efficient and inefficient sides of the dotted line. The ciliary beat frequency was 17–20 Hz. Scale bar = 20 µm.

 
Differentiation of Ciliated Cells Was Inhibited by Serum-Containing Medium and Promoted by KSR Medium
From the results presented thus far, we predicted three possibilities for differentiation of ciliated cells: KSR contains promoting factors for differentiation of ciliated cells from ES cells; FBS contains inhibitory factors that suppressed the differentiation; or a combination of these two. To examine these possibilities, EBs were cultured in medium containing either KSR or FBS at concentrations of 1%–20%. As shown in Fig. 4AGo (lanes 1–5), the expression of HFH-4 was not significantly affected by the concentration of KSR. When EBs were cultured in the medium containing low concentration of FBS, the expression of HFH-4 was slightly increased, but it was still lower than that of the EBs cultured in KSR medium (Fig. 4AGo, lanes 6–10). Moreover, EBs cultured in 10% KSR medium containing 0.1%–10% FBS showed a dose-dependent decrease in the expression of HFH-4 (Fig. 4AGo, lanes 11–16). Thus, we concluded that FBS contains inhibitory factors for the differentiation of ciliated cells. However, we could not exclude the possibility that KSR contains promoting factors, since the expression of HFH-4 in the EBs cultured in medium containing a low concentration of FBS was lower than that of the EBs cultured in KSR medium. Negative control data was not obtained, as EBs could not survive for more than 3 days in medium containing 0% FBS and 0% KSR.


Figure 4
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  		Figure 4. Examination of the culture conditions for ciliated cell differentiation. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of HFH-4, a ciliary marker gene in EBs cultured in medium containing KSR or FBS at 1%–20%, serum-free medium other than KSR medium, and KSR medium containing 0.1%–10% FBS. HFH-4 expression was not dose-dependent for KSR (lanes 1–5), but it was slightly increased in the medium containing low concentrations of FBS (lanes 6–10). The expression of HFH-4 in the EBs cultured in KSR medium containing FBS was decreased in an FBS dose-dependent manner (lanes 11–16). The expressions of HFH-4 in the EBs cultured in medium containing supplement (ITS-A and B-27) were higher than that of EBs cultured in 10% FBS medium but lower than that of EBs cultured in 10% KSR medium (lanes 17–20). (B): The schema of the culture conditions. White lines and gray lines indicate culturing in KSR medium and FBS medium, respectively. EBs were cultured in the KSR or FBS medium with different timings during the initial 5 days of attachment culture. Total culturing time was 15 days. (C): RT-PCR analysis of HFH-4 in the EBs cultured under the various culture conditions. The expression of HFH-4 in EBs cultured in KSR medium for more than the initial 2 days of attachment culture (lanes 3–6) was as high as that of the EBs cultured in KSR medium throughout (lane 7). In the EBs cultured in FBS medium for more than the initial 2 days of attachment culture (lanes 9–12), the expression of HFH-4 was as low as that of the EBs cultured in FBS medium throughout (lane 1). When EBs were cultured in KSR medium for the 1st day of attachment culture and then switched to FBS medium from the 2nd day of attachment culture, the expression of HFH-4 was low (lane 2). When EBs were cultured in FBS medium day 1 of attachment culture and then switched to KSR medium, the expression level of HFH-4 was high (lane 8). Abbreviations: EB, embryoid body; FBS, fetal bovine serum; HFH-4, hepatocyte nuclear factor-3/fork-head homolog-4; ITS-A, insulin-transferrin-selenium A; KSR, knockout serum replacement; RT, reverse transcription.

 
To further examine the possibility that KSR contains inducing factors, EBs were cultured in a serum-free medium different from KSR medium. When EBs were cultured in DMEM containing ITS-A supplement or B-27 supplement, HFH-4 expression was slightly increased, but it was still less than that of the EBs cultured in KSR medium (Fig. 4AGo, lanes 17–20). This result indicates that KSR contains factors for inducing the differentiation of ciliated cells, since the efficiency of differentiation of ciliated cells by culturing in serum-free medium containing KSR was higher than that for spontaneous differentiation in serum-free medium other than KSR medium.

KSR Medium During the Initial 2 Days of Attachment Culture of EBs Is Important for Differentiation of Ciliated Cells
To examine the timing of the KSR induction and FBS inhibition of ciliated cell differentiation, we used different times of culturing in KSR or FBS medium during the initial 5 days of attachment culture (Fig. 4BGo). The expression of HFH-4 in the EBs cultured in FBS medium for more than the initial 2 days of attachment culture was as low as that of the EBs cultured in FBS medium continuously (Fig. 4CGo). In contrast, the expression of HFH-4 in the EBs cultured in KSR medium for more than the initial 2 days of attachment culture was as high as that of the EBs cultured in KSR medium continuously (Fig. 4CGo). These results indicate that during the initial 2 days of culture attachment, KSR medium had inducing activity for ciliated cells even when the culture medium was replaced with FBS medium, which showed inhibitory effects on the differentiation, after that time. However, after 2 days of attachment culture with FBS, KSR did not induce the differentiation of ciliated cells, indicating a time dependence for the differentiation mechanism.

Respiratory Marker Genes Were Expressed in the Induced EBs
During normal mouse development, ciliated cells appear in various tissues, including embryonic node, cerebral ventricles, auris interna, respiratory system, kidneys, and reproductive organs, such as oviduct and vas deferens [22–25]. To ascertain the tissue type containing the differentiated ciliated cells in our system, we examined gene expressions by RT-PCR (Fig. 5AGo). Respiratory marker genes TTF-1 and SP-C were more highly expressed in the EBs cultured in KSR medium than in those cultured in FBS medium. Ovgp1, an oviduct marker gene, was not expressed in the EBs cultured in either KSR medium or FBS medium. Immunohistochemical analysis revealed that some of the TTF-1-positive cells were localized near the ß-tubulin IV-positive cells (Fig. 5B–5EGo), but some of the TTF-1-positive cells and ß-tubulin IV-positive cells were located independently (data not shown). These results indicated that the induced ciliated cells and differentiated epithelium had several features of respiratory tissue.


Figure 5
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  		Figure 5. Respiratory marker genes were expressed in the induced embryoid bodies (EBs). (A): Analysis of gene expression by reverse transcription-polymerase chain reaction (RT-PCR). TTF-1 and SP-C are respiratory marker genes. Ovgp1 is an oviduct marker gene. The expression of HFH-4, SP-C, and TTF-1 in the EBs cultured in KSR medium were higher than those in the EBs cultured in FBS medium. Ovgp1 was not expressed in the EBs cultured in both KSR and FBS medium. The tissues from an 8-week-old mouse were used as controls. (B–E): Double immunostaining for ß-tubulin IV and TTF-1. (B): Anti-ß-tubulin IV antibody staining. (C): Anti-TTF-1 antibody staining. (D): Merged fluorescent image for ß-tubulin IV and TTF-1. TTF-1-positive cells were located near the ß-tubulin IV-positive cells. (E): DAPI staining. Scale bar = 100 µm (B–E). Abbreviations: DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; FBS, fetal bovine serum; HFH-4, hepatocyte nuclear factor-3/forkhead homolog-4; KSR, knockout serum replacement; Ovgp1, oviductal glycoprotein1; SP-C, surfactant protein-C; TTF-1, thyroid transcription factor-1.

 
The Effect of Growth Factors on Differentiation of Ciliated Cells
Growth factors are known to affect the differentiation of the respiratory tract. Therefore, it is likely that growth factors also affect the differentiation of ciliated cells in this inductive system. To test this hypothesis, EBs were cultured in KSR medium supplemented with a range of different growth factors during the initial 5 days of attachment culture, then moved to KSR medium without supplements for a further 10 days of culturing. The supplemented growth factors were BMP-2, BMP-4, BMP-7, noggin, TGF-ß1, activin, nodal, FGF-2, FGF-10, EGF, and Shh. Treatment of EBs with BMPs (BMP-2, BMP-4, and BMP-7) drastically decreased the expression of HFH-4 in a dose-dependent manner (Fig. 6AGo). However, no other growth factor that was added affected the HFH-4 expression. To clarify whether the differentiation of ciliated cells from ES cells was actually inhibited by the treatment with BMPs, EBs treated with BMP-4 were immunostained using anti-ß-tubulin IV antibody. No ß-tubulin IV-positive cells were observed in the BMP-4-treated EBs (Fig. 7Go), suggesting that BMPs inhibit the differentiation process for ciliated cells. As ciliated cells were rarely induced when EBs were cultured in FBS medium, we predicted that the FBS contained small amounts of BMP that acted to suppress ciliated cell differentiation. To test this idea, EBs were cultured in FBS medium supplemented with noggin, which is an antagonist of BMP-2, BMP-4, and BMP-7. However, EBs cultured in FBS medium supplemented with noggin showed no increase in HFH-4 expression (Fig. 6AGo). Therefore, the present results suggest that factors in the FBS other than BMPs inhibited the ciliated cell induction, perhaps acting in concert with BMPs.


Figure 6
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  		Figure 6. The effect of growth factors on differentiation of embryoid bodies (EBs) into ciliated cells. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression in EBs treated with various growth factors. Supplemented growth factors were BMP-2, BMP-4, BMP-7, noggin, TGF-ß1, activin, nodal, FGF-2, FGF-10, EGF, and Shh. Addition of BMPs (BMP-2, BMP-4, and BMP-7) decreased the expression of HFH-4 and increased the expression of CC10. The addition of growth factors other than BMPs had no effect on the expression of either HFH-4 or CC10. The expression of mucus cell marker gene MUC5AC was increased by addition of TGF-ß superfamily proteins such as TGF-ß1, BMPs, activin, and nodal, and EBs cultured in FBS medium also expressed MUC5AC. The expression of MUC5AC in the EBs cultured in FBS medium was decreased by the addition of high concentrations of noggin. (B): RT-PCR analysis of HFH-4 and CC10 in the EBs that were altered by treatment with BMP-4 (50 ng/ml) during the initial 5 days of attachment culture. Number indicates the day of treatment beginning with BMP-4. The expressions of HFH-4 and CC10 were examined at day 15 of attachment culture. BMP-4 treatment from days 0, 1, and 2 of attachment culture decreased the expression of HFH-4 and increased the expression of CC10. However, treatment with BMP-4 after the 3rd day of attachment culture had no effect on the expression of HFH-4 or CC10. Abbreviations: BMP, bone morphogenetic protein; CC10, clara cell 10-kDa protein; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; HFH-4, hepatocyte nuclear factor-3/forkhead homolog-4; KSR, knockout serum replacement; Shh, sonic hedgehog; TGF, transforming growth factor.

 

Figure 7
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  		Figure 7. BMP-4 inhibits differentiation of ciliated cells from embryonic stem cells. (A): Immunostaining of nontreated embryoid bodies (EBs) with anti-ß-tubulin IV antibody. (B): Nontreated EBs stained with DAPI. (C): Immunostaining of BMP-4 (50 ng/ml)-treated EBs with anti-ß-tubulin IV antibody. (D): BMP-4-treated EBs stained with DAPI. ß-Tubulin IV-positive cells were not observed in EBs treated with BMP-4. Scale bar = 500 µm (A–D). Abbreviations: BMP, bone morphogenetic protein; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride.

 
EBs treated with the BMP-supplemented medium showed a dose-dependent increase in the expression of CC10, a marker gene of Clara cells, which are nonciliated cells of the respiratory tract. In contrast, the expression of HFH-4 was decreased in EBs treated with BMPs (Fig. 6AGo). CC10 was not expressed in nontreated EBs or EBs treated with other growth factors, suggesting that ciliated cells and clara cells are induced selectively by BMPs.

Expression of the mucus cell-specific gene MUC5AC was increased in EBs cultured in the presence of TGF-ß superfamily proteins such as TGF-ß1, BMP-2, BMP-4, BMP-7, and nodal (Fig. 6AGo). MUC5AC was also expressed in the EBs cultured in FBS medium but was decreased by the addition of high doses of noggin (Fig. 6AGo). Since BMP-4 inhibited the differentiation of ciliated cells but at the same time stimulated the expression of clara-cell marker genes (CC10), we treated EBs with BMP-4 over different times of the initial 5 days of attachment culture. When EBs were treated with BMP-4 for 5 days from days 0, 1, and 2 after the beginning of attachment culture, the expression of HFH-4 was decreased (Fig. 6BGo). In contrast, the expression of CC10 was increased. However, when EBs were treated with BMP-4 from 3 days after the beginning of attachment culture, the expressions of HFH-4 and CC10 were not changed compared with the EBs that were cultured without BMP-4. Under these culturing conditions, the action of BMP-4 was required during the initial 2 days of attachment culture for the suppression of HFH-4 and the upregulation of CC10.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we report the successful in vitro differentiation of ciliated cells from ES cells. The ciliated cells induced in the present study had a 9 + 2 microtubule structure and had multiple cilia. In vivo, ciliated cells are differentiated in the embryonic node, cerebral ventricle, auris interna, respiratory system, kidney, and reproductive organs, such as oviduct and vas deferens [22–25]. However, the ciliated cells of embryonic node, kidney, and auris interna are mono-cilium, and sensory cilia have a 9 + 0 arrangement of microtubules. The ciliary beat frequency of the induced ciliated cells was 17–20 Hz, which is comparable to the ciliated cells of normal respiratory tract (approximately 20 Hz) [26]. Moreover, respiratory tissue marker genes, such as TTF-1 and SP-C, were expressed in the EBs cultured in KSR medium, but there was no expression of Ovgp1, a marker gene for the oviduct. In some areas of outgrowth from the induced EBs, TTF-1-positive cells were colocated with ß-tubulin IV-positive cells. Based on these and previously reported results, we suggest that the ciliated epithelial cells induced in the present study in vitro most closely represent those found in respiratory tissues such as trachea or lung in vivo. However, the absence of Clara-cell markers and the fact that the ß-tubulin IV-positive and TTF-1-positive cells were not completely colocalized preclude a definitive conclusion as to the type of ciliated cell-containing tissue induced in our experiments.

Ciliated cells were only induced in significant numbers when the EBs were cultured in KSR medium, and not when they were cultured in FBS medium. The effect of KSR was not dose-dependent, and EBs cultured in medium containing a low concentration of FBS showed a slightly increased expression of HFH-4. However, further experiments revealed that the expression of HFH-4 in EBs cultured in the medium containing low concentrations of FBS was apparently lower than that of the EBs cultured in KSR medium. Moreover, when EBs were cultured in other serum-free medium, containing ITS-A or B-27 supplement, the expression of HFH-4 was slightly increased, although still lower than that of EBs cultured in KSR medium. Together, these results indicate that KSR contains promoting factors for the differentiation of ciliated cells that could not be components of ITS-A or B-27 and that FBS contains factors that can inhibit differentiation.

The present study also suggested that the initial 2 days of attachment culture are critical for cell fate determination in ES cells cultured in KSR medium. Respiratory tract develops by interactions of epithelial cells and mesenchymal cells, which are derived from endoderm and mesoderm, respectively [27]. These interactions are mediated by several growth factors [28–30]. Among them, BMP-4 is important in determining distal-proximal polarity of respiratory cells [30]. In embryonic lung, FGF-10 is secreted from distal mesenchymal cells and induces expression of BMP-4 in epithelial cells. During lung bud extension in early embryonic development, the epithelial cells, which are distant from the cells secreting FGF-10, are not affected by FGF-10. As a result, the expression of BMP-4 in such cells is decreased, and these cells differentiate into proximal cells such as ciliated cells and clara cells [30]. Taken together, cells expressing high levels of BMP-4 differentiate into distal cells, whereas cells with low expression of BMP-4 differentiate into proximal cells, including ciliated cells. In the present study, BMP-4 was inhibitory for the differentiation of ES cells into ciliated cells. This result is consistent with the model that BMP-4 determines the distal-proximal polarity of respiratory tract. However, in the present study, FGF-10 did not affect the expression of ciliated-cell marker genes, and the expression of CC10, which is a proximal-cell marker gene, was increased by BMP-4 treatment. These results suggest that the block in differentiation of ciliated cells occurred at a different stage from that predicted from the model. In an embryo, clara cells are found near ciliated cells in respiratory tract, and they can even differentiate into ciliated cells under certain conditions [31]. These reports support the possibility that the BMP-4 drives the differentiation into clara cells and, as a result, induces the inhibition of ciliated-cell differentiation. TGF-ß superfamily proteins such as TGF-ß1, BMP-2, BMP-4, BMP-7, and nodal tended to increase the expression of MUC5AC, a marker gene of mucus cells. This result is consistent with the report that TGF-ß2 can induce the expression of MUC5AC in trachea [32]. Here, only BMP-2, BMP-4, and BMP-7 had an inhibitory effect on differentiation into ciliated cells, hinting that these growth factors were the factors in FBS responsible for its inhibitory activity. However, the expression of HFH-4 was not increased when EBs were cultured in FBS medium supplemented with noggin, which is an antagonist against BMP-2, BMP-4, and BMP-7. MUC5AC was expressed in the presence of both FBS and KSR medium supplemented with BMPs, and this expression was inhibited by high concentrations of noggin (300 ng/ml). This indicates that FBS contains not only BMP-2, BMP-4, and BMP-7 but also other factors active in the inductive mechanism.

Delaying the treatment of BMP-4 (until after day 3) had no effect on the gene expressions measured over days 0–2, which is the time frame in which BMP suppressed the expression of HFH-4 (ciliated cells) and increased the expression of CC10 (clara cells). This suggests that the cell fate for these two lineages was determined during the initial 2 days of attachment culture. These findings coincide with the result that KSR medium is essential during the initial 2 days of attachment culture.

The inductive method for ciliated cells reported here is considered to mimic the conditions under which differentiation occurs in vivo during early development. Thus, this system provides a useful in vitro means of studying differentiation-mechanisms for ciliated cells and potentially for other respiratory cells. In addition, the system could be used to assay the effects of harmful substances on fetal ciliated epithelial cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Akira Kurisaki (International Cooperative Research Project) of Japan Science and Technology for helpful comments and discussion. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Ministry of Health, Labour and Welfare 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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