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Keywords:

  • Induced pluripotent stem cells;
  • embryoid bodies;
  • ciliated cells;
  • respiratory epithelium

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Objectives/Hypothesis

We have previously demonstrated the potential use of induced pluripotent stem (iPS) cells for regeneration of respiratory epithelium by culturing embryoid bodies (EBs). The aim of the present study was to determine the most effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium.

Study Design

Experimental study.

Methods

iPS cells cultured on a gelatin-coated dish were seeded on low-attachment plates for generating EBs. Under several conditions including the air–liquid interface (ALI) method, with varying cell numbers and suspension times, EBs were transferred to a gelatin-coated dish supplemented with growth factors. The shape, size, aggregation, and adhesion of EBs for iPS cell differentiation were evaluated, and the cultured tissue was histologically examined.

Results

EBs appropriate for differentiation were observed using 1,000 cells after 5 days of suspension culture. Respiratory epithelium-like tissue was histologically observed. The ciliary epithelium was confirmed immunohistologically.

Conclusions

Based on the varying suspension times and cell numbers with the ALI method, this study presented effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium.

Level of Evidence

NA. Laryngoscope, 124:E8–E14, 2014


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Some patients require tracheal reconstruction after the resection of malignancies or stenotic inflammation of the trachea. In conventional treatments, tracheal defects are reconstructed by either end-to-end anastomosis or autologous tissue implantation using skin or cartilage from the nasal septum, auricle, or costal cartilage. However, these techniques are both invasive and unstable. Recently, tissue engineering techniques for various tissues and organs have been reported in experimental studies.[1-3] Our group successfully rebuilt tracheal defects in humans resulting from excision of malignant tumors of the thyroid gland or laryngotracheal stenotic lesions using an artificial graft made from a collagen sponge scaffold with polypropylene mesh.[4] However, it was noted that the delay in epithelial regeneration on the luminal surface of the prosthesis remained a problem.

Recently, it was reported that mouse embryonic stem cells (ESCs) can differentiate into ciliated epithelial cells.[5] However, ethical issues exist in relation to the clinical application of ESCs lines. Building on the findings of previous studies, Takahashi and Yamanaka generated induced pluripotent stem cells (iPSCs) from mouse skin fibroblasts by introducing four transcription factors.[6] These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue engineering applications. In addition, the use of iPSCs obtained from patient-derived somatic cells can prevent transplant rejection. We have previously demonstrated the potential use of iPSCs for regeneration of respiratory epithelium by culturing embryoid bodies (EBs). EB formation is a crucial step in the differentiation of many ESCs and iPSCs for induction. The purpose of the present study was to evaluate the most effective conditions for EB formation from iPSCs for regeneration of respiratory epithelium.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

iPSC Culture

iPSCs from the iPS-MEF-Ng-20D-17 line (20D-17; RIKEN BioResource Center, Tsukuba, Japan) were routinely cultured on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts, SNL 76/7 (DS Pharma Biomedical Co., Ltd., Osaka, Japan), in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen, Grand Island, NY) supplemented with 15% fetal bovine serum (SAFC Biosciences, Lenexa, KS), 2 mmol/L L-glutamine (Gibco, Invitrogen), 0.4 mL β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and nonessential amino acids (Gibco, Invitrogen). iPSC lines were passaged every second day with 0.25% trypsin-ethylenediaminetetraacetic acid (Gibco, Invitrogen). To maintain pluripotency of iPSCs during selection of undifferentiated cells, iPSCs were cultured on puromycin-resistant SNL 76/7 fibroblasts in culture medium containing 1.0 μg/mL puromycin (Sigma-Aldrich), then green fluorescent protein (GFP)-expressing undifferentiated cells in a gelatin-coated dish (6-cm diameter) were selected. GFP expression was negative when differentiation was induced, whereas it remained positive in undifferentiated cells.[7]

To prepare puromycin-resistant SNL 76/7 cells, the Streptomyces alboniger puromycin-N-acetyl-transferase gene was subcloned into a retrovirus shuttle vector, pCX4bsr,[8] then introduced into SNL 76/7 cells by infecting the recombinant retrovirus vector as described previously.[9] Puromycin-resistant SNL cells were selected by culturing in DMEM in the presence of 2 μmol/L puromycin.

EB Formation Phase: Suspension Culture

iPSCs were cultured on a gelatin-coated dish in the presence of serum. To induce differentiation, undifferentiated cells were completely dissociated to single cells. The dissociated iPSCs were then seeded onto a culture dish and incubated for 25 minutes at 37°C to remove the feeder cells. To create aggregates of the iPSCs, dissociated iPSCs were seeded onto a low-attachment 96-well plate (500, 1,000, 2,000, and 4,000 cells per well) and incubated in Knockout DMEM (Gibco, Invitrogen) containing 10% Knockout Serum Replacement (KSR) (Gibco) (Fig. 1). Four groups with different cell numbers were as follows: group A, 500 cells/well; group B, 1,000 cells/well; group C, 2,000 cells/well; group D, 4,000 cells/well. The aggregates were cultured for 3, 5, 7, and 9 days on a 96-well plate to form EBs; namely, serum-free culture of embryoid body-like aggregates (Table 1).[10]

image

Figure 1. Differentiation of embryoid bodies (EBs). To generate EBs, induced pluripotent stem cells (iPSCs) were seeded in low-attachment plates in serum-free medium (EB formation phase). Under varying conditions of cell numbers and suspension times, EBs were transferred to a gelatin-coated dish supplemented with growth factors and cultured under the condition of the air–liquid interface. b-FGF = basic fibroblast growth factor. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1. Four Groups With Different Cell Numbers for Embryoid Body Formation.
GroupCell Numbers
A500 cells/well
B1,000 cells/well
C2,000 cells/well
D4,000 cells/well

EB Differentiation Phase: Submerged Culture

The EBs were then transferred to a gelatin-coated Transwell 12-insert/12-well plate (Corning Inc., Corning, NY) (both 0.4 μm, 12 diameter, polyester; 4 EBs per well) and cultured in differentiation medium for 5 days of attachment culture. Serum-free differentiation medium was used in the present study: Knockout DMEM supplemented with 10% KSR, 100 ng/mL Activin A and basic fibroblast growth factor (b-FGF).

EB Differentiation Phase: Air–Liquid Interface Culture

After the 5th day of attachment, medium containing growth factors from the apical side of the EBs was removed for air–liquid interface (ALI) cultivation and medium on the basal side of the EBs was replaced with 10% KSR medium. EBs at the ALI were further cultivated for 15 days (total attachment culture time, 20 days).

Morphologic Examination In Vitro

Cultured tissues were examined after 21 days of culture and observed under a bright field.

Antibodies

The primary antibodies used for immunologic studies were mouse monoclonal antibodies against β-tubulin IV (1:100; Sigma-Aldrich) and rabbit monoclonal antibodies against Forkhead Box J1(FOXJ1) (1:100; Biorbyt, San Francisco, CA). The secondary antibodies were anti-mouse and anti-rabbit immunoglobulin G heavy chain an light chain (H+L) conjugated with fluorescein isothiocyanate (1:100; FI-2000, Vector Laboratories, Burlingame, CA).

Immunohistochemistry and Analysis

The samples were fixed with ethyl alcohol in phosphate-buffered saline (PBS) for 10 minutes at room temperature, rinsed three times with PBS, then treated with PBS containing 1% bovine serum albumin, 0.2% gelatin, and 0.05% saponin for 30 minutes to block nonspecific binding. The samples were then immunostained with primary antibodies overnight at 4°C. They were then incubated with secondary antibody for 30 minutes at room temperature, rinsed three times with PBS, then incubated with PBS containing 1 μg/mL (final concentration) of 4′,6-diamidine-2′-phenylidole dihydrochloride for 10 minutes. They were then washed again and mounted for observation by epifluorescence microscopy (Olympus IX 71 with ORCA-AG camera; Olympus, Tokyo, Japan).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Morphologic Examination In Vitro

EB formation was evaluated under several conditions of different suspension times and cell numbers (Table 1). The shape, size, adhesion, and aggregation of EBs for iPSCs differentiation were determined.

At day 3 of suspension culture, the shape of the EBs was spheroid in all four groups (Fig. 2), and the size was from 375 μm to 700 μm (Fig. 3). The adhesion of EBs to the plate was stable, whereas the aggregation of EBs was poor. The EBs were not able to maintain the shape of a spheroid.

image

Figure 2. Morphological examination in vitro after 3, 5, 7, and 9 days of suspension culture. From left to right is a gradual growth and transformation with time of simple, densely packed embryoid bodies (EBs) into cystic EBs. Bars: 100 μm [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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image

Figure 3. Diameter of embryoid bodies (EBs) versus suspension time. The diameter (major axis) of EBs was measured. Group A: 500 cells/well; group B: 1,000 cells/well; group C: 2,000 cells/well; group D: 4,000 cells/well. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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At day 5 of suspension culture, the shape of the EBs was spheroid in all four groups (Fig. 2), and the size reached a plateau from 600 μm to 1,000 μm (Fig. 3). The adhesion of EBs to the plate was stable, whereas the aggregation of EBs was good. The EBs were able to maintain the shape of a spheroid.

At day 7 of suspension culture, the shape of the EBs became uneven in all four groups (Fig. 2), and the size was gradually increased from 800 μm to 1,100 μm (Fig. 3). The adhesion of EBs to the plate was unstable, whereas the aggregation of EBs was good.

At day 9 of suspension culture, the shape of the EBs became lobular in a disorderly manner (Fig. 2), and the size was from 1,050 μm to 1,400 μm (Fig. 3) . The adhesion of EBs to the plate was unstable, whereas the aggregation of EBs was good.

These results are summarized in Table 2. Suspension culture for 5 days was the most effective conditions for EB formation (Table 2).

Table 2. Embryoid Body Formation for Different Suspension Times.
  Day 3Day 5Day 7Day 9
Suspension cultureShapeSpheroidSpheroidUnevenLobular
Attachment cultureAdhesionStableStableUnstableUnstable
AggregationPoorGoodGoodGood

Next, EB formation was evaluated under four conditions of cell numbers (500, 1,000, 2,000, or 4,000 cells/well) at day 5 of suspension culture (Table 3 and Fig. 4).

Table 3. Embryoid Body Formation for Different Cell Numbers.
  Group A, 500 Cells/WellGroup B, 1,000 Cells/WellGroup C, 2,000 Cells/WellGroup D, 4,000 Cells/Well
Suspension cultureShapeSpheroidSpheroidSpheroidSpheroid
Attachment cultureAdhesionStableStableStableUnstable
AggregationPoorGoodGoodGood
Cilia-like structureAbsentPresentAbsentAbsent
image

Figure 4. Morphological examination in vitro after 5 days of suspension and 7 days of adhesion culture (total, 12 days culture). (A) 500 cells/well. (B) 1,000 cells/well. (C) 2,000 cells/well. (D) 4,000 cells/well. (E) Enlarged view of group B. Bars: 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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In group A, the adhesion of EBs to the plate was stable, whereas the aggregation of EBs was poor. The EBs were not able to maintain the shape of spheroid. No cilia were found on the surface of the EBs by light microscopy analysis.

In group B and group C, the adhesion of EBs to the plate was stable, whereas the aggregation of EBs was good. The EBs were able to maintain the shape of a spheroid. Cilia-like structure was found on the surface of the EBs in group B but not in group C.

In group D, the adhesion of EBs to the plate was unstable, whereas the aggregation of EBs was good. No cilia were found on the surface of the EBs.

These results are summarized in Table 3. Suspension culture with 1,000 cells/well was the most effective condition for EB formation.

Immunohistochemical Analysis

Tracheal epithelium, a multilayered pseudostratified columnar epithelium, is composed of ciliated cells, goblet cells, intermediate cells, basal cells, and basement membrane. Our previous experiments confirmed that iPSCs have the potential to differentiate into respiratory epithelium-like tissue, as shown by immunohistochemical examination.[11] As cytokeratin AE1/3 is a specific marker of basal cells, antibodies against cytokeratin AE1/3 were used to identify differentiated cells. Basal cells stained with the antibodies were observed on the basement membrane of the epithelium. However, their differentiation into ciliated cells could not be confirmed immunohistochemically. In the present study, immunohistochemical analysis was performed to clarify the presence of differentiated ciliated cells (Fig. 5). As β-tubulin IV and FOXJ1 are both ciliary marker proteins, antibodies against them were used to identify differentiated ciliated cells. Respiratory epithelial cells stained with these antibodies were observed on the upper side of the epithelium after 5 days suspension and 16 days adhesion culture in group B. These results show that iPS-derived cells did indeed differentiate into epithelium-like tissue.

image

Figure 5. Immunological examination in vitro after 5 days suspension and 16 days adhesion culture in group B (total, 21 days culture). Immunostaining of induced embryoid bodies (EBs) for β-tubulin IV, a ciliated cell marker protein. (A) β-tubulin IV antibody staining. (B) 4′,6-diamidine-2′-phenylidole dihydrochloride (DAPI) staining. (C) Merged fluorescent image of β-tubulin IV and DAPI. Immunostaining of induced EBs for Foxj1, a ciliated cell marker protein. (D) Foxj1 antibody staining. (E) DAPI staining. (F) Merged fluorescent image of Foxj1 and DAPI. Bars: 200 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

The trachea is indispensable for maintaining respiratory function in the living body. Because it is involved in several functions, such as breathing and phonation, tracheal defects can impair multiple functions. Surgical management of tracheal reconstruction is one of the most difficult problems associated with tracheal resection in patients with malignant or stenotic inflammatory lesions of the trachea. Generally, tracheal lesions are treated by resection followed by mobilization and reconstruction of the remaining tracheal segments using end-to-end anastomoses. However, unsatisfactory tracheal reconstruction can result in severe complications such as fistula and stenosis. Recent experimental and clinical studies on the development of alloplastic tracheal prostheses have demonstrated that the lack of an epithelial lining on the luminal surface is the leading cause of failure.

Our group developed an artificial scaffold for use in tracheal regeneration therapy, and our product obtained good clinical results.[4] However, the artificial graft requires about 2 months for epithelial regeneration. Promotion of epithelialization is important for the prevention of infections during the period of regeneration.

Numerous studies have focused on the in vitro differentiation of ciliated epithelial cells by using ESC lines as potential cell sources.[12, 13] However, ESCs are prone to transplant rejection and are surrounded by ethical issues. iPSCs were generated in 2006 from mouse skin fibroblasts by the introduction of four transcription factors.[6] The use of iPSCs obtained from patient-derived somatic cells can prevent transplant rejection and ethical issues.

Imaizumi et al.[14, 15] demonstrated the potential use of iPSCs as a new cell source for tracheal regeneration. They reported that iPSCs have the potential to differentiate into chondrogenic cells; however, no report has revealed differentiation into respiratory epithelium. Here, we showed successful in vitro differentiation of iPSCs into ciliated-like cells by the EB formation method.

As iPSCs and ESCs differentiate in suspension culture, they typically form three-dimensional aggregates of cells termed EBs. Over time, EBs increase in cell number, and three-dimensional aggregates that include endoderm, ectoderm, and mesoderm are formed. Subsequently, the endoderm gives rise to the epithelial lining of the respiratory and digestive tracts and lineage-specific organs, including thyroid, lungs, liver, pancreas, intestines, prostate, and bladder. Generation of a wide variety of cell types (e.g., cardiac myocytes, hematopoietic cells, neurons, pancreatic islet cells) from ESCs has been reported. Kamiya et al.[16] reported ESC-derived cardiomyocytes (mesodermal differentiation), and Shimoji et al.[17] reported neurons (ectodermal differentiation) by the EB method.

However, differentiation into respiratory epithelium (endodermal differentiation) has not been well examined or established in iPSCs. Only a few studies have addressed the possibility of respiratory epithelium differentiation from ESCs.[5] We have previously reported the potential use of iPSCs for regeneration of respiratory epithelium by culturing EBs to resolve the difficulties associated with conventional methods.[10] In previous histological experiments, we confirmed that iPSCs have the potential to differentiate into respiratory epithelium-like tissue.[11] Respiratory epithelium-like tissue was observed in EBs by culturing in serum-free differentiation medium supplemented with activin A and bFGF. However, their differentiation into ciliated cells could not be confirmed immunohistologically. Phenotypic analysis is also essential. In addition, we could not determine the most effective conditions for EB formation for iPSCs differentiation into respiratory epithelium-like tissue. To resolve these issues, in the present study, the effective conditions for EB formation from iPSCs for regeneration of respiratory epithelium were examined. In our study, the aggregation, shape, and adhesion of EB for iPSCs differentiation were evaluated. The aggregation of EBs was good at 5, 7, and 9 days, the shape was spheroid at 3 and 5 days, and the adhesion was stable at 5 days. EBs with good aggregation, spheroidal shape, and stable adhesion were obtained by suspension culture at 5 days.

Coucouvanis et al.[18] demonstrated that mature EBs were formed from an outer layer of primitive endoderm surrounding an interior of differentiating cells. Dang et al.[19] showed that ESCs on the outer surface of the developing EBs differentiate into primitive endoderm that regulates the cell fate decisions of remaining pluripotent cells. In our study, the formation of mature EBs, which were spheroidal shaped with good aggregation and adhesion, depended on the seeding cell number and suspension time. Krista et al.[20] evaluated varying cell seeding densities to determine the effects on EB formation. Koike et al.[21] demonstrated that various types of EBs were formed from mouse ESCs under various culture conditions, and they characterized these EBs in terms of gene expression pattern to estimate the differentiation status of the bodies. When they increased the seeding cell number from 1,000 to 4,000 ESCs, the gene expression pattern changed. Itskovitz-Eldor et al.[22] demonstrated that in vitro aggregation of ESCs results in formation of EBs, with regional differentiation into embryonically distinct cell types. In addition, human ESCs, when differentiating in suspension in vitro, acquire molecular markers specific to the three embryonic germ layers. In our current studies, dissociated iPSCs (500–4,000 cells/well) were cultured in a low cell-adhesion 96-well plate, and 1,000 cells per well with 5 days in suspension were found to be the most effective conditions for EB formation for differentiation into ciliated cells.

Other parameters can also have a significant influence on iPSCs differentiation. For example, EB size may be an important factor not only with regard to the reproducibility of iPSCs differentiation experiments, but also during regulation of endogenously influenced cell type-specific differentiation, as reported recently.[23] Dissociated iPSCs (1,000 cells per well and 5 days in suspension) formed uniformly sized cell masses (about 750 μm), and selectively differentiated into the target cells (i.e., endoderm-derived cells).

Culture condition is essential for tissue regeneration in vitro. For example, selecting the appropriate medium is known to be critical for airway epithelial cell culture to promote differentiation and to form suitable epithelial barriers with tight junctions. Respiratory epithelial cells face highly variable conditions regarding pressure, mechanical stress, and oxygen supply. Therefore, we used an oxygen-enriched ALI culture method. Primary airway epithelial cells are composed of mixed cell types, such as goblet cells, basal cells, and both ciliated and nonciliated epithelial cells. However, there is no well-established in vitro respiratory epithelium model to date. Lee et al.[24] have established an ALI culture method of passaged human nasal epithelial cell monolayers for drug transport studies.

Further studies to induce efficient differentiation of iPSCs into airway epithelial cells are expected using chemically defined cell culture systems. Rodin et al.[25] reported that extracellular matrix proteins, particularly basement membrane components, are an important part of in vivo niches for differentiation. These proteins have been shown to influence cellular differentiation, adhesion, proliferation, migration, and self-renewal of cells. For instance, Hahn et al.[26] reported that laminin and collagen type IV promote the differentiation of epithelial cells.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

iPSCs have the potential to differentiate into respiratory epithelium-like tissue. In this study, based on the varying suspension times and cell numbers with ALI method, effective conditions for EB formation from iPSCs for regeneration of respiratory epithelium was demonstrated.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

The authors thank Ms. Etsuko Sato for her technical assistance.

BIBLIOGRAPHY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY