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Accelerated generation of human induced pluripotent stem cells from human oral mucosa using episomal plasmid vectors and maternal transcription factor Glis1

July 4, 2016 / Categories: Digital Dentistry, Implant Dentistry

Kashiwagi, Takahiro

Hashimoto, Yoshiya

Tanaka, Masahiro

Baba, Shunsuke

Induced pluripotent stem cells (iPSCs) possess high pluripotency and differentiation potential and may constitute a possible source of autologous stem cells for clinical applications. However, the lengthy reprogramming process (up to one month) remains one of the most significant challenges facing standard virus-mediated methodology. The Gli-like transcription factor Glis1 is highly expressed in unfertilized eggs and one-cell-stage embryos. In this study, iPSCs were generated using a combination of primary human oral mucosal fibroblasts (HOFs) and episomal plasmid vectors expressing transcription factors, including Glis1.

Introduction

The successful reprogramming of human and mouse somatic cells into induced pluripotent stem cells (iPSCs) via ectopic overexpression of pluripotency-associated transcription factors is considered a major scientific breakthrough.1Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K. Generation of human induced pluripotent stem cells from dermal fibroblasts. → Proc Natl Acad Sci U S A. 2008 Feb;105(8):2883–8.2Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. → Nature. 2008 Jan;451(7175):141–6.3Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. → Cell. 2007 Nov;131(5):861–72.4Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. → Cell. 2006 Aug;126(4):663–76.5Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. → Science. 2007 Dec;318(5858):1917–20.

Similar to the characteristics of embryonic stem (ES) cells,6Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. → Nature. 1981 Jul;292(5819):154–6.7Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. → Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634–8.8Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. → Science. 1998 Nov;282(5391):1145–7. human iPSCs can proliferate indefinitely, while retaining pluripotency, and can differentiate into all cell types found in the body. IPSCs have been generated from dermal fibroblasts,9Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. → Cell. 2007 Nov;131(5):861–72. peripheral blood,10Su RJ, Neises A, Zhang XB. Generation of iPS cells from human peripheral blood mononuclear cells using episomal vectors. In: Turksen K, Nagy A, editors. Induced pluripotent stem (iPS) cells: methods and protocols. → New York: Springer; 2016. p. 57–69. (Walker JM, editor. Methods in molecular biology; vol. 1357). dental pulp cells,11Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, Takeda-Kawaguchi T, Iida K, Kunisada T, Shibata T, Yamanaka S, Tezuka K. Dental pulp cells for induced pluripotent stem cell banking. → J Dent Res. 2010 Aug;89(8):773–8. gingival fibroblasts,12Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, Matsumoto T, Yamanaka S, Yatani H. Gingival fibroblasts as a promising source of induced pluripotent stem cells. → PLoS One. 2010 Sep;5(9):e12743. periodontal ligaments,13Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts. → J Periodontal Res. 2011 Aug;46(4):438–47. oral mucosa14Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K, Noma T. Generation of human induced pluripotent stem cells from oral mucosa. → J Biosci Bioeng. 2010 Sep;110(3):345–50. and mesenchymal stromal cells.15Oda Y, Yoshimura Y, Ohnishi H, Tadokoro M, Katsube Y, Sasao M, Kubo Y, Hattori K, Saito S, Horimoto K, Yuba S, Ohgushi H. Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. → J Biol Chem. 2010 Sep;285(38):29270–8.

Gingival tissue is routinely resected during general dental treatments, such as tooth extraction, periodontal surgery and dental implantation, and generally treated as biomedical waste.16Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. Egusa et al. successfully derived iPSCs from human gingival fibroblasts (HGFs) using retroviral transduction of transcription factors; they also reported that the reprogramming efficiency of mouse gingival fibroblasts was higher than that of dermal fibroblasts.17Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, Matsumoto T, Yamanaka S, Yatani H. Gingival fibroblasts as a promising source of induced pluripotent stem cells. → PLoS One. 2010 Sep;5(9):e12743. However, retroviral integration increases the risk of tumor formation, while integration-free methods decrease this potential risk.18Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. The development of novel approaches to generating integration-free iPSCs has eliminated the concern of integrating virus-associated genotoxicity in clinical applications.19Su RJ, Baylink DJ, Neises A, Kiroyan JB, Meng X, Payne KJ, Tschudy-Seney B, Duan Y, Appleby N, Kearns-Jonker M, Gridley DS, Wang J, Lau KH, Zhang XB. Efficient generation of integration-free iPS cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. → PLoS One. 2013 May;8(5):e64496. Integration-free human iPSCs have been generated using several methods.20Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. Okita et al. reported a simple method that uses p53 suppression and nontransforming L-MYC to generate human iPSCs with episomal plasmid vectors.21Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. Our recent study demonstrated that iPSCs could be generated from a combination of primary HGFs and an episomal plasmid vector.22Umezaki Y, Hashimoto Y, Nishishita N, Kawamata S, Baba S. Human gingival integration-free iPSCs; a source for MSC-like cells. → Int J Mol Sci. 2015 Jun;16(6):13633–48. However, the lengthy reprogramming process (up to one month) remains one of the most significant challenges facing standard virusmediated methodology.

Maekawa et al. reported that the Gli-like transcription factor Glis1 (Glis family zinc finger 1) markedly enhances the generation of iPSCs from both mouse and human somatic fibroblasts when it is expressed together with three transcription factors collectively known as OSK (OCT3/4, SOX2 and KLF4) using retroviral transduction.23Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, Kawamura Y, Mochizuki H, Goshima N, Yamanaka S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. → Nature. 2011 Jun;474(7350):225–9. However, little is known regarding whether Glis1 can effectively promote direct reprogramming during iPSC generation using an episomal plasmid vector. In the current study, iPSCs were generated by combining primary human oral mucosal fibroblasts (HOFs) with episomal plasmid vectors expressing OCT3/4, short-hairpin RNA (shRNA) against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1.

Materials and methods

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Ethical statement

Approval for the sampling of human oral mucosa tissue, establishing iPSCs and genome/gene analysis was obtained from the Ethics Committee of Osaka Dental University, Hirakata, Japan (authorization No.: 110783; approval date: 30 September 2013) and the DNA Recombination Experiment Safety Committee of Osaka Dental University (authorization No.: 54; approval date: 18 July 2014). Written informed consent was obtained from the participant. The animal experiments followed a protocol approved by the Animal Committee of Osaka Dental University (authorization No.: 14-06002; approval date: 8 July 2014).

Cell culturing

HOFs were established from oral mucosal tissue 3 mm in diameter obtained using a skin trephine (derma punch, Maruho, Osaka, Japan) from a 23-year-old Asian male. Human oral mucosal tissue was placed in 35 mm tissue culture dishes and cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) containing 10% fetal bovine serum at 37 °C and 5% CO2.24Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, Matsumoto T, Yamanaka S, Yatani H. Gingival fibroblasts as a promising source of induced pluripotent stem cells. → PLoS One. 2010 Sep;5(9):e12743. The medium was replaced every three days. Once the HOFs had proliferated, the tissue was removed. When the cells reached subconfluence, they were dissociated using 0.25% trypsin (Invitrogen, Carlsbad, Calif., U.S.) and transferred to 60 mm tissue culture dishes (passage 1). HOFs were regularly passaged at a 1:3 ratio every three to four days.

Generation of iPSCs from HOFs with episomal vectors

One microgram of an expression episomal plasmid mixture containing pCXLE-hOCT3/4-shp53-F that expresses OCT3/4 and shRNA against p53, pCXLE-hSK that expresses SOX2 and KLF4, pCXLE-hUL that expresses L-MYC and LIN28, and pCXLE-hGlis1 that expresses Glis1 (Addgene, Cambridge, Mass., U.S.) was electroporated into 6 × 105 primary HOFs (passage 5) with the Amaxa 4D-Nucleofector (Lonza, Basel, Switzerland) according to the manufacturer’s instructions using program DT-130 (Lonza). These cells were then transferred on to mitomycin C-treated SNL 76/7 cells (cat. No. 07032801, lot No. 08F009; European Collection of Authenticated Cell Cultures, Porton Down, U.K.) at 5 × 104 cells per 100 mm dish. The following day, the culture medium was replaced with embryonic stem cell (ESC) culture medium consisting of DMEM/F12 medium (Sigma-Aldrich, St. Louis, Mo., U.S.) supplemented with 20% Knock-Out Serum Replacement (Gibco, Grand Island, N.Y., U.S.), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1% nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Gibco) and 5 ng/ mL fibroblast growth factor-2 (ReproCELL, Kanagawa, Japan). Thirty days subsequent to transduction, a number of colonies were mechanically picked and transferred to a 24-well plate. After several passages, ESC-like colonies were selected for further cultivation and characterization. IPSCs were generated and maintained in ESC culture medium. For routine passaging, iPSC colonies were detached with CTK solution (2.5 μg/mL trypsin, 1 mg/mL collagenase IV, 20% KSR, 1 mM CaCl2/PBS, and 70% PBS) and split at a 1:3 ratio every four to five days.

Quantitive real-time reverse transcription-polymerase chain reaction

Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Limburg, Netherlands) according to the manufacturer’s protocol. Single-stranded complementary DNA was synthesized from a total of 500 ng RNA (DNase-treated) using the PrimeScript RT Master Mix (Takara, Shiga, Japan). KhES-1 RNA was provided by the Foundation for Biomedical Research and Innovation (Kobe, Japan). Quantitative real-time reverse transcription-polymerase chain reaction (qRTPCR) was conducted in triplicate using SYBR Select Master Mix (Life Technologies, Grand Island, N.Y., U.S.) with a StepOnePlus system (Life Technologies) and the following PCR program: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 15 s. Specific primers are listed in Table 1. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was co-amplified as an internal standard. Gene expression was measured using the ΔΔCT method.25Gimbel M, Ashley RK, Sisodia M, Gabbay JS, Wasson KL, Heller J, Wilson L, Kawamoto HK, Bradley JP. Repair of alveolar cleft defects: reduced morbidity with bone marrow stem cells in a resorbable matrix. → J Craniofac Surg. 2007 Jul;18(4):895–901. Differences in gene expression between KhES-1, HOF-iPSCs and HOFs were evaluated by variance analysis with the Tukey test.

Table1

Table 1
List of primers used for qRT-PCR.

Surface antigen test

Cells (5 × 105) were obtained after treatment with 0.025% trypsin (Life Technologies). Cell surface antigen staining was performed in phosphate-buffered saline (PBS) with 2% human serum albumin (Mitsubishi-Tanabe Pharma, Osaka, Japan). The cell suspension was incubated with the antibodies listed in Table 2 for 30 min at 4 °C. Murine anti-human antibodies were used at the recommended concentrations. Primary antibodies and isotype controls are listed in Table 2. The stained cells were analyzed with FACSAria II (Becton Dickinson, Franklin Lakes, N.J., U.S.) and the data were analyzed using the FlowJo software (Tree Star, Ashland, Ore., U.S.).

Table2

Table 2
Antibodies used for flow cytometry and immunochemical staining of HOF-iPSCs

Immunochemistry

For fixed staining of differentiation-specific markers, cells were fixed for 30 min in 4% paraformaldehyde at 4 °C, followed by washing in PBS. The cells were then permeabilized for 15 min with 2% bovine serum albumin and 0.1% Triton X-100 (Sigma-Aldrich) and incubated overnight at 4 °C with the primary antibodies diluted in PBS containing 2% bovine serum albumin. The cells were then washed and incubated for 1 h with the appropriate fluorescence-conjugated secondary antibodies. Primary antibodies and secondary antibodies are listed in Table 2. The staining images were acquired with a ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, Calif., U.S.).

In vivo differentiation (teratoma formation)

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Jackson Laboratory, Bar Harbor, Maine, U.S.) were anesthetized and iPSCs (1 × 106) were transplanted under the epidermal space of the neck. Two hundred microliters of saline was injected into a second epidermal space as a negative control. Mice were euthanized 12 weeks later and teratoma samples were collected and subjected to histological analysis. Teratomas were processed according to standard paraffin embedding and hematoxylin and eosin staining procedures by the Business Support Center for Biomedical Research Activities (Kobe, Japan).

Karyotype analysis

Chromosome G-band analysis was performed at Nihon Gene Research Laboratories (Sendai, Japan). At least 15 metaphases were analyzed.

Results

Generation of iPSCs from HOFs using episomal plasmid vectors

Three lines of HOFs were established from the oral mucosa of the 23-year-old Asian male (Fig. 1). Homogeneous fibroblasts emerged from the oral mucosal tissue one week after the start of culturing. HOFs were exponentially expanded up to 30 passages; cells were counted at each passage and plated at 1.5 × 104 cells/cm2. Colonies with a flat human ESC-like morphology and non-ESC-like colonies were counted at around day 20 after HOF transfection with episomal plasmid vectors expressing human OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1. The average number of ESC-like colonies from three experiments was 54.7 ± 3.05, with a reprogramming efficiency of approximately 1%; the average number of non-ESC-like colonies was 25.3 ± 3.21 (Table 3). A number of colonies obtained from the HOF cells were mechanically picked at passage 1. Several days later, four ESClike colonies were selected and expanded. All colonies were similar to ESCs in morphology and proliferative capacity and were named “HOF-iPSCs”.

Table3

Table 3
ESC-like colonies obtained from HOFs. The number of colonies per 5 × 104 cells after cell reprogramming with episomal vectors. These data were obtained from three independent induction experiments using HOFs from a donor.
Article3-1Fig. 1
Excision of oral mucosal tissue by punch biopsy.
Figs. 2 a–c
Generation of HOF-iPSCs.
(a) Time course for HOF reprogramming.
(b) Microscopy image of original HOFs in culture.
(c) Generated HOF-iPSC colonies on SNL feeder cells.

Expression of ESC-specific marker genes in HOF-iPSCs

HOF-iPSCs were selected for characterization from among the picked clones after 23 passages based on their higher level of proliferation and stability of the ESC-like morphology. The expression of the ESC-specific marker genes OCT3/4, NANOG, SOX2, TERT, KLF4 and C-MYC in HOF-iPSCs was analyzed using qRT-PCR (Fig. 3). Expression of NANOG and SOX2 was significantly higher and that of C-MYC and TERT was lower in KhES-1 cells compared with that in HOF-iPSCs (Figs. 3b–e). No significant difference was observed between KhES-1 and HOF-iPSCs for OCT3/4 and KLF4 expression (Figs. 3a & f). KLF4 was the only gene to exhibit higher expression in HOF cells compared with both the KhES-1 cells and HOF-iPSCs (Fig. 3f).

Fig. 2a

Fig. 2
Figs. 2b & c
Fig. 3a-c
Fig. 3d-f
Figs. 3a–f
QRT-PCR analysis of the expression of six pluripotency-related genes in HOF-iPSCs:
(a) OCT3/4,
(b) NANOG,
(c) SOX2,
(d) C-MYC,
(e) TERT
and (f) KLF4.KhES-1 cells (passage 23) were used as the positive control and HOFs (passage 6) as the negative control.

Characterization of HOf-iPSCs

HOF-iPSCs were selected for characterization from among the picked clones after 20 passages based on increased proliferation and stability of the ESC-like morphology. Expression of the ESC-specific surface markers SSEA-3, SSEA-4 and TRA-1-60 in HOF-iPSCs was analyzed using flow cytometry; all three markers were expressed (Fig. 4). HOF-iPSCs could be maintained beyond 20 passages and still demonstrated ESC-like morphology. In addition, HOF-iPSCs expressed ESC-specific surface markers, such as OCT3/4, SSEA-4, TRA-1-60 and TRA-1-81 (Fig. 5). Tumor formation was observed three months after the injection of HOF-iPSCs under the epidermal space in the neck of immunodeficient mice. Histological examination showed that the tumor contained various tissues, including cartilage (mesoderm), melanocytes (ectoderm), gut-like tube tissue (endoderm) and neural tissue (ectoderm; Fig. 6). Karyotype analysis of the tested clones showed a normal human karyotype (Fig. 7).

Fig. 4a-c
Flow cytometry analysis of pluripotent markers in HOF-iPSCs: (a) SSEA-3, (b) SSEA-4 and (c) TRA-1-60.

 

Fig. 5
Fig. 5a-d
Generated HOF-iPSCs stained for (a) OCT3/4, (b) SSEA-4, (c) TRA-1-60 and (d) TRA-1-81; scale bar = 100 μm.
Fig. 6a-c
IPSCs have the potential to differentiate into three germ layers in vivo. Hematoxylin and eosin staining of teratomas derived from iPSCs at passage 20 revealed the presence of (a) cartilage (mesoderm; red arrow), melanocytes (ectoderm; black arrow), (b) gut-like tube tissue (endoderm; red arrow) and (c) neural tissue (ectoderm; red arrow).

Fig. 7

Fig. 7 Karyotype analysis of iPSCs at passage 20 using G-band staining.

Discussion

Many strategies have been proposed for the management of large defects in oral tissue or organs such as due to congenital abnormalities, trauma or cancer treatment. Autogenous bone grafts are the gold standard for such reconstruction because of their osteoconductive, osteoinductive and nonimmunogenic properties.26Gimbel M, Ashley RK, Sisodia M, Gabbay JS, Wasson KL, Heller J, Wilson L, Kawamoto HK, Bradley JP. Repair of alveolar cleft defects: reduced morbidity with bone marrow stem cells in a resorbable matrix. → J Craniofac Surg. 2007 Jul;18(4):895–901.27Sakamoto F, Hashimoto Y, Kishimoto N, Honda Y, Matsumoto N. The utility of human dedifferentiated fat cells in bone tissue engineering in vitro. → Cytotechnology. 2015 Jan;67(1):75–84. Recently, cell therapy using stem cells combined with osteoconductive biomaterials or scaffolds has become a promising alternative to autogenous bone grafts.28Levi B, Glotzbach JP, Wong VW, Nelson ER, Hyun J, Wan DC, Gurtner GC, Longaker MT. Stem cells: update and impact on craniofacial surgery. → J Craniofac Surg. 2012 Jan;23(1):319–22. In order for cell therapy to efficiently treat large defects in oral tissue or organs, it is important to produce a sufficient number of cells that function similarly to primary islets. IPSCs, referred to as pluripotent stem cells, have been generated via retrovirus-mediated introduction of four transcription factors29Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. → Cell. 2007 Nov;131(5):861–72.30Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. → Cell. 2006 Aug;126(4):663–76. and represent a potentially unlimited source of cells. IPSCs that can be efficiently generated from tissue easily accessible to dentists have great potential;31Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry—part I: stem cell sources. → J Prosthodont Res. 2012 Jul;56(3):151–65. iPSCs have been generated from various oral mesenchymal cells32Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry—part I: stem cell sources. → J Prosthodont Res. 2012 Jul;56(3):151–65. and these cells have been reported to possess higher reprogramming efficiency than skin fibroblasts do.33Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, Takeda-Kawaguchi T, Iida K, Kunisada T, Shibata T, Yamanaka S, Tezuka K. Dental pulp cells for induced pluripotent stem cell banking. → J Dent Res. 2010 Aug;89(8):773–8. Oral mucosal tissue is easily accessible and can be harvested by a simple and safe procedure.

Oral mucosal wounds are characterized by rapid re-epithelialization and remodeling and are known to heal quickly compared with other skin injuries. This rapid re-epithelialization and remodeling is due to the increased production of active MMP-2 in oral mucosal fibroblasts compared with skin fibroblasts; MMP-2 may play an important role in rapid extracellular matrix reorganization and scarless wound healing.34Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K, Noma T. Generation of human induced pluripotent stem cells from oral mucosa. → J Biosci Bioeng. 2010 Sep;110(3):345–50.35Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. → Stem Cells. 2009 Nov;27(11):2667–74.36Stephens P, Davies KJ, Occleston N, Pleass RD, Kon C, Daniels J, Khaw PT, Thomas DW. Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity. → Br J Dermatol. 2001 Feb;144(2):229–37. Therefore, we hypothesized that HOFs generated from patient tissue might provide a superior cell source for iPSCs. In the present study, we found that the endogenous expression level of KLF4 was higher in HOFs than in ESCs or HOF-iPSCs. Endogenous KLF4 has been shown to be expressed in gingival and periodontal fibroblasts derived from oral tissue.37Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts. → J Periodontal Res. 2011 Aug;46(4):438–47. Miyoshi et al. also found that HOFs express not only KLF4 and C-MYC but also NANOG and OCT4 at low levels, suggesting that HOFs possess a number of epigenetic advantages for reprogramming.38Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K, Noma T. Generation of human induced pluripotent stem cells from oral mucosa. → J Biosci Bioeng. 2010 Sep;110(3):345–50.

Integrating virus-associated genotoxicity and tumor formation in iPSCs is of concern for clinical application.39Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. Integration-free human iPSCs have been generated using several methods.40Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. → Stem Cells. 2009 Nov;27(11):2667–74.41Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. → Nature. 2009 Apr;458(7239):766–70.42Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC. A nonviral minicircle vector for deriving human iPS cells. → Nat Methods. 2010 Mar;7(3):197–9.43Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. → Cell Stem Cell. 2010 Nov;7(5):618–30.44Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. → Cell Stem Cell. 2009 Jun;4(6):472–6.45Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. → Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348–62. Okita et al. used two of their findings to improve reprogramming efficiency using episomal plasmids;46Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. → Nat Methods. 2011 May;8(5):409–12. iPSC generation is markedly enhanced by p53 suppression47Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. → Nature. 2009 Aug;460(7259):1132–5. and L-MYC is more potent and specific than C-MYC during human iPSC generation.48Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S. Promotion of direct reprogramming by transformation- deficient Myc. → Proc Natl Acad Sci U S A. 2010 Aug;107(32):14152–7. In our previous study,49Umezaki Y, Hashimoto Y, Nishishita N, Kawamata S, Baba S. Human gingival integration-free iPSCs; a source for MSC-like cells. → Int J Mol Sci. 2015 Jun;16(6):13633–48. iPSCs were generated from HGFs using the above-mentioned method. The generated iPSCs expressed ESC-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratomas were formed from the iPSCs, demonstrating their ability to differentiate into three germ layers. However, 50 ESC-like colonies were obtained only 30 days post-HGF transfection. This lengthy reprogramming process (up to one month) is comparable to that of the standard virus-mediated methodology.50Shimada H, Hashimoto Y, Nakada A, Shigeno K, Nakamura T. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia. → Biochem Biophys Res Commun. 2012 Jan;417(2):659–64.

The maternal Gli-like transcription factor Glis1 is highly expressed in unfertilized eggs and onecell- stage embryos.51Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, Kawamura Y, Mochizuki H, Goshima N, Yamanaka S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. → Nature. 2011 Jun;474(7350):225–9. Maekawa et al. showed that Glis1, but not C-MYC, increased iPSC tumorigenicity and markedly enhanced the generation of iPSCs from both mouse and human fibroblasts when expressed together with OCT3/4, SOX2 and KLF4.52Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, Kawamura Y, Mochizuki H, Goshima N, Yamanaka S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. → Nature. 2011 Jun;474(7350):225–9. In the present study, we observed 50 colonies of human ES-like cells as early as 20 days after initial episomal plasmid vector transduction. These results demonstrate that Glis1 enhances the efficiency of iPSC generation using episomal plasmid vectors expressing OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC and LIN28. However, iPSC generation from multiple donors will be required to establish the application of iPSC technology to biomedical research.

Conclusion

Oral mucosal tissue can be conveniently obtained using a simple and safe procedure and possesses epigenetic advantages for reprogramming. We have successfully established a technique for rapidly and safely generating human iPSCs from oral mucosa using episomal plasmid vectors expressing OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1. In order to repair large bone defects caused by trauma, tumors or congenital deficiency, it is necessary to combine sufficient cell numbers and biomaterials. The accelerated generation of integration-free human iPSCs would facilitate the application of clinical-grade iPSC technology for the treatment of large oral tissue or organ defects.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

We wish to thank Takako Yamamoto of the Foundation for Biomedical Research and Innovation for her support with flow cytometry analysis. This study was supported by a MEXT/JSPS KAKENHI grant (No. 25463041).

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