に作れば、哺乳動物の臓器や組織も両生類のイモリのように再生復元させることが出来るというメカニズムを医学 に応用するものです。このような生体内再生 “ Tissue Engineering” は世界に先駆けて我々が提唱してきた方 法であり、人工気管や人工神経などはすでに臨床応用され、これによって救われる患者さんの数も着々と増えてき ています。この新しい技術は 21 世紀の医療の中心的柱になるものと考えています。
Tissue Engineering: We have devised a completely new approach to the development of artificial organs. The main procedure using tissue engineering for tissues and internal organs involves the removal of the cell component from auto- or allo-organs to obtain only the extracellular matrix, so-called refined extracellular matrix(ECM)and reconstitutes the solid structure from the extracted collagen. This ECM or reconstituted structure is then employed as scaffolding, which after implantation into the patients is used for the regeneration or re-differentiation of tissue. Organs made of self-cells thus regenerate. Organs that regenerate in this manner not only possess highly differentiated tissue structures, but also show functional recovery, because all the cells are derived from the patients themselves. Whether or not our new method is practicable will depend mainly on the intrinsic regeneration capacity of each tissue. Up to now, in higher mammals including man, it has been believed that highly differentiated organs lose their ability to regenerate. We consider that mammals do not, in fact, lose this potential, and that the potential is hidden by excessively rapid wound healing around the failing tissues. In this sense, if we can provide good conditions using refind ECM, we can induce this hidden potential even in higher mammals. We have already carried out succeessful trials at regenerating peripheral nerves, the esophagus, the trachea, and blood vessels with this method. A similar method is also applicable to other soft tissue organs such as the liver, heart, and lung, as well as the spinal cord. These results will be welcomed by patients who are dependent on palliative life-support systems, or transplantation candidates who are waiting for suitable donors. An additional benefit is that patients will be freed from the side effects of immunosuppressive drugs. The judgment of the brain death can then be discussed separately from the issue of transplantation, and will become a personal problem. Further more, this new approach help to reduce ever-expanding medical costs, which are in danger of destroying our health insurance system in the near future.
No study based on these concepts has ever been done either in Japan or abroad. In this sense, our pioneering work is expected to be a major area of medical science for the coming generation.
Strategy and targets of our study
The target organs currently being considered for this development project are the heart, heart valves, esophagus, stomach, intestine, gallbladder, trachea, lung, liver, kidney, peripheral nerves, spinal cord, cornea, tendons, ligaments, cartilage, bone, fatty tissue, periodontal tissue, and permanent teeth. We plan to employ the two majour methods as described below.
ECM Method
To obtain the purified extracellular matrix, cell components are completely removed from homo or allo-organs. The solid structure is reconstituted from the ECM and extracted collagen. Growth factors are then applied to facilitate cell proliferation. Then this ECM-collagen-growth factor composite is implanted into the
living body as a temporary scaffolding for new organ regeneration. Besides this, bioabsorbable materials will also be applied instead of purified ECM as a bulk structure for organ regeneration. Both extracted collagen and growth factors are should facilitate cell proliferation and cell redifferentiation, leading to regeneration of organs completely composed of cells derived from patients.
Tissue Engineering and Field theory
Cells(or living tissues)of patients are complexed(mixed)with purified ECM or bioabsorbable material.
Using this complex, reconstruction of the failing tissues or organs will be attempted. Mesenchymal stem cell
(MSC)obtained from the bone marrow is now applied to this method.
・iPS 細胞誘導法に関する研究
体細胞に特定の多能性関連遺伝子を導入することで人工多能性幹細胞(iPS 細胞)が誘導される。iPS 細胞は多能 性を維持しながら無限に増殖し、胎盤以外の生体を構成するすべての細胞へと分化することが可能である。iPS 細胞 技術を応用することにより、患者個人の体細胞から大量の移植用細胞を確保することが可能となるだけでなく、医 学研究に必要な十分量の疾患特異的細胞を得ることができる。しかしながら、レトロウィルスを用いた標準的樹立 法ではヒト iPS 細胞誘導までに約 1 ヶ月間必要である。このことは治療、研究の両面における主な問題点の一つで ある。本年度は誘導条件の最適化を試みることで、ヒト iPS 細胞樹立に要する期間を大幅に短縮することに成功し た。ヒト脂肪幹細胞(ADS 細胞)に濃縮レトロウィルス上清を用いて Oct4, Sox2, Klf4, L-Myc を導入し、生理的低 酸素下(5% O2)において 5 種類の化学阻害薬(GSK3beta 阻害薬 CHIR99021、MAPK/ERK 阻害薬 PD0325901、
TGF-beta 阻害薬 A83-01、ヒストン脱アセチル化阻害薬 酪酸、ROCK 阻害薬 Y-27632)を一定期間作用させた。遺 伝子導入後 6 日目からヒト ES 細胞様のコロニーが出現し、10 日目には導入遺伝子の抑制(サイレンシング)と多 能性マーカー Tra-1-81 の発現が確認された。樹立された細胞は増殖能、形態、全遺伝子発現パターン、多能性関連 マーカー、全ゲノムレベルでのメチル化状態、生体内外での分化能において ES 細胞と類似していた。我々が開発 した手法を用いることにより短期間で高品質なヒト iPS 細胞を樹立することが可能となる。
図 1:低酸素環境下で化学阻害薬を併用することにより樹立されたヒト iPS 細胞と誘導スケジュール
Induced pluripotent stem(iPS)cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process(up to 1 month)remains one of the most significant challenges facing standard virus-mediated methodology. This year, we reported the accelerated generation of human iPS cells from adipose-derived stem(ADS)cells, using a new combination of chemical inhibitors(GSK3beta inhibitor CHIR99021, MAPK/ERK inhibitor PD0325901, TGF-beta inhibitor A83-01,HDAC inhibitor sodium butyrate、ROCK ihbitor Y-27632)under a setting of physiological hypoxia(5% O2)in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem(ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method offers a powerful tool for rapidly generating bona fide human iPS cells.
【業 績 目 録】
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誌上発表▣
1. 原著論文
中村達雄:肺における再生医療.Clinical Engineering. 22:32-36(2011)
Nakamura, T., Omori, K., Kanemaru, S.: Tissue-engineered airway and“in situ tissue engineering”. Gen Thorac Cardiovasc Surg. 59:91-97(2011)
Ohno, S., Hirano, S., Tateya, I., Kojima, T., Ito, J.: Management of vocal fold lesions in difficult laryngeal exposure patients in phonomicrosurgery. Auris Nasus Larynx. 38:373-80(2011)
Ohno, S., Hirano, S., Kanemaru, S., Kitani, Y., Kojima, T., Tateya, I.,Nakamura, T., Ito, J.: Implantation of an atelocollagen sponge with autologous bone marrow-derived mesenchymal stromal cells for treatment of vocal fold scarring in a canine model. Ann Otol Rhinol Laryngol. 120:401-8(2011)
Ohno, S., Hirano, S., Kanemaru, S., Tateya, I., Kitani, Y., Kojima, T., Nakamura, T., Ito, J.: Prevention of buccal mucosa scarring with transforming growth factor β3. Laryngoscope. 121:1404-9(2011)
Ohno, S., Hirano, S., Kanemaru, S., Kitani, Y., Kojima, T., Ishikawa, S., Mizuta, M., Tateya, I., Nakamura, T., Ito, J.:
Transforming Growth Factor β3 for the prevention of vocal fold scarring. Laryngoscope.(in press)
Honda, M., Nakamura, T., Hori, Y., Shionoya, Y., Yamamoto, K., Nishizawa, Y., Kojima, F., Shigeno, K.: Feasibility study of corticosteroid treatment for esophageal ulcer after EMR in a canine model. Journal of Gastroenterology. 46:866-72(2011)
Honda, M., Hori, Y., Nakada, A., Uji, M., Nishizawa, Y., Yamamoto, K., Kobayashi, T., Shimada, H., Kida, N.,
Sato, T., Nakamura, T.: Use of adipose-derived stromal cells for prevention of esophageal stricture after circumferential EMR in a canine model. Gastrointestinal Endoscopy. 73:777-84(2011)
Honda, M., Hori, Y., Shionoya, Y., Yamamoto, K., Kida, N., Kojima, F., Nakamura, T.: Fluid overload deteriorate the chylothorax. Disease of Esophagus.(in press)
Yamamoto, K., Kojima, F., Tomiyama, K., Nakamura, T., Hayashino, Y.: Meta-analysis of therapeutic procedures for acquired subglottic stenosis in adults. Ann Thorac Surg. 91:1747-53(2011)
Yamamoto, K., Tomiyama, K., Mitsuoka, M.: Total cricoidectomy and laryngotracheal reconstruction for subglottic stenosis with glottic involvement. Interact Cardiovasc Thorac Surg. 13:351-3(2011)
Shimada, 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.
BBRC.(in press)
Hirasaki, Y., Fukunaga, M., Kidokoro, A., Hashimoto, A., Nakamura, T., Tsujimoto, H., Hagiwara, A.: Development of a novel antiadhesive material, alginate flakes, ex vivo and in vivo. Surg today. 41: 970-7(2011)
Kojima, T., Kanemaru, S., Hirano, S., Tateya, I., Ohno, S., Nakamura, T., Ito, J.: Regeneration of radiation damaged salivary glands with adipose-derived stromal cells. Laryngoscope. 121: 1864-1869(2011)
Kojima, T., Kanemaru, S., Hirano, S., Tateya, I., Suehiro, A., Kitani, Y., Kishimoto, Y., Ohno, S., Nakamura, T., Ito, J.:
The protective efficacy of basic fibroblast growth factor in radiation-induced salivary gland dysfunction in mice. Laryngoscope. 121: 1870-1875(2011)
Hashimoto, A., Kuwabara, M., Hirasaki, Y., Tsujimoto, H., Torii, T., Nakamura, T., Hagiwara, A.: Reduction of air leaks in a canine model of pulmonary resection with a new staple-line buttress. J Thorac Cardiovasc Surg.
142: 366-71(2011)
東高志、中井隆介、渡邊誠、茂野啓示:多次元 MRI を用いた顎機能診断への期待.歯界展望 . 117: 795-816(2011)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(1) 治療計画が頭に浮か ぶ歯周組織審査チャート&採り方のコツ! . デンタルハイジーン . 31:648-654(2011)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(2) 長期症例に学ぶすべ ての治療の前に必要なモチベーションとその実際. デンタルハイジーン . 31:768-774(2011)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(3) 歯周治療を成功させ るキュレットワーク「押さえる」「探る」「確かめる」〜長期にわたり歯周組織を管理する.デンタルハイジー ン . 31:880-886(2011)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(4) 〜再評価とチーム医 療が支える〜治療が活きる高度な DH ワーク . デンタルハイジーン . 31:1002-1008(2011)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(5) 歯周外科処置におけ る歯科衛生士の役割−歯周外科の知識から患者説明のポイントまで− . デンタルハイジーン .(in press)
佐野明美、金子真弓、茂野啓示:極めたい!長期症例における DH ワーク&テクニック(6) 完 長期メンテナン スの極意〜患者教育と手技のポイント〜 . デンタルハイジーン .(in press)
2. 著書
中村達雄:感染性心内膜炎.「疾患を有する高齢者が来院したら?̶歯科医師・スタッフが知っておきたいポイン
ト̶」(編著:佐藤田鶴子、発行:株式会社ヒョーロン・パブリッシャーズ、全 197 頁)73-78(2011)
中村達雄:感染性心内膜炎.「疾患・病態を有する高齢者への歯科における対応」(編著:佐藤田鶴子、発行:株式 会社ヒョーロン・パブリッシャーズ、全 197 頁)74-78(2011)
稲田有史:複合性局所疼痛症候群(CRPS)の診断と治療 Ⅴ.CRPS の手術療法 .「日整会広報室ニュース」(社団 法人日本整形外科学会)84:6(2011)
稲田有史:神経再生療法 . 「神経障害性疼痛」(編 : 眞下節、克誠堂出版株式会社)380-387(2011)
3. 総説
稲田有史、諸井慶七郎、中村達雄、森本茂、古家仁:人工神経を中心とする生体内再生治療を用いた複合性局所疼 痛症候群(CRPS)の治療.整形外科.62: 809-814(2011)
稲田有史:機能再建のマイスターは何をみているのか.DOCTOR'S NETWORK.45: 23-27(2011)
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学会等の発表▣
1. 学会・研究発表会
中村達雄:生体内再生(in situ Tissue Engineering)と末梢神経の再生医療.第 11 回長崎障害者支援再生医療研究 会(2011.4.26 長崎)特別講演
Nakada, A., Shigeno, K.: Utility of a Slightly Modified Collagen Scaffold. Bio Japan 2011 World Business Forum
(2011.10.5-7 Yokohama)招待講演
大野覚:組織工学的手法を用いた瘢痕声帯治療:骨髄由来間葉系幹細胞とアテロコラーゲンスポンジの有用性.第 18 回京都耳鼻咽喉科研究会(2011.4.2 京都)
大野覚、平野滋、金丸眞一、木谷芳晴、児嶋剛、楯谷一郎、北村守正、伊木健浩、石川征司、伊藤壽一:Transforming Growth Factor β3 による瘢痕声帯の予防.第 23 回日本喉頭科学会総会・学術講演会(2011.4.22 旭川)
Ohno, S., Hirano, S., Kanemaru, S., Kitani, Y., Kojima, T., Tateya, I., Nakamura, T., Ito, J.: Implantation of an Atelocollagen Sponge with Bone Marrow-Derived Stromal Cells for the Treatment of Vocal Fold Scarring.
The 91st Annual Meeting of The American Broncho-EsophagologicalAssociation.(2011.4.28 Chicago)
大野 覚、平野 滋、金丸眞一、楯谷一郎、北村守正、木谷芳晴、児嶋 剛、伊木健浩、石川征司、伊藤壽一:ア テロコラーゲンスポンジを足場材料とした骨髄由来間葉系幹細胞移植による瘢痕声帯治療.第 112 回日本耳 鼻咽喉科学会総会・学術講演会(2011.5.20 京都)
Ohno, S., Hirano, S., Kanemaru, S., Tateya, I., Kada, S., Ito, J.: Transforming Growth Factor beta3 for the prevention of vocal fold scarring. The Voice Foundation's 40th Annual Symposium: Care of the Professional Voice
(2011.6.2 Philadelphia)
楯川幸弘、中田顕、中村達雄:胆管内無水エタノール注入によるラット肝硬変モデルの作製:胆道閉鎖症の病態に 近づけるか? . 第 38 回日本胆道閉鎖症研究会(2011.12.10 名古屋)
本多通孝:バイオスカホールドを用いた全周性・血管茎付き人工小腸の作成.第 7 回日本消化管学会総会(2011.2 京都)
本多通孝:脂肪由来幹細胞治療による食道 ESD 後狭窄の予防.第 10 回日本再生医療学会総会(2011.3 東京)
本多通孝:全周性人工小腸の作成と消化吸収機能の評価.第 66 回日本消化器外科学会総会(2011.7 名古屋)