KEIO RESEARCHERS INFORMATION SYSTEM

Profile Summary 【 Display / hide 】

Profile Summary 【 Display / hide 】

• 角膜上皮の再生医療分野で研究していました。
  現在は慶應義塾大学病院細胞培養加工施設（KHCPC）に在職しています。

Career 【 Display / hide 】

Career 【 Display / hide 】

• 2003.04

  -

  2005.03

  東京歯科大学, 市川総合病院　角膜センター, 臨時職員（研究員）

• 2009.04

  -

  2017.03

  慶應義塾大学, 医学部　眼科学教室, 特任助教（研究）

• 2017.04

  -

  2017.09

  慶應義塾大学, 医学部　眼科学教室, 訪問研究員

• 2017.04

  -

  2017.09

  株式会社セルージョン, 研究員

• 2017.10

  -

  2019.03

  慶應義塾大学, 医学部　眼科学教室, 特任助教（研究）

display all >>

Academic Background 【 Display / hide 】

Academic Background 【 Display / hide 】

• 2000.04

  -

  2002.03

  Chiba University, 自然科学研究科, 生命地球科学専攻

  Graduate School, Completed, Master's course

• 2002.04

  -

  2003.03

  Chiba University, 自然科学研究科, 生命資源科学専攻

  Graduate School, Withdrawal before completion, Doctoral course

• 2005.04

  -

  2009.03

  Keio University, 医学研究科

  Graduate School, Completed, Doctoral course

Academic Degrees 【 Display / hide 】

Academic Degrees 【 Display / hide 】

• 博士（医学）, Keio University, Coursework, 2009.03

  A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor population （培養角膜上皮シートにおけるサイトケラチン15陽性前駆細胞群の増加を誘導する新たなNIH/3T3二層フィーダー培養系）

Licenses and Qualifications 【 Display / hide 】

Licenses and Qualifications 【 Display / hide 】

• 日本再生医療学会　臨床培養士, 日本再生医療学会の認定資格。2年ごと更新。, 2016.01

Papers 【 Display / hide 】

Papers 【 Display / hide 】

• Corneal Endothelial Regeneration Using Mesenchymal Stem Cells Derived from Human Umbilical Cord

  Yamashita K., Inagaki E., Hatou S., Higa K., Ogawa A., Miyashita H., Tsubota K., Shimmura S.

  Stem Cells and Development (Stem Cells and Development)  27 ( 16 ) 1097 - 1108 2018.08

  ISSN  15473287

  　View Summary

  © Copyright 2018, Mary Ann Liebert, Inc. Corneal blindness is the third leading cause of blindness in the world, and one of the main etiologies is dysfunction of the corneal endothelium. Current treatment of corneal endothelial disease is allogenic corneal transplantation, which is limited by the global shortage of donor corneas and immunological rejection. The corneal endothelium consists of a monolayer of cells derived from the neural crest and mesoderm. Its main function is to prevent corneal edema by tight junctions formed by zonular occludens-1 (ZO-1) and Na, K-ATPase pump function. The human umbilical cord (UC) is a rich source of mesenchymal stem cells (MSCs). UC-MSCs that have multi-lineage potential may be an accessible allogenic source. After inducing differentiation with medium containing glycogen synthase kinase (GSK) 3-β inhibitor, UC-MSCs formed polygonal corneal endothelial-like cells that functioned as tissue-engineered corneal endothelium (UTECE). Expressions of major corneal endothelial markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR). Western blotting confirmed the expression of Na,K-ATPase and PITX2, the functional and developmental markers of corneal endothelial cells. Immunohistochemistry revealed the localization of Na,K-ATPase and ZO-1 in cell-cell junctions, suggesting the presence of tight junctions. In vitro functional analysis revealed that UTECE had significantly high pump function compared with UC-MSCs. Moreover, UTECE transplanted into a rabbit model of bullous keratopathy successfully maintained corneal thickness and transparency. Our findings suggest that UTECE may be used as a source of allogenic cells for the treatment of corneal endothelial disease.

  • Access to Document (DOI)

• Long-term homeostasis and wound healing in an in vitro epithelial stem cell niche model.

  Miyashita H, Niwano H, Yoshida S, Hatou S, Inagaki E, Tsubota K, Shimmura S.

  Sci Rep. 7   43557 2017.02

  Research paper (scientific journal), Joint Work, Accepted

  　View Summary

  Cultures of epithelial cells are limited by the proliferative capacity of primary cells and cell senescence. Herein we show that primary human epithelial cell sheets cultured without dermal equivalents maintained homeostasis in vitro for at least 1 year. Transparency of these sheets enabled live observation of pigmented melanocytes and Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) labeled epithelial cells during wound healing. Cell turn over and KRT15 expression pattern stabilized within 3 months, when KRT15 bright clusters often associated with niche-like melanocytes became apparent. EdU labels were retained in a subset of epithelial cells and melanocytes after 6 months chasing, suggesting their slow cell cycling property. FUCCI-labeling demonstrated robust cell migration and proliferation following wounding. Transparency and long-term (1 year) homeostasis of this model will be a powerful tool for the study of wound healing and cell linage tracing.

  • Access to Document (DOI)

• Long-term maintenance of limbal epithelial progenitor cells using rho kinase inhibitor and keratinocyte growth factor.

  Miyashita H, Yokoo S, Yoshida S, Kawakita T, Yamagami S, Tsubota K, Shimmura S.

  Stem Cells Transl Med. 2 ( 10 ) 758 - 765 2013.10

  Research paper (scientific journal), Joint Work, Accepted

  　View Summary

  Corneal epithelial stem cells are located in the limbus, the junction between the cornea and the conjunctiva. A limbal epithelium model in vitro would be useful for the study of epithelial stem cells, as well as improving the quality of cultivated epithelial sheets for the treatment of limbal stem cell deficiency. In this study, we succeeded in constructing a limbal epithelium-like structure that could be maintained for at least 5 months in vitro. We modified conventional medium by replacing epidermal growth factor with keratinocyte growth factor (KGF) and adding Y-27632, a rho kinase inhibitor. Using this medium, epithelial cells freshly isolated from human limbus were cocultured with human mesenchymal stem cell-derived feeder cells. Cells formed a stratified layer without air exposure, and both basal and suprabasal layers maintained their unique morphologies for up to 5 months. Basal layers expressed the progenitor marker p63 uniformly and K15 heterogeneously. Expressions of PAX6, K3, and K12 indicated that cell sheets underwent normal differentiation in the corneal epithelium lineage. Although medium was changed daily after day 7, cell debris was observed every day, suggesting that cell sheets underwent turnover. Furthermore, secondary colonies were observed from cells dissociated from 1-month and 3-month cultured sheets. In conclusion, human limbal epithelial cell sheet cultures with KGF and Y-27632 maintained stratification, high expression of both stem/progenitor markers and differentiation markers, and colony-forming cells long-term. This protocol may be useful as an in vitro limbal epithelial model for basic studies.

  • Access to Document (DOI)

• A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations.

  Miyashita H, Shimmura S, Higa K, Yoshida S, Kawakita T, Shimazaki J, Tsubota K.

  Tissue Eng Part A. 14 ( 7 ) 1275 - 1282 2008.07

  Research paper (scientific journal), Accepted

  　View Summary

  Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.

  • Access to Document (DOI)

• Hypoxia enhances the expansion of human limbal epithelial progenitor cells in vitro.

  Miyashita H, Higa K, Kato N, Kawakita T, Yoshida S, Tsubota K, Shimmura S.

  Invest Ophthalmol Vis Sci. 48 ( 8 ) 3586 - 3593 2007.08

  Research paper (scientific journal), Accepted

  　View Summary

  PURPOSE:
  To demonstrate the effects of hypoxia on proliferation and differentiation of human limbal epithelial cells in vitro.
  
  METHODS:
  Primary human limbal epithelial cells were harvested from the rim of donor corneas. Colony-forming efficiency (CFE) and cell proliferation were observed in standard (20% O(2)) or hypoxic (2% O(2)) culture conditions. Cell cycle, forward scatter (FSC) and side scatter (SCC) of cells were analyzed by flow cytometry. Proliferating cells were also observed by pulse labeling (2 hours) with BrdU and Ki67 staining. Apoptosis was detected by TUNEL assay. Isolated colonies were examined by immunohistochemistry against K15, p63, involucrin, and K3. Involucrin expression was also analyzed by Western blot analysis.
  
  RESULTS:
  Both CFE and proliferation of limbal epithelial cells was significantly enhanced in hypoxia. Flow cytometry revealed a higher fraction of hypoxic cells in the G(0)/G(1)-phase and fewer cells in the S-phase, compared with normoxia. However, there was no difference in the uptake of BrdU during a 2-hour pulse, suggesting that hypoxic colonies contained rapidly cycling cells. Apoptotic cells were sparse in both groups, and hypoxic cells showed lower FSC compared with normoxic cells. Although there was no difference in the staining pattern of K15, p63, and Ki67, cells cultivated in normoxia expressed higher levels of the differentiation markers involucrin and K3. Significantly higher involucrin expression was also observed by Western blot.
  
  CONCLUSIONS:
  Hypoxic culture (2%) enhances proliferation while inhibiting differentiation of limbal epithelial cells in vitro.

  • Access to Document (DOI)

Papers, etc., Registered in KOARA 【 Display / hide 】

Papers, etc., Registered in KOARA 【 Display / hide 】

• Label retaining cells in long term cultured confluent epithelial cell sheets

  Miyashita, Hideyuki

  科学研究費補助金研究成果報告書 2016

• Melanocyte stem cells as a niche for corneal epithelial stem cells

  Miyashita, Hideyuki

  科学研究費補助金研究成果報告書 2014

• Identification of feeder cell surface factor that inhibit epithelial cell differentiation

  Miyashita, Hideyuki

  科学研究費補助金研究成果報告書 2009

Research Projects of Competitive Funds, etc. 【 Display / hide 】

Research Projects of Competitive Funds, etc. 【 Display / hide 】

• 角膜上皮幹細胞ニッチにおけるメラノサイト幹細胞の役割

  2013.04

  -

  2014.03

  Grant-in-Aid for Scientific Research, Hideyuki Miyashita, Principal investigator

  　View Summary

  メラノサイトは輪部では幹細胞と共局在するため、ニッチ候補の一つとして考えられている。我々は以前の研究において、少なくとも1年にわたり安定的に初代培養上皮およびメラノサイトが維持されること、このとき培養１か月目までは均一に上皮幹細胞マーカーK15を発現している基底層が、培養３カ月目以降はK15陽性細胞クラスターと陰性細胞に自己組織化されることを見出した（Miyashita H, 2013）。そこで、メラノサイトもしくはメラノサイト幹細胞が上皮幹細胞のニッチとして機能していると仮説を立てた。もしメラノサイトがニッチであるならば、培養メラノサイトを培養系に添加することで上皮幹細胞が増加し、逆にメラノサイトを培養系から除去することで上皮幹細胞が減少するはずである。しかしながら実際には、培養メラノサイトの添加、およびFCMによるメラノサイトの除去、どちらも培養上皮シートにおけるK15の発現に変化は与えなかった。なお、自己組織化までは論文としてまとめ、2017年に論文として発表した(Miyashita et al, 2017)。

• フィーダー細胞表面に存在する上皮分化抑制因子の同定

  2008.04

  -

  2009.03

  Grant-in-Aid for Scientific Research, Hideyuki Miyashita, Principal investigator

  　View Summary

  この研究では、上皮細胞と3T3フィーダー細胞と直接接触する際の効果がどういった因子によるものかを、マイクロアレイ解析と、フィーダー細胞への発現ベクター及びshRNA発現ベクターの導入により解析した。予備実験段階で上皮細胞とフィーダー細胞との接触効果を確認しており、これについては論文として発表することができた（Miyashtia H, 2008）。マイクロアレイ解析ではNIH3T3に高発現する候補因子の中から、qRT-PCR及びWBを用いて発現の高い細胞表面分子を同定することはできたものの、当該cDNA発現ベクターやshRNA発現ベクターの導入による上皮未分化性維持への顕著な効果は認められなかった。

• ヒトフィーダー細胞を用いたヒト培養上皮シートの評価

  2006.04

  -

  2008.03

  Grant-in-Aid for Scientific Research, Hideyuki Miyashita, Principal investigator

  　View Summary

  この研究では臨床応用を見据え、移植用ヒト上皮細胞を培養する際に頻用されるマウス由来の3T3に替えて、同種であるヒト骨髄間葉系幹細胞がフィーダー細胞としてどの程度有用化を解析した。その結果、上皮とフィーダーを直接接触させて共培養する場合には上皮がフィーダーを排除しにくい問題があったものの、臨床で用いられるような間接共培養の場合は3T3フィーダーを用いた場合と同等の上皮シートを作成できることを明らかにした。この結果はのちに、ウサギへの移植実験を担当した大本雅弘医師との共著で論文にまとめるとともに（Omoto M, Miyashita H, 2009）、厚生労働省のヒト幹細胞臨床研究指針に則った培養上皮シートの臨床研究（H21年医政第0191004号）に直接つながった。