ヒトiPS 由来血管内皮前駆細胞の機能評価の構築

• Project/Area Number: 25505009
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Regenerative medicine
• Research Institution: Foundation for Biomedical Research and Innovation
• Principal Investigator: 秋丸 裕司 公益財団法人先端医療振興財団, その他部局等, 研究員 (70241247)
• Co-Investigator(Kenkyū-buntansha): 浅原 孝之 東海大学, 医学部, 教授 (20246200) ; 川本 篤彦 公益財団法人先端医療振興財団, その他部局等, その他 (00275330)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Discontinued (Fiscal Year 2014)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: ヒトiPS / 血管内皮前駆細胞 / EPC / シングルセルqPCR / single cell qPCR

Outline of Annual Research Achievements

1. ヒトiPS細胞由来CD34陽性EPC(iPS-CD34+EPC)の解析
ヒトiPS細胞をfeeder-free且つ、ウシserum-free条件下にて３つの添加因子を用いることで、CD34陽性血管内皮前駆細胞(EPC)を分化誘導する系を確立した。CD34抗体で分離したiPS由来EPCは生体のEPCと同様に以下の細胞活性を持つことが明らかになった。①EPCコロニー形成能②血管内皮細胞特異的tube形成能 ③血管内皮細胞への分化能④血管内皮細胞関連遺伝子マーカーの発現。
2. iPS-CD34+EPCの体外増幅培養
分化誘導Day15で分離したiPS-CD34+EPCはFACS分離後に3~7%(~5 x 104細胞)程なので、これらの細胞を体外増幅培養した結果、StemSpan (serum free)培養液にVEGF, SCL, IL-6, Flt3 ligand, TPOを添加して非接着状態で1週間培養すると4～6 倍の増幅効果が得られた。分化誘導Day15で接着細胞と浮遊細胞から別々にFACS分離したiPS-CD34+EPCはEPCコロニー形成、さらにHUVECへのmigration活性を有していた。
3. iPS-EPCと生体EPCの比較のためのシングルセルqPCRによる細胞解析
重症下肢虚血5検体と健常者2検体の各96細胞の191遺伝子のシングルセルqPCR解析とSpearman相関検定の結果、治療効果の最も高い検体(+6)のみがCD34+CD45-分画でCD34とVEGF、CXCL2、血管内皮マーカーとの相関を示すのに対して、治療効果の低い検体(+3 ~ +1)ではCD34+CD45-分画においてCD34と相関がある遺伝子群は検出されず、CD34+CD45+分画において+6検体と同様に血管新生関連遺伝子群と相関とそれら以外に血管平滑筋、神経分化、肝分化、及び細胞増殖抑制因子との相関がCD34と顕著に見られた。これらのことは、CD34細胞が血管新生因子群を発現しているにもかかわらず、同時に血管系譜以外の遺伝子が発現し、細胞増殖活性が低下していることが治療効果の減弱の一因となっていることが示唆された。

Research Products

• [Journal Article] Sonic Hedgehog signaling regulates vascular differentiation and function in human CD34 positive cells: vasculogenic CD34(+) cells with Sonic Hedgehog. (2015)
  • Author(s): Kanaya K, Masaaki I, Okazaki T, Nakamura T, Horii-Komatsu M, Alev C, Akimaru H, Kawamoto A, Akashi H, Tanaka H, Asahi M, Asahara T.
  • Journal Title: Stem Cell Res Volume: 14(2) Issue: 2 Pages: 165-76
  • DOI: 10.1016/j.scr.2015.01.003
  • Peer Reviewed / Open Access
• [Journal Article] SDF-1/CXCR4 Axis in Tie2-lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing. (2015)
  • Author(s): Kawakami Y, Ii M, Matsumoto T, Kuroda R, Kuroda T, Kwon SM, Kawamoto A, Akimaru H, Mifune Y, Shoji T, Fukui T, Kurosaka M, Asahara T.
  • Journal Title: J Bone Miner Res. Volume: 30(1) Issue: 1 Pages: 95-105
  • DOI: 10.1002/jbmr.2318
  • Peer Reviewed
• [Journal Article] Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential. (2014)
  • Author(s): H. Masuda, R. Tanaka, S. Fujimura, M. Ishikawa, H. Akimaru, T. Shizuno, A. Sato, Y. Okada, Y. Iida, J. Itoh, Y. Itoh, H. Kamiguchi, A. Kawamoto, T. Asahara.
  • Journal Title: J Am Heart Assoc. Volume: 3 Issue: 3
  • DOI: 10.1161/jaha.113.000743
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion Following Bone Fracture. (2014)
  • Author(s): Fukui T, Mifune Y, Matsumoto T, Shoji T, Kawakami Y, Kawamoto A, Ii M, Akimaru H, Kuroda T, Horii M, Yokoyama A, Alev C, Kuroda R, Kurosaka M, Asahara T.
  • Journal Title: Cell Transplant Volume: May 2
  • Peer Reviewed
• [Journal Article] Selective interference targeting of lnk in umbilical cord-derived late endothelial progenitor cells improves vascular repair, following hindlimb ischemic injury, via regulation of JAK2/STAT3 signaling. (2014)
  • Author(s): Lee SH, Lee KB, Lee JH, Kang S, Kim HG, Asahara T, Kwon SM.
  • Journal Title: Stem Cells Volume: Dec 28 Issue: 5 Pages: 1490-1500
  • DOI: 10.1002/stem.1938
  • Peer Reviewed
• [Journal Article] Tauroursodeoxycholic Acid, a Bile Acid, Promotes Blood Vessel Repair by Recruiting Vasculogenic Progenitor Cells. (2014)
  • Author(s): Cho JG, Lee JH, Hong SH, Lee HN, Kim CM, Kim SY, Yoon KJ, Oh BJ, Kim JH, Jung SY, Asahara T, Kwon SM, Park SG.
  • Journal Title: Stem Cells Volume: - Issue: 3 Pages: 792-805
  • DOI: 10.1002/stem.1901
  • Peer Reviewed
• [Journal Article] Cross talk with hematopoietic cells regulates the endothelial progenitor cell differentiation of CD34 positive cells. (2014)
  • Author(s): Kwon SM, Lee JH, Lee SH, Jung SY, Kim DY, Kang SH, Yoo SY, Hong JK, Park JH, Kim JH, Kim SW, Kim YJ, Lee SJ, Kim HG, Asahara T.
  • Journal Title: PLoS One Volume: 9(8) Issue: 8 Pages: e106310-e106310
  • DOI: 10.1371/journal.pone.0106310
  • Peer Reviewed
• [Journal Article] Dextran induces differentiation of circulating endothelial progenitor cells. (2014)
  • Author(s): S. Obi, H. Masuda, H. Akimaru, T. Shizuno, K. Yamamoto, J. Ando, and T. Asahara
  • Journal Title: Physiol. Rep. Volume: 2 Issue: 3 Pages: e00261-e00261
  • DOI: 10.1002/phy2.261
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Local transplantation of granulocyte colony stimulating factor-mobilized CD34+ cells for patients with femoral and tibial nonunion: Pilot clinical trial. (2014)
  • Author(s): Kuroda R, Matsumoto T, Niikura T, Kawakami Y, Fukui T, Lee SY, Mifune Y, Kawamata S, Fukushima M, Asahara T, Kawamoto A, Kurosaka M.
  • Journal Title: Stem Cells Transl Med Volume: 3(1) Issue: 1 Pages: 128-34
  • DOI: 10.5966/sctm.2013-0106
  • Peer Reviewed
• [Journal Article] A phase II clinical trial of CD34+ cell therapy to explore endpoint selection and timing in patients with critical limb ischemia. (2014)
  • Author(s): Fujita Y, Kinoshita M, Furukawa Y, Nagano T, Hashimoto H, Hirami Y, Kurimoto Y, Arakawa K, Yamazaki K, Okada Y, Katakami N, Uno E, Matsubara Y, Fukushima M, Nada A, Losordo DW, Asahara T, Okita Y, Kawamoto A
  • Journal Title: Circ J Volume: 78(2) Pages: 490-501
  • NAID: 130003382211
  • Peer Reviewed
• [Journal Article] Human cardiac stem cells with reduced notch signaling show enhanced therapeutic potential in a rat acute infarction model. (2013)
  • Author(s): Matsuda T, Miyagawa S, Fukushima S, Kitagawa-Sakakida S, Akimaru H, Horii-Komatsu M, Kawamoto A, Saito A, Asahara T, Sawa Y.
  • Journal Title: Circ J Volume: 78(1) Pages: 222-231
  • NAID: 130003382177
  • Peer Reviewed
• [Journal Article] A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization (2013)
  • Author(s): Kawakami Y, Ii M, Matsumoto T, Kawamoto A, Kuroda R, Akimaru H, Mifune Y, Shoji T, Fukui T, Asahi M, Kurosaka M, Asahara T.
  • Journal Title: Laboratory Investigation Volume: 93 Issue: 9 Pages: 1036-1053
  • DOI: 10.1038/labinvest.2013.93
  • Peer Reviewed
• [Journal Article] Improvement of cardiac stem cell-sheet therapy for chronic ischemic injury by adding endothelial progenitor cell transplantation: analysis of layer-specific regional cardiac function. (2013)
  • Author(s): Kamata S, Miyagawa S, Fukushima S, Nakatani S, Kawamoto A, Saito A, Harada A, Shimizu T, Daimon T, Okano T, Asahara T, Sawa Y
  • Journal Title: Cell Transplant Volume: Epub ahead of print
  • Peer Reviewed
• [Book] 図説 分子病態学 改訂5版(6．細胞の増殖・分化と再生医学) (2014)
  • Author(s): 秋丸裕司 浅原孝之(一瀬白帝 編著)
  • Total Pages: 408
  • Publisher: 中外医学社