JP6339943B2 - 3次元培養による細胞の再プログラミング - Google Patents
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Description
RT4およびヒト胎児由来腎臓(HEK)293細胞を、それぞれ、McCOY’5A(HYCLONE)およびDMEM(HYCLONE)において培養した。
ヒト膀胱パピローマ細胞系RT4細胞を、単層接着培養(2D)および球培養(3D)、それぞれにより培養した。本発明者らは、成長因子を追加された無血清培地の代わりに、10%ウシ胎仔血清を含む完全成長培地を用いた。
図1A(左)に示されているように、ヒト膀胱パピローマ細胞系RT4細胞は、通常の付着ディッシュ上での単層接着培養により培養され、上皮形態を示した。対照的に、図1A(右)において、低付着ディッシュ上で培養されたRT4細胞は、プレートから剥離され、浮遊する3D球を形成し、その球は数週間、持続した。
幹細胞マーカーの発現レベルを、定量的PCR(Q−PCR)を用いて決定した。簡単に述べれば、全RNAを、Trizol LS試薬(Invitrogen)を用いて細胞から単離し、その後、逆転写酵素反応混合物(SuperScript III First−Strand Synthesis System、Invitrogen)およびオリゴdTプライマー(Invitrogen)と混合した。Q−PCRを、Power SYBR Green RT−PCR Kit(Applied Biosystems)を用いて実施した。GADPHを、cDNAインプットを標準化するための対照として用いた。結果を、試験細胞培養物における試験マーカーの発現レベルの、2D培養における発現レベルに対する、倍数変化として示した。データを、平均±標準偏差、N=3として示した。スチューデントT検定を用いて、3D培養細胞についての結果が、2D培養細胞と統計学的に異なるかどうかを決定した。図において、統計的有意性を有する差を、1個のアスタリスク(*、P<0.05)または2個のアスタリスク(**、P<0.01)でマークした。実施例および図における全てのQ−PCR研究を、他に規定がない限り、類似した方法を用いて実施した。
幹細胞のパーセンテージを、「サイドポピュレーション(Side Population)」(SP)識別アッセイ(幹細胞の色素流出性質を用いた、幹細胞パーセンテージの検出のためのフローサイトメトリー方法)により検出した。簡単に述べれば、細胞をトリプシン処理し、単独かまたは50μg/mlベラパミル(Sigma)と共の5μg/ml Hoechst 33342(Sigma)と37℃で90分間、インキュベートした。細胞を遠沈し、死細胞を排除した後、FACSVantage SE(BD Biosciences)でのフローサイトメトリー細胞ソーティング(FACS)分析に供した。球培養群におけるSP細胞のパーセンテージは、単層培養群における0.37±0.12%と比較して、0.83±0.32%であった。
3.a. HEK293細胞の細胞形態
HEK293は、もともとヒト胎児由来腎臓細胞に由来した非癌性細胞系である。細胞培養ディッシュに接着したHEK293細胞は、単層上皮形態を示したが(図3A左)、低接着性プレート上で培養されたHEK293は、浮遊3D球を形成した(図3A右)。
いくつかの幹細胞マーカーの発現を、2D培養HEK293細胞および3D培養HEK293細胞の両方を用いて、Q−PCRおよびウェスタンブロッティングにより決定した。公知のiPS誘導因子のQ−PCR分析により、OCT4、NANOG、SOX2、KLF4、およびLIN28が、単層培養と比較して、5日目および10日目で、球培養において全てアップレギュレートされることが示された(図3B、Q−PCR分析に用いられたプライマーは下記の表3に列挙されている)。ウェスタンブロッティング分析によってもまた、球培養群におけるNANOGおよびSOX2のタンパク質レベルが、単層群と比較して高いことが示された(図3C)。一次抗体および二次抗体は、実施例2におけるこれらと同じであった。
この実施例において、HEK293細胞を、腎臓幹細胞表現型への再プログラミング活性について、およびまた、腎臓における高度に分化した細胞の別個の型への分化転換再プログラミング活性について試験した。
球培養の初代細胞への効果をさらに評価するために、MEFを球へ培養した。MEFは、それらが細胞培養プレートに接着している場合、楕円形核の周囲に分枝状細胞質を有する典型的な形態を示したが、低接着性プレート上で培養された場合、それらは3D球を形成した(図5A)。iPS誘導因子および数個の未分化ES細胞マーカー遺伝子の発現を、Q−PCRにより調べた。Sox2、Gdf3、Dax1、およびSlc2a3の発現は、7日間および12日間、培養された後、単層MEFよりMEF球において高かった(図5B、Q−PCR分析に用いられたプライマーは下記の表3に列挙されている)。Oct4、Nanog、およびKlf4はMEF球においてアップレギュレートされなかったが(図5Bおよび5C)、MEF球における高レベルのSox2タンパク質が、ウェスタンブロッティングにより確認され、それは、テラトーマ細胞系PA−1におけるレベルに匹敵した。対照的に、単層MEFにおいてウェスタンブロッティングによりSox2の発現は検出されなかった(図5D)。アルカリフォスファターゼの発現を決定するためにAP染色を実施した。図5Eに示されているように、陽性AP染色が、MEF球において見出されたが、単層MEFにおいて見出されなかった。これらのデータより、MEFの球培養が、いくつかの胚性幹細胞遺伝子の発現を促進し得ることが明らかにされた。
MEF球におけるSox2の高発現を考慮して、本発明者らは、MEF球がニューロスフェア性質を獲得するかどうかを試験した。図6A〜Bに示されているように、Nestinが、7日間および12日間培養されたMEF球においてアップレギュレートされた。本発明者らはまた、MEF球において、分化したニューロン、アストロサイト、およびオリゴデンドロサイトに関連した遺伝子の発現を分析した。ニューロンマーカー(Map2、NeuN、Tuj1、NF−L、NF−M、およびNF−H)、アストロサイトマーカー(Gfap、S100A、およびS100B)、およびオリゴデンドロサイトマーカー(MbpおよびNg2)は全て、MEF球においてアップレギュレートされた(図6A〜B、データを、平均±標準偏差、N=4として示した)。数個の神経特異的転写因子(Pax6、Sox1、Otx2、Zic1、NeuroD1、およびOlig2)もまた、MEF球においてより高かった(図6A〜B)。Tuj1およびGfapタンパク質が、1日間、3日間、5日間、および7日間培養されたMEF球において検出され、時間と共に増加したが、単層MEFにおいては発現しなかった(図6C)。
ヒト乳癌細胞系である、MCF−7細胞系を、DMEM−高グルコース培地(HYCLONE)において培養した。
ラット骨芽細胞をDMEM−高グルコース培地(HYCLONE)において培養した。ラット骨芽細胞を、生後1日のラット頭蓋からの一次分離(primary isolation)により取得した。
ラットニューロン幹細胞をDMEM/F12(HYCLONE)において培養した。ラットニューロン幹細胞を、生後1日のラット脳からの一次分離により取得した。
Anokye-Danso, F., Trivedi, C.M., Juhr, D., Gupta, M., Cui, Z., Tian, Y., Zhang, Y., Yang, W., Gruber, P.J., Epstein, J.A., et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376-388.
Birgersdotter, A., Sandberg, R., and Ernberg, I. (2005). Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol 15, 405-412.
Cukierman, E., Pankov, R., Stevens, D.R., and Yamada, K.M. (2001). Taking cell-matrix adhesions to the third dimension. Science 294, 1708-1712.
Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K., Adachi, T., and Sasai, Y. (2011). Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51-56.
Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S., Matsumura, M., Wataya, T., Nishiyama, A., Muguruma, K., and Sasai, Y. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519-532.
Episkopou, V. (2005). SOX2 functions in adult neural stem cells. Trends Neurosci 28, 219-221.
Eshghi, S., and Schaffer, D.V. (2008). Engineering microenvironments to control stem cell fate and function.
Fischbach, C., Chen, R., Matsumoto, T., Schmelzle, T., Brugge, J.S., Polverini, P.J., and Mooney, D.J. (2007). Engineering tumors with 3D scaffolds. Nat Methods 4, 855-860.
Golebiewska, A., Brons, N.H., Bjerkvig, R., and Niclou, S.P. Critical appraisal of the side population assay in stem cell and cancer stem cell research. Cell Stem Cell 8, 136-147.
Griffith, L.G., and Swartz, M.A. (2006). Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol 7, 211-224.
Hendrix, M.J., Seftor, E.A., Seftor, R.E., Kasemeier-Kulesa, J., Kulesa, P.M., and Postovit, L.M. (2007). Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer 7, 246-255.
Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A.E., and Melton, D.A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 26, 795-797.
Ingber, D. (1991). Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis. J Cell Biochem 47, 236-241.
Jensen, U.B., Lowell, S., and Watt, F.M. (1999). The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis. Development 126, 2409-2418.
Jones, P.H., Harper, S., and Watt, F.M. (1995). Stem cell patterning and fate in human epidermis. Cell 80, 83-93.
Keung, A.J., Kumar, S., and Schaffer, D.V. (2010). Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues. Annu Rev Cell Dev Biol 26, 533-556.
Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., et al. (2009). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472-476.
Kim, J., Efe, J.A., Zhu, S., Talantova, M., Yuan, X., Wang, S., Lipton, S.A., Zhang, K., and Ding, S. (2011). Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci U S A 108, 7838-7843.
Li, R., Liang, J., Ni, S., Zhou, T., Qing, X., Li, H., He, W., Chen, J., Li, F., Zhuang, Q., et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51-63.
Liu, J., Kuznetsova, L.A., Edwards, G.O., Xu, J., Ma, M., Purcell, W.M., Jackson, S.K., and Coakley, W.T. (2007). Functional three-dimensional HepG2 aggregate cultures generated from an ultrasound trap: comparison with HepG2 spheroids. J Cell Biochem 102, 1180-1189.
Liu, Y., Clem, B., Zuba-Surma, E.K., El-Naggar, S., Telang, S., Jenson, A.B., Wang, Y., Shao, H., Ratajczak, M.Z., Chesney, J., et al. (2009). Mouse fibroblasts lacking RB1 function form spheres and undergo reprogramming to a cancer stem cell phenotype. Cell Stem Cell 4, 336-347.
Manasek, F.J., Burnside, M.B., and Waterman, R.E. (1972). Myocardial cell shape change as a mechanism of embryonic heart looping. Dev Biol 29, 349-371.
Mani, S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., et al. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704-715.
McBeath, R., Pirone, D.M., Nelson, C.M., Bhadriraju, K., and Chen, C.S. (2004). Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6, 483-495.
Nelson, C.M., and Bissell, M.J. (2006). Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 22, 287-309.
Osafune, K., Takasato, M., Kispert, A., Asashima, M., and Nishinakamura, R. (2006). Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay. Development 133, 151-161.
Pampaloni, F., Reynaud, E.G., and Stelzer, E.H. (2007). The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8, 839-845.
Shi, Y., Do, J.T., Desponts, C., Hahm, H.S., Scholer, H.R., and Ding, S. (2008). A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2, 525-528.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.
Vazin, T., and Schaffer, D.V. (2010). Engineering strategies to emulate the stem cell niche. Trends Biotechnol 28, 117-124.
Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandal, P.K., Smith, Z.D., Meissner, A., et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-630.
Yamada, K.M., and Cukierman, E. (2007). Modeling tissue morphogenesis and cancer in 3D. Cell 130, 601-610.
Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381-384.
Zhu, S., Li, W., Zhou, H., Wei, W., Ambasudhan, R., Lin, T., Kim, J., Zhang, K., and Ding, S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7, 651-655.
Claims (11)
- 線維芽細胞を神経細胞に分化転換する方法であって、
前記線維芽細胞を、細胞付着を低下させる材料を含む低接着性基質上で培養して3次元細胞凝集体を形成し、該3次元細胞凝集体の形成が前記線維芽細胞を前記神経細胞に分化転換させる手順を含み、
前記材料が、0.2%から5.0%までの範囲の濃度のアガロースからなるハイドロゲル層を含む、
方法。 - アガロースが0.5%の濃度である、請求項1に記載の方法。
- アガロースが培地と混合される、請求項1に記載の方法。
- 材料が、低接着性基質の表面としてコーティングされる、請求項1に記載の方法。
- 前記線維芽細胞が、支持基質または構造が培地内に配置されていない状態で、培養される、請求項1〜4のいずれかに記載の方法。
- 前記線維芽細胞がマウス胎仔由来線維芽細胞または尾端線維芽細胞である、請求項1〜5のいずれかに記載の方法。
- 3次元細胞凝集体が多細胞性かつ多層性の細胞凝集体である、請求項1〜6のいずれかに記載の方法。
- 3次元細胞凝集体が球状の形を有する、請求項7に記載の方法。
- 3次元細胞凝集体が、少なくとも10μmの直径を有する、請求項8に記載の方法。
- 培地へ誘導剤としてブチル化ヒドロキシアニソールを導入する手順をさらに含む、請求項1〜9のいずれかに記載の方法。
- 前記線維芽細胞がインビトロで培養される、請求項1に記載の方法。
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