過去と現在のMao and Hu Labのすべてのメンバーに、興味深い議論とプロジェクトへの多大な貢献に心から感謝しています。国立成育医療研究センターの多大なるご支援に感謝いたします。本研究は、中国国家自然科学基金会(U20A20351は毛建華、胡立丹82200784)、中国浙江省自然科学基金会(No.LQ22C070004 Lidan Hu へ)。

1. iPS細胞のメンテナンス
<ol><li>iPS細胞の解凍<ol><li>液体窒素から細胞を取り出し、37°Cのウォーターバスで細胞を速やかに融解します。融解細胞を、3 mLのiPS細胞維持培地(材料表)で調製した15 mLのチューブに移します。培地を静かに混ぜます。</li>
		<li>300 x g で5分間遠心分離します。上清を取り除き、10 μM Y-27632を含む1 mLのiPS細胞維持培地に細胞を静かに再懸濁します(細胞を上下に2〜3回ピペットで移動させます)。</li>
		<li>成長因子低減(GFR)細胞外マトリックスゲル(1:100)でコーティングした6ウェル組織培養プレートに細胞懸濁液を移し、10 μM Y-27632をあらかじめ添加したiPS細胞維持培地2 mL(約4 x 104 cells/cm2)に細胞懸濁液を移します。</li>
		<li>細胞を37°C、5%CO2 で5〜6日間(80%〜90%コンフルエント)培養し、iPS細胞維持培地を毎日交換します。</li>
	</ol></li>
	<li>ヒトiPS細胞の通過<ol><li>iPS細胞維持培地を6ウェルプレートから取り出します。iPS細胞をDPBSで一度洗浄します。</li>
		<li>700〜800μLの0.48 mM EDTA溶液を加え、室温(RT)で1分間インキュベートした後、分解溶液を除去します。37°Cの温度で3〜5分間インキュベートし続けます。</li>
		<li>細胞がシート状に消化されたら(細胞を単一細胞に消化しない)、10 μM Y-27632を含むiPS細胞維持培地1 mLを加えて消化を終了します。細胞を慎重に上下に2〜3回ピペットで移動させます。</li>
		<li>細胞懸濁液を、成長因子低減(GFR)細胞外マトリックスゲル(1:100)と10 μM Y-27632をあらかじめ添加したiPS細胞維持培地2 mLでコーティングした6ウェル組織培養プレートに移します。 注:継代比は1:6〜1:20(約4×104 細胞/cm2)の範囲で、平均凝集サイズは約50〜200μmである。</li>
		<li>37°Cで80%〜90%のコンフルエント(5〜6日)まで細胞を培養し、iPS細胞の維持培地を毎日 交換します。</li>
	</ol></li>
</ol>2. EB形成による間葉系幹細胞とiPS細胞の分化
注:この方法は、以前の文献21、22、23、24から導出されたものである。この方法の概要を 図1に示します。この方法の特徴を 表1にまとめた。
<ol><li>培地の調製<ol><li>α-MEMに1%(v/v)のGlutaMAX、10%(v/v)のFBS、10 ng/mLのFGF2、および5 ng/mLのTGFβを添加して、MSC分化培地を調製します。</li>
		<li>α-MEMに1%(v/v)のGlutMAX、10%(v/v)のFBS、および1 ng/mLのFGF2を添加して、MSC維持培地を調製します。</li>
	</ol></li>
	<li>iPS細胞の作製<ol><li>成長因子還元(GFR)細胞外マトリックスゲル(1:100)でコーティングした6ウェル組織培養プレート上のiPS細胞維持培地中で、37°C、5%CO2 で80%〜90%のコンフルエントになるまで培養hiPS細胞ライン(融解後少なくとも2〜3回継代)。</li>
	</ol></li>
	<li>0日目:EB形成<ol><li>iPS細胞維持培地を6ウェルプレートから取り出します。iPS細胞をDPBSで一度洗浄します。</li>
		<li>700〜800 μLの0.48 mM EDTA溶液を加え、室温で1分間インキュベートした後、消化溶液を除去します。37°Cの温度で3〜5分間インキュベートし続けます。</li>
		<li>細胞がシート状に消化されたら(細胞を単一細胞に消化しないでください)、iPS細胞維持培地(10 μMのRock阻害剤と1:100のGFR-細胞外マトリックスゲルを含む)2 mLを添加して消化を終了します。</li>
		<li>細胞を6ウェル低接着プレートに移します。組織培養プレートの2ウェルと低接着プレートのウェル1ウェルの比率で細胞を播種します。細胞をシェーカー(60 rpm/min)で37°C、5% CO2 で24時間インキュベートし、球状のEBを形成します。</li>
	</ol></li>
	<li>1日目-7日目:EBの分化<ol><li>EBをパスツールピペットで遠心分離管に移し(EBを破壊せずに)、室温でEBを5〜10分間自然に沈殿させます。次に、上澄み液を取り除きます。</li>
		<li>EB を 2 mL の MSC 分化培地を含む 6 ウェル低アタッチメントプレートに移します。EBをシェーカーで37°C、5%CO2 で7日間培養し、培地を1回交換します。</li>
	</ol></li>
	<li>8日目-17日目:EB接種<ol><li>EBをパスツールピペットで遠心チューブに移し(EBを破壊せずに)、EBを5〜10分間自然に沈殿させます。次に、上澄み液を取り除きます。</li>
		<li>EBを、2 mLのMSC維持培地を含むGFR細胞外マトリックスゲルコーティングされた6ウェルプレートに移します。細胞接着後、定期的な培地交換で90%のコンフルエント(~10日)まで培養します。</li>
	</ol></li>
	<li>18日目-27日目:MSCの成熟と拡大<ol><li>EB由来の培養物を37°Cの解離溶液で5〜10分間処理します(消化時間は細胞の種類によって異なります)。</li>
		<li>細胞の一部が単一細胞に消化されたら、MSC維持培地2 mLを添加して消化を終了させ、細胞懸濁液を遠心分離チューブに移します。残った未消化細胞をDPBSで一旦洗浄し、再度消化します。すべての細胞が単一の細胞に消化されるまで、数回繰り返します。細胞懸濁液を250 x g で5分間遠心分離し、上清を除去します。</li>
		<li>ゼラチンでコーティングした培養プレート(約2×105 細胞/cm2)に細胞を播種します。培地を定期的に交換するMSC維持培地で90%のコンフルエントになるまで培養します。この時点で、この世代のセルを継代0(P0)として指定します。</li>
		<li>MSCを純粋で成熟させるために、約6日間で1:3の分割比で細胞を2回継代し続けます。ほとんどの細胞は線維芽細胞様の形態(紡錘形)を示します。</li>
	</ol></li>
</ol>3. 単層培養による間葉系幹細胞とiPS細胞の分化
注:この方法は、以前の文献25、26、27、28から導出されたものである。この方法の概要を 図1に示します。この方法の特徴を 表1にまとめた。
<ol><li>培地の調製<ol><li>α-MEMに1%(v/v)のGlutMAX、10%(v/v)のFBS、および1 ng/mLのFGF2を添加して、MSC維持培地を調製します。</li>
	</ol></li>
	<li>iPS細胞の作製<ol><li>成長因子還元(GFR)細胞外マトリックスゲル(1:100)でコーティングした6ウェル組織培養プレート上のiPS細胞維持培地中で、50%〜60%のコンフルエントになるまで50%〜60%の濃度になるまで培養 hiPS細胞ライン(融解後少なくとも2〜3回継代)。</li>
	</ol></li>
	<li>0日目-13日目:直接単層培養による分化<ol><li>iPS細胞維持培地を6ウェルプレートから取り出します。</li>
		<li>MSC維持培地2 mLを直接添加し、培地を毎日交換しながら、37°C、5%CO2 で14日間培養します。</li>
	</ol></li>
	<li>14日目-35日目:反復継代によるMSCの成熟<ol><li>単層培養物を37°Cの解離溶液で5〜10分間処理します(消化時間は細胞の種類によって異なります)。</li>
		<li>細胞が単一細胞に消化されたら、2 mLのMSC維持培地を添加して消化を終了させ、細胞懸濁液を遠心分離チューブに移します。残った未消化細胞をDPBSで一旦洗浄し、再度消化します。すべての細胞が単一の細胞に消化されるまで、数回繰り返します。細胞懸濁液を250 x g で5分間遠心分離し、上清を除去します。</li>
		<li>ゼラチンでコーティングした培養皿(約2 x 105 cells/cm2)に細胞を播種します。培地とMSC維持培地で90%のコンフルエントになるまで培養し、定期的に交換します。この時点で、この世代のセルを継代0(P0)として指定します。</li>
		<li>MSCを純粋で成熟させるために、約18日間に1:3の分割比で細胞を6回継代し続けます。ほとんどの細胞は線維芽細胞様の形態(紡錘形)を示します。</li>
	</ol></li>
</ol>4. フローサイトメトリーによるヒト-PS細胞増殖幹細胞の表面抗原解析
注:骨髄由来MSCの表面抗原と同様に、iPS細胞を駆動するMSCはCD105、CD73、CD90を発現するが、CD45、CD34は発現しない29。また、ヒトiPS細胞はネガティブコントロール細胞としても利用することができます。フローサイトメトリーによるヒトiPS細胞駆動型幹細胞およびiPS細胞の表面抗原解析を 図2に示します。
<ol><li>iPS細胞駆動型MSCを解離液で37°Cで2〜3分間処理します。ヒトiPS細胞を0.48 mM EDTA解離試薬で処理し、室温で1分間インキュベートした後、解離試薬を除去します。37°Cの温度で3〜5分間インキュベートし続けます。</li>
	<li>細胞が単一細胞に消化されたら、培地(MSCにはMSC維持培地、iPS細胞にはiPS細胞維持培地)を加えて消化を終了します。</li>
	<li>細胞懸濁液を350 x g で5分間遠心分離し、上清を除去します。</li>
	<li>細胞を冷たいDPBSで一度洗浄し、350 x g で5分間遠心分離した後、上清を除去します。</li>
	<li>細胞をカウントし、10 x 106 cells/mL の冷たい 10% FBS-DPBS (v/v) 溶液に再懸濁し、100 μL/チューブの細胞懸濁液 (1 x 106 cells/tube) を 1.5 mL 遠心チューブに分配します。</li>
	<li>100 μLの細胞懸濁液に5 μLのヒトFc受容体遮断溶液を加えます。室温で5〜10分間インキュベートして、非特異的ブロッキングを実行します。</li>
	<li>350 x g で5分間遠心分離し、上清を除去します。細胞を冷たい2%FBS-DPBS(v/v)溶液で2回洗浄します。</li>
	<li>細胞を100μLの2%FBS-DPBS(v/v)溶液に再懸濁します。</li>
	<li>100 μLの細胞に5 μL(1試験)の抗CD34-FITCおよび5 μL(1試験)の抗CD45-APCを加えます 停止。抗CD73-APC5 μL(1試験)、抗CD90-FITC5 μL(1試験)、抗CD105-PE5 μL(1試験)を別の100 μL細胞懸濁チューブに加えます。5 μL のヒト IgG1 アイソタイプコントロール FITC、ヒト IgG1 アイソタイプコントロール PE、およびヒト IgG1 アイソタイプコントロール APC を 3 本目の 100 μL 細胞懸濁チューブに加えます。暗闇の中で氷の上で15〜20分間インキュベートします。その間に、単一染色ビーズを準備します。</li>
	<li>細胞を冷たい2%FBS-DPBS(v/v)溶液で2回洗浄します。</li>
	<li>300 μLの冷たい2%FBS-DPBS(v/v)溶液を加えて細胞を再懸濁し、200メッシュスクリーンフィルターでろ過します。</li>
	<li>フローサイトメトリーを用いてサンプルを解析します。アイソタイプコントロール抗体で染色した細胞懸濁液を分析し、ゲートを設定します。次に、標的抗体で染色した細胞懸濁液を分析します。</li>
</ol>5. ヒトリヒトiPS細胞を駆動する間葉系幹細胞の骨形成分化
注:ヒトiPS細胞を駆動するMSCは、骨形成分化能を有する(図3A、B)。骨形成分化のプロトコルは以下に示されています。
<ol><li>α-MEMに10%FBS、100 nMデキサメタゾン、10 mMベータ-グリセロリン酸、および100 μMアスコルビン酸を添加して、骨形成誘導培地を調製します。</li>
	<li>1 x 105 MSCをゼラチンでコーティングした48ウェルプレートに播種し、60%〜70%のコンフルエントになるまで培養します。</li>
	<li>培地を除去し、0.3 mLの骨形成誘導培地を添加します。細胞を骨形成誘導培地中で37°C、5%CO 2で14日間培養し、 培地を3日ごとに交換します。</li>
	<li>培地を取り出し、一度DPBSで洗浄してください。</li>
	<li>0.3 mL の 4% PFA を加え、室温で 30 分間インキュベートします。</li>
	<li>PFA を除去します。細胞を0.3 mLのDPBSで10分間室温で洗浄し、3回繰り返します。</li>
	<li>DPBSを除去し、0.2 mLのアリザリンレッド染色溶液を加え、室温で30分間インキュベートします。</li>
	<li>染色液を除去し、0.3 mL DPBSで細胞を室温で10分間リンスし、3回繰り返します。</li>
	<li>カルシウム沈着の染色を光学顕微鏡で観察します。</li>
</ol>6. ヒトiPS細胞駆動型幹細胞の脂肪分化
注:ヒトiPS細胞を駆動する間葉系幹細胞は、脂肪分化能を持っています(図3C、D)。脂肪形成の微分のためのプロトコルは次与えられる。
<ol><li>α-MEMに10%FBS、1 μMデキサメタゾン、1μMのIBMX、10 μg/mLのインスリン、および100 μMのインドメタシンを添加して、脂肪誘発培地を調製します。α-MEMに10%FBSおよび10μg/mLのインスリンを添加することにより、脂肪形成維持培地を調製します。</li>
	<li>1 x 105 MSCをゼラチンでコーティングした48ウェルプレートに播種し、細胞密度が90%に達するまで培養します。</li>
	<li>培地を取り除き、0.3 mLの脂肪誘発培地を加えます。細胞を脂肪誘導培地中で37°C、5%CO2で4日間培養し続けます。</li>
	<li>培地を除去し、0.3 mLの脂肪形成維持培地を添加します。細胞を脂肪形成維持培地中で37°C、5%CO2で3日間培養し続けます。</li>
	<li>脂肪細胞の分化と成熟まで、手順6.3と6.4を2〜3回繰り返します。</li>
	<li>培地を取り出し、一度DPBSで洗浄してください。</li>
	<li>0.3 mL の 4% PFA を加え、室温で 10 分間インキュベートします。PFAを除去し、DPBSで2回すすぎます。</li>
	<li>メーカーの指示に従ってOli Red O染色剤を塗布し、光学顕微鏡で脂質滴を観察します。</li>
</ol>7. ヒトiPS細胞を駆動する間葉細胞の軟骨分化
注:ヒトiPS細胞を駆動する間葉系幹細胞は、軟骨形成分化能を有する(図3E、F)。軟骨形成分化のプロトコールは以下に示す。
<ol><li>α-MEMに10%FBS、100 nMデキサメタゾン、1%インスリン-トランスフェリン-セレン(ITS)、10 μMアスコルビン酸、1 mMピルビン酸ナトリウム、50 μg/mLプロリン、0.02 nMトランスフォーミング成長因子β3(TGFβ3)、および0.5 μg/mL骨形成タンパク質6(BMP-6)を添加して軟骨芽細胞誘導培地を調製します。</li>
	<li>2.5 x 105 iPS細胞駆動型MSCを採取し、150 x g で5分間遠心分離した後、上清を除去します。細胞を1 mLのα-MEMに懸濁し、150 x g で5分間遠心分離します。</li>
	<li>上清を除去し、細胞を0.5 mLの軟骨形成誘導培地に懸濁します。細胞を150 x g で5分間遠心分離します。</li>
	<li>蓋を直接緩め、軟骨誘発培地を3日ごとに37°C、5%CO2で21日間培養します。遠心分離管を静かにはじいて、軟骨分化中は毎日軟骨形成ペレットを(軟骨形成ペレットを破壊することなく)懸濁させます。</li>
	<li>上清を取り除き、0.5 mLのPBSで2回洗浄します。</li>
	<li>0.3 mL の 4% PFA を加え、24 時間固定します。</li>
	<li>パラフィンは軟骨形成ペレットを包埋し、それらを切片化します(3 μm)。切片をトルイジンブルー(1%)で30分間染色します。</li>
	<li>細胞外軟骨細胞マトリックスを光学顕微鏡で観察します。</li>
</ol>

hiPSC駆動MSCにおける表面抗原と増殖能の特性評価

<ol>
	<li>Weng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem/stromal+cell+senescence:+Hallmarks,+mechanisms,+and+combating+strategies.">Mesenchymal stem/stromal cell senescence: Hallmarks, mechanisms, and combating strategies.</a> Stem Cells Translational Medicine. 11 (4), 356-371 (2022).</li><li>Soliman, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stromal+cells:+One+name,+multiple+identities.">Multipotent stromal cells: One name, multiple identities.</a> Cell Stem Cell. 28 (10), 1690-1707 (2021).</li><li>Friedenstein, A. J., Chailakhyan, R. K., Gerasimov, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+osteogenic+stem+cells:+in+vitro+cultivation+and+transplantation+in+diffusion+chambers.">Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers.</a> Cell and Tissue Kinetics. 20 (3), 263-272 (1987).</li><li>Friedenstein, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursors+for+fibroblasts+in+different+populations+of+hematopoietic+cells+as+detected+by+the+in+vitro+colony+assay+method.">Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.</a> Experimental Hematology. 2 (2), 83-92 (1974).</li><li>Friedenstein, A. J., Gorskaja, J. F., Kulagina, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+precursors+in+normal+and+irradiated+mouse+hematopoietic+organs.">Fibroblast precursors in normal and irradiated mouse hematopoietic organs.</a> Experimental Hematology. 4 (5), 267-274 (1976).</li><li>El Agha, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+fibrotic+disease.">Mesenchymal stem cells in fibrotic disease.</a> Cell Stem Cell. 21 (2), 166-177 (2017).</li><li>Mushahary, D., Spittler, A., Kasper, C., Weber, V., Charwat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation,+and+characterization+of+human+mesenchymal+stem+cells.">Isolation, cultivation, and characterization of human mesenchymal stem cells.</a> Cytometry A. 93 (1), 19-31 (2018).</li><li>Ullah, M., Liu, D. D., Thakor, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell+homing:+Mechanisms+and+strategies+for+improvement.">Mesenchymal stromal cell homing: Mechanisms and strategies for improvement.</a> iScience. 15, 421-438 (2019).</li><li>Regmi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+viability+and+function+of+mesenchymal+stromal+cell+spheroids+is+mediated+via+autophagy+induction.">Enhanced viability and function of mesenchymal stromal cell spheroids is mediated via autophagy induction.</a> Autophagy. 17 (10), 2991-3010 (2021).</li><li>Hoang, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapy+for+human+diseases.">Stem cell-based therapy for human diseases.</a> Signal Transduction and Targeted Therapy. 7 (1), 272 (2022).</li><li>Jiang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Mesenchymal+stem+cells+derived+from+human+pluripotent+cells,+an+unlimited+and+quality-controllable+source+for+therapeutic+applications.">Concise review: Mesenchymal stem cells derived from human pluripotent cells, an unlimited and quality-controllable source for therapeutic applications.</a> Stem Cells. 37 (5), 572-581 (2019).</li><li>Soontararak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+(MSC)+derived+from+induced+pluripotent+stem+cells+(iPSC)+equivalent+to+adipose-derived+MSC+in+promoting+intestinal+healing+and+microbiome+normalization+in+mouse+inflammatory+bowel+disease+model.">Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model.</a> Stem Cells Translational Medicine. 7 (6), 456-467 (2018).</li><li>Di Baldassarre, A., Cimetta, E., Bollini, S., Gaggi, G., Ghinassi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cell+technology+and+cardiomyocyte+generation:+Progress+and+clinical+applications.">Human-induced pluripotent stem cell technology and cardiomyocyte generation: Progress and clinical applications.</a> Cells. 7 (6), 48 (2018).</li><li>Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology:+a+decade+of+progress.">Induced pluripotent stem cell technology: a decade of progress.</a> Nature Reviews. Drug Discovery. 16 (2), 115-130 (2017).</li><li>Levy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shattering+barriers+toward+clinically+meaningful+MSC+therapies.">Shattering barriers toward clinically meaningful MSC therapies.</a> Science Advances. 6 (30), eaba6884 (2020).</li><li>Zhao, C., Ikeya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+applications+of+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells.">Generation and applications of induced pluripotent stem cell-derived mesenchymal stem cells.</a> Stem Cells International. 2018, 9601623 (2018).</li><li>Path, G., Perakakis, N., Mantzoros, C. S., Seufert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+treatment+of+diabetes+mellitus+-+Focus+on+mesenchymal+stem+cells.">Stem cells in the treatment of diabetes mellitus - Focus on mesenchymal stem cells.</a> Metabolism. 90, 1-15 (2019).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+derivation+of+functional+mesenchymal+stem+cells+from+human+pluripotent+stem+cells.">One-step derivation of functional mesenchymal stem cells from human pluripotent stem cells.</a> Bio-Protocol. 8 (22), e3080 (2018).</li><li>Hua, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-intensity+pulsed+ultrasound+promotes+osteogenic+potential+of+iPSC-derived+MSCs+but+fails+to+simplify+the+iPSC-EB-MSC+differentiation+process.">Low-intensity pulsed ultrasound promotes osteogenic potential of iPSC-derived MSCs but fails to simplify the iPSC-EB-MSC differentiation process.</a> Frontiers in Bioengineering and Biotechnology. 10, 841778 (2022).</li><li>Dupuis, V., Oltra, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+produce+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells:+Mesenchymal+stem+cells+from+induced+pluripotent+stem+cells.">Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells.</a> World Journal of Stem Cells. 13 (8), 1094-1111 (2021).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+stem+cells.+A+Werner+syndrome+stem+cell+model+unveils+heterochromatin+alterations+as+a+driver+of+human+aging.">Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.</a> Science. 348 (6239), 1160-1163 (2015).</li><li>Liu, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+Fanconi+anemia+pathogenesis+and+therapeutics+using+integration-free+patient-derived+iPSCs.">Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.</a> Nature Communications. 5, 4330 (2014).</li><li>Kubben, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+the+antioxidant+NRF2+pathway+in+premature+aging.">Repression of the antioxidant NRF2 pathway in premature aging.</a> Cell. 165 (6), 1361-1374 (2016).</li><li>Duan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+deficiency+reprogrammes+human+neural+stem+cells+towards+a+glioblastoma+stem+cell-like+phenotype.">PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.</a> Nature Communications. 6, 10068 (2015).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+released+from+human+induced+pluripotent+stem+cells-derived+MSCs+facilitate+cutaneous+wound+healing+by+promoting+collagen+synthesis+and+angiogenesis.">Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis.</a> Journal of Translational Medicine. 13, 49 (2015).</li><li>Hu, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+secreted+by+human-induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+attenuate+limb+ischemia+by+promoting+angiogenesis+in+mice.">Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.</a> Stem Cell Research &amp; Therapy. 6 (1), 10 (2015).</li><li>Kang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+human+induced+pluripotent+stem+cells+retain+adequate+osteogenicity+and+chondrogenicity+but+less+adipogenicity.">Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.</a> Stem Cell Research &amp; Therapy. 6 (1), 144 (2015).</li><li>Wang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mesenchymal+stem+cells+from+human+induced+pluripotent+stem+cells+results+in+downregulation+of+c-Myc+and+DNA+replication+pathways+with+immunomodulation+toward+CD4+and+CD8+cells.">Differentiation of mesenchymal stem cells from human induced pluripotent stem cells results in downregulation of c-Myc and DNA replication pathways with immunomodulation toward CD4 and CD8 cells.</a> Stem Cells. 36 (6), 903-914 (2018).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Kim, S., Kim, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem-like+cells+for+producing+extracellular+vesicles.">Generation of mesenchymal stem-like cells for producing extracellular vesicles.</a> World Journal of Stem Cells. 11 (5), 270-280 (2019).</li></ol>

著者は何も開示していません。

このプロトコルでは、ヒトiPS細胞をMSCに鑑別する2つの代表的な方法を検討しました20,21,22,23,24,25,26,27,28,30。どちらの方法も、iPS細胞からMSC?...

間葉系間質細胞(MSC)は、中胚葉由来の成体多能性幹細胞です1。MSCは、ほとんどすべての結合組織に存在します2。MSCが1970年代に初めて発見され、1987年にFriedensteinらによって骨髄からの単離に成功して以来3,4,5、骨、軟骨、腱、筋肉、脂肪組織、造血支持間質などのさまざまなヒト体細胞組織(胎児および成人を含む)がMSCの単離に使用されてきました1,2,6,7.MSCは、多くの体細胞系統に分化するための高い増殖能力と可塑性を示し、損傷組織や炎症組織に移動する可能性があります2,8,9。これらの特性により、MSCは再生医療の候補となる可能性があります10。しかし、体細胞組織由来MSC(st-MSC)は、提供が限られていること、細胞増殖能力が限られていること、品質のばらつき、およびドナーからの病原体の伝播の可能性に対するバイオセーフティー上の懸念によって制限されている11,12。
ヒト人工多能性幹細胞(iPS細胞)は、胚性幹細胞と同様の機能を持つ転写因子(Oct4、Sox2、Klf4、c-Myc)でリプログラミングした成体細胞に由来します13,14。iPS細胞は、ST-MSCと比較して、供給量が無制限で、低コスト、高純度、品質管理の利便性、スケール生産や遺伝子改変が容易であるという利点があります15,16,17。
iPS細胞-MSCのこれらの利点により、iPS細胞からMSCを駆動する方法がいろいろと報告されています。これらの分化法は、(1)胚様体(EB)の形成と(2)単層培養の使用という2つの培養方法論を中心としています11,18,19,20。本明細書では、2つの方法論のそれぞれに対する代表的なアプローチを特徴づけた。さらに、時間、コスト、増殖能、MSCバイオマーカーの発現、およびin vitroでの分化能力に基づく2つの代表的なアプローチの比較もアクセスしました。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alizarin red staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0148S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD105 (PE)</td>
			<td>Biolegend</td>
			<td>323206</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD34 (FITC)</td>
			<td>Biolegend</td>
			<td>343503</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD45 (APC)</td>
			<td>Biolegend</td>
			<td>304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD73( APC)</td>
			<td>Biolegend</td>
			<td>344006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD90 (FITC)</td>
			<td>Biolegend</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Solarbio</td>
			<td>A8100</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-6</td>
			<td>Novoprotein</td>
			<td>C012</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide level shaker</td>
			<td>Crystal</td>
			<td>CO-06UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation Beads</td>
			<td>BioLegend</td>
			<td>424601</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>STEMCELL Technology</td>
			<td>07959</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160; medium</td>
			<td>Servicebio</td>
			<td>G4610</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HAKATA</td>
			<td>HS-FBS-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Stemcell</td>
			<td>78003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control APC</td>
			<td>BioLegend</td>
			<td>403505</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control FITC</td>
			<td>BioLegend</td>
			<td>403507</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control PE</td>
			<td>BioLegend</td>
			<td>403503</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#946;1</td>
			<td>Stemcell</td>
			<td>78067</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX&#160;</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Solarbio</td>
			<td>SI9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>P3376</td>
			<td></td>
		</tr>
		<tr>
			<td>iPSC maintenance medium</td>
			<td>STEMCELL Technology</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Media Supplement</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0341-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>BD Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Oli Red O staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0158S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Solarbio</td>
			<td>P0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;3</td>
			<td>Novoprotein</td>
			<td>CJ44</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue staining kit</td>
			<td>Solarbio</td>
			<td>G2543</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme(1x)&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 6 Well Plate</td>
			<td>Costar</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemcell</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone</td>
			<td>SH30265</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Solarbio</td>
			<td>G8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of two representative methods for differentiation of human induced pluripotent stem cells into mesenchymal stromal cells

間葉系間質細胞(MSC)は、再生医療で広く利用されている成体多能性幹細胞です。体細胞組織由来のMSCは、提供が限られていること、品質のばらつきがあること、バイオセーフティーによって制限されているため、過去10年間で、ヒト人工多能性幹細胞(iPS細胞)からMSCを作製する取り組みが大幅に増加しています。ヒトiPS細胞を間幹細胞に分化させるためのこれまでの取り組みは、(1)胚様体(EB)の形成と(2)単層培養の2つの培養方法が中心でした。このプロトコルでは、iPS細胞からMSCを誘導する際のこれら2つの代表的な方法について説明します。それぞれの方法には、時間、コスト、細胞増殖能、MSCマーカーの発現、 in vitroでの分化能力など、長所と短所があります。このプロトコルは、どちらの方法もiPS細胞から成熟した機能的なMSCを導き出すことができることを示しています。単層法は低コスト、操作の簡便さ、骨形成の分化の容易さを特徴とし、EB法は時間消費が少ないという特徴があります。

プロトコール(図1A)に従い、iPS細胞をEB形成法および単層培養法でMSCに分化させました。分化中、細胞は異なる代表的な形態を示しました(図1B、C)。
図1Bに示すように、iPS細胞コロニーは、分化前は典型的なコンパクトな形態を示し、細胞が密集した明確な境界を示しています。iPS細胞が解離?...

Watch this Scientific Journal Video about Advancements in iPSCs and Genetic Disease Research at JoVE.com

著者スポットライト:iPS細胞と遺伝性疾患研究の進歩 

このプロトコルでは、ヒトiPS細胞を間葉系間質細胞(MSC)に分化させるための2つの代表的な方法を説明し、比較します。単層法は、低コスト、操作の簡便さ、骨形成の分化の容易さを特徴としています。胚様体(EB)法は、時間消費が少ないという特徴があります。

ヒト人工多能性幹細胞から間葉系間質細胞への分化のための2つの代表的な方法の比較

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

本研究は、遺伝性疾患のメカニズムと治療戦略の研究です。私たちの研究は、細胞株、iPS細胞、オルガノイド、マウスなどのマルチレベルの疾患モデルに基づいています。iPS細胞には、他のモデルに比べて明確な利点があります。患者さんのiPS細胞、およびその後の派生細胞やオルガノイドは、患者さんと同じゲノムを共有しているため、多くの疾患の患者細胞やモデルが豊富に揃っています。また、患者さんからのiPS細胞の確立方法もかなり成熟しています。iPS細胞を特定の細胞やオルガノイドに分化させる方向には多くの課題が残されており、現在の開発における発生メカニズムの理解が深まることが現場では起こっています。

comparison-two-representative-methods-for-differentiation-human

Research

JoVE Journal

Biology

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

795 ビュー.  The Children's Hospital, Zhejiang University School of Medicine. 本研究は、遺伝性疾患のメカニズムと治療戦略の研究です。私たちの研究は、細胞株、iPS細胞、オルガノイド、マウスなどのマルチレベルの疾患モデルに基づいています。iPS細胞には、他のモデルに比べて明確な利点があります。患者さんのiPS細胞、およびその後の派生細胞やオルガノイドは、患者さんと同じゲノムを共有しているため、多くの疾患の患者細胞や?...

ビデオ: 著者スポットライト:iPS細胞と遺伝性疾患研究の進歩 

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

生物学

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

ヒト人工多能性幹細胞をトランスフェクトしNucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

運動ニューロンにマウス胚性幹細胞の効率的な分化

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

ヒト心筋細胞の生成：フィーダーフリーヒト人工多能性幹細胞からの分化プロトコル

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.