この作品は、ジョアンナM.ニコライ財団からのメラノーマ研究者のためのフェローシップによってルースL.キルシュシュタイン国立研究サービス賞F31下の国立衛生研究所によってサポートされていました。この作品は、さらにNYSTEMからの助成金やトライ機関幹細胞イニシアチブ（スター基金）を通じてサポートされていました。

注：ここで説明メラノサイトプロトコルが最初雲母らによって実証されました。 1.培養培地の調製、コーティングされた料理とhPSCsのメンテナンス<ol><li>ミディアム準備 注：最大2週間、暗所で4℃ですべてのメディアを保管してください。殺菌のためのすべてのメディアをフィルタリングします。 <ol><li> DMEM / 10％FBSを準備します。 885 mlのDMEM、100mlのFBS 10mlのペニシリン/ストレプトマイシン、5mlのL-グルタミンを混合します。殺菌のためのフィルタ。 </li><li> hESCの培地を準備します。 800 mlのDMEM / F12を200ml KSR、5mLのL-グルタミン10mlのMEM、最小必須アミノ酸溶液、βメルカプトエタノールに、1ml、5mlのペニシリン/ストレプトマイシンを混ぜます。 10ng / mLのFGF-2を追加し濾過した後。 </li><li> KSR-分化培地を準備します820ミリリットルノックアウトDMEM、150ミリリットルKSR、10ミリリットルのL-グルタミン、10ミリリットルペニシリン/ストレプトマイシン、10ミリリットルMEM最小必須アミノ酸溶液およびβメルカプトエタノール1ミリリットルを混ぜます。殺菌のためのフィルタ。 </li><li> N2-分化培地を準備します。 12グラムDMEM / F12粉末Iを溶解しnは980ミリリットルのdH 2 O 1.55グラムのグルコース、2グラムの重炭酸ナトリウムおよび100 mgのアポヒトトランスフェリンを追加します。 25 mgのヒトインスリンおよび40μlの1N NaOHで2ミリリットルのdH 2 Oを混ぜます。溶解した後、培地に混合物を追加します。 100μlのプトレシン二塩酸塩、60μlの亜セレン、100μlのプロゲステロンを追加します。フィルタリング前のdH 2 Oで1リットルに最終容量を持参。 </li><li>フルメラノサイト媒体を準備します。 50％Neurobasal培地、30％の低グルコースDMEM、および20％MCDB201を兼ね備えています。この追加するには：0.8％ITS +、250 nMのL-グルタミン、100μMのアスコルビン酸（L-AA）、50 ngの/ mlのコレラ毒素、50 ngの/ mlのSCF、0.05μMのデキサメタゾン、100 nMのEDN3、4 ngの/ mlのFGF2 。滅菌フィルターその後、残りの試薬を追加：2％B27サプリメント、25 ngの/ mlのBMP4、3μMCHIR99021、500μMのキャンプを。 </li></ol></li><li>培養皿のコーティング<ol><li>このようなマトリゲルとしてゼラチン状タンパク質を用いて塗布行います。 1 mlの部品への開口部、アリコートを凍結マトリゲル時繰り返し凍結融解cを避けるためにycles。解凍し、19ミリリットルDMEM / F12と1ミリリットル凍結アリコートを再サスペンド。 10cmの皿にプレート5 mlです。 RTで1時間の皿をインキュベートします。すぐに細胞をプレーティングする前に、ゲル状のタンパク質を吸引し、DMEM / F12で洗浄します。 </li><li>ポリ-L-オルニチン臭化水素酸塩/マウスラミニン-I /フィブロネクチン（PO /ラム/ FN）を使用してコーティングを行います。含むPBSでコーティング皿を15μg/ mlのポリ-L-オルニチン臭化水素酸塩。加湿インキュベーター中、37℃でO / Nインキュベートします。吸引液とを1μg/ mlのマウスラミニンIおよび2μg/ mlのフィブロネクチンを含むPBSでコーティングする前にPBSで3回プレートを洗浄します。加湿インキュベーター中、37℃で皿O / Nインキュベートします。 <ol><li>細胞をプレーティングする前に、溶液を吸引し、プレートを組織培養フード内蓋なしで完全に乾燥してみましょう。プレートは約10〜15分間乾燥することができます。 注：結晶構造が目で表面に現れたときに料理が乾燥し、細胞播種の準備ができています。区画ESは限り液体が蒸発しないように、PBSで2週間培養器でLAM / FNを含有して維持することができます。プレートは、数時間室温で乾燥させ維持することができます。 </li></ol></li></ol></li><li> hPSCsのメンテナンス 注：hPSCsは、ヒトES細胞培地中の0.1％ゼラチンと、有糸分裂不活性化したマウス胚性線維芽細胞（MEF）上で維持されています。細胞は、すべての6-8日に分割する必要があります。 <ol><li>被覆5分間室温でPBS中の0.1％ゼラチンで10cmディッシュ。 </li><li> 37℃の水浴中で急速に凍結したMEFを解凍します。 </li><li>ゼラチンを吸引し、細胞をプレー。 DMEM / 10％FBS中〜50,000細胞/ cm 2の密度でプレートしたMEF。 hPSCsを追加する前に37°CO / NでのMEFをインキュベートします。 </li><li>準備されたMEFプレートからDMEM / 10％FBSを吸引し、PBSでプレートを洗浄し、hPSCsと10ミリリットルのhESC培地を追加します。通路1の割合でhPSCs：5/10前継代の密度に応じて。 注：hPSCsを融解または単細胞として継代されている場合、SUP最初の継代まで、10μMY-27632二塩酸塩（ROCKI）でplement。 </li><li> mlの10新鮮なhESCの培地で毎日細胞を養います。 </li><li>分化した細胞、不規則な境界線、または透明の中心28を含んでいるように思われる任意の多能性コロニーを削除分化を開始する前に。機械的に除去すると、解剖顕微鏡を用いて層流フードの下にピペットで不規則なコロニーを吸引します。 </li></ol></li></ol>分化のためのhPSCsの2めっき注：分化条件は、10cmの皿に記載されています。 <ol><li>ステップ1.3.1で説明したように分化を開始する前に、10センチメートルマトリゲル料理を準備します。 </li><li>分化のためのメッキhPSCsは、通路の準備密度（〜80％コンフルエント）、ヒトES細胞培地を吸引、PBSで洗浄し、細胞に、0.05％トリプシン-EDTAの3ミリリットルを追加でhPSCsの板を起動すると。 </li><li>精力的に皿horizo​​ntallを振ります2分間のyのMEFは、単一細胞としてリフトオフまで顕微鏡下で可視化しながら。 HPSCコロニーをコロニーとして付着したままでなければなりません。 </li><li> MEFのが持ち上げられた後、トリプシンを吸引するがhPSCsのコロニーをデタッチする前に。 </li><li>プレートに10μMのY-27632の二塩酸塩を含有したhESC培地10mlを追加し、コロニーの上に上下にピペッティングして細胞を切り離します。 </li><li>ステップ2.1で調製した10cmディッシュからマトリゲル液を吸引し、塊を除去するために、DMEM / F12で洗浄します。マトリゲルプレート上に2：1の比率でhPSCsをプレート。 10ミリリットルまで、10μMY-27632二塩酸塩を含有したhESC培地を追加します。 37°CO / Nでインキュベートします。 注：このめっきは/ cm 2で約100,000細胞をもたらすはずです。 </li></ol>神経分化の3誘導注：hPSCsが80％コンフルエントである場合に分化が（0日目）に開始されるべきです。細胞は、ヒトES細胞培地を毎日供給することができます。分化を開始するまで、10μMのY-27632の二塩酸塩を含みます。 <ol><li> 0日目および1日目に、100 nmのLDN193189と10μMのSB431542を含むKSR-分化培地10mlで細胞を養います。 </li><li> 2日目に、100 nmのLDN193189、10μMのSB431542および3μMのCHIR99021を含むKSR-分化培地10mlで細胞を養います。 </li><li> 3日目に、10μMのSB431542および3μMCHIR99021を含むKSR-分化培地10mlで細胞を養います。 </li><li> 4および5日目フィード15 75％のKSR-分化培地のmlおよび3μMCHIR99021を含む25％N2-分化培地での細胞上。 注意：浮遊細胞の点で細胞死を大量に見ることが心配しないでください。これは正常であり、予想されること。 </li><li>日に6と7フィード50％KSR-分化培地および50％N2-分化培地の15ミリリットルを持つ細胞は3μMCHIR99021、25 ngの/ mlのBMP4、および100nM EDN3を含む両方。 </li><li>日8日そして9飼料25％KSR-分化培地と75％のN2-分化培地20mlを持つ細胞は3μMCHIR99021、25 ngの/ mlのBMP4、および100nM EDN3を含む両方。 </li><li> 11日目の細胞の再メッキに1.2.2に示すように日9および10にPO /ラム/ FNの料理を準備します。 </li><li> 3μMCHIR99021、25 ngの/ mlのBMP4、および100nM EDN3を含むN2-分化培地20mlで一日10フィード細胞上。 </li></ol> NC仕様のための液滴4.再プレート<ol><li> 11日目吸引ラム上/完全に準備されたPO / LAM / FNプレートと乾燥からFN。 </li><li> 、11日目の細胞から培地を除去し、PBSで洗浄し、そのような10cmの皿当たりのAccutaseとして4ミリリットルの細胞剥離液を追加します。 37℃で25分間インキュベートします。 </li><li>皿にフルメラノサイト培地5mlを追加し、すべての細胞がプレートから浮き上がるまで、手動で10ミリリットルピペットでピペッティングして細胞を再懸濁します。 15ミリリットルチューブにサスペンションを転送します。 </li><li>スピン200×gで5分間、細胞ダウンとフルメラノサイト培地1ml当たり2×10 6個の細胞で細胞を再懸濁します。血球計数器または同等の技術上のトリパンブルー排除を用いて細胞数を計測します。 </li><li>乾燥PO /ラム/ FN 10cmの皿に（彼らは触れず）互いに近接板10μlの滴。プレートは十分に乾燥された場合は、液滴が明確に定義されたエッジを持って実行しないでください。 注：これは、拡張用の皿の中の部屋を維持しながら、細胞を再播種する際に重要である細胞のための高密度ローカル環境を作成します。 </li><li>ゆっくり（付着細胞を乱さない）全メラニン細胞培地10mlを追加し、液滴は、細胞が付着することを可能にするために10から20分間室温で放置します。インキュベーターに皿を移動します。 </li></ol> 5.メラニン前駆細胞を拡大<ol><li> 2〜3日ごとにフルメラノサイト培地を供給し続けます。 注：色素沈着がワット表示されるように開始する必要があります最初の週の終わりまでに、細胞のクラスターをithinと第二週によって非常に明確になります。このようなこれらの初期の小さな、暗いクラスタを識別するために一枚の紙のように白い背景の上にプレートを表示します。プレート全体が均一に着色さになるまで、細胞は、（ 図2Bを参照）、時間をかけて徐々に着色さになります。 </li><li> 〜1の割合で週に一度継代細胞：6（液滴としてメッキが、この段階ではもはや必要ではありません）。細胞を解離し、フルメラノサイト培地中で再めっきの前に、プレーン神経基本培地で2回洗浄するためのAccutaseを使用してください。 PO / LAM / FNプレート上の細胞を維持します。 注：我々は、全メラノサイト媒体は維持し、ヒトES細胞とIPSC由来メラノサイトを拡大するのに最適であることを見出しました。しかし、細胞は、市販のM254培地中で簡単に培養することができるが、簡略化されたメディアは死んで、プレートをリフトオフ細胞のリスクを増大させます。 </li></ol>

<ol>
	<li>Ebert, A. D., 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=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457, 277-280 (2009).</li><li>Lee, G., 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+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461, 402-406 (2009).</li><li>Lee, G., 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=Large-scale+screening+using+familial+dysautonomia+induced+pluripotent+stem+cells+identifies+compounds+that+rescue+IKBKAP+expression.">Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression.</a> Nat biotechnol. 30, 1244-1248 (2012).</li><li>Lee, G., Studer, L. <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+for+the+study+of+human+disease.">Induced pluripotent stem cell technology for the study of human disease.</a> Nat methods. 7, 25-27 (2010).</li><li>Zimmer, 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=Evaluation+of+developmental+toxicants+and+signaling+pathways+in+a+functional+test+based+on+the+migration+of+human+neural+crest+cells.">Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells.</a> Environ health persp. 120, 1116-1122 (2012).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neural+crest+induction,+melanocyte+specification,+and+disease-related+pigmentation+defects+in+hESCs+and+patient-specific+iPSCs.">Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs.</a> Cell rep. 3, 1140-1152 (2013).</li><li>Mariani, 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=FOXG1-Dependent+Dysregulation+of+GABA/Glutamate+Neuron+Differentiation+in+Autism+Spectrum+Disorders.">FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders.</a> Cell. 162, 375-390 (2015).</li><li>Doulatov, 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=Modeling+Diamond+Blackfan+Anemia+in+Vivo+Using+Human+Induced+Pluripotent+Stem+Cells.">Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells.</a> Blood. 124, (2014).</li><li>Cherry, A. B. C., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogrammed+Cells+for+Disease+Modeling+and+Regenerative+Medicine.">Reprogrammed Cells for Disease Modeling and Regenerative Medicine.</a> Annu Rev Med. 64, 277-290 (2013).</li><li>Inoue, H., Nagata, N., Kurokawa, H., 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=iPS+cells:+a+game+changer+for+future+medicine.">iPS cells: a game changer for future medicine.</a> Embo J. 33, 409-417 (2014).</li><li>Kim, C., 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=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494, 105-110 (2013).</li><li>Fang, D., 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=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem cells. 24, 1668-1677 (2006).</li><li>Huang, X., Saint-Jeannet, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+neural+crest+and+the+opportunities+of+life+on+the+edge.">Induction of the neural crest and the opportunities of life on the edge.</a> Dev biol. 275, 1-11 (2004).</li><li>White, R. M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocytes+in+development,+regeneration,+and+cancer.">Melanocytes in development, regeneration, and cancer.</a> Cell stem cell. 3, 242-252 (2008).</li><li>Morrison, S. J., White, P. M., Zock, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification,+isolation+by+flow+cytometry,+and+in+vivo+self-renewal+of+multipotent+mammalian+neural+crest+stem+cells.">Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.</a> Cell. 96, 737-749 (1999).</li><li>Elworthy, S., Pinto, J. P., Pettifer, A., Cancela, M. L., Kelsh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phox2b+function+in+the+enteric+nervous+system+is+conserved+in+zebrafish+and+is+sox10-dependent.">Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent.</a> Mech develop. 122, 659-669 (2005).</li><li>Harris, M. L., Baxter, L. L., Loftus, S. K., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+proteins+in+melanocyte+development+and+melanoma.">Sox proteins in melanocyte development and melanoma.</a> Pigm cell melanoma r. 23, 496-513 (2010).</li><li>Herbarth, 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=Mutation+of+the+Sry-related+Sox10+gene+in+Dominant+megacolon,+a+mouse+model+for+human+Hirschsprung+disease.">Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease.</a> PNAS. 95, 5161-5165 (1998).</li><li>Southard-Smith, E. M., Kos, L., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox10+mutation+disrupts+neural+crest+development+in+Dom+Hirschsprung+mouse+model.">Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model.</a> Nat genetics. 18, 60-64 (1998).</li><li>Dupin, E., Glavieux, C., Vaigot, P., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin+3+induces+the+reversion+of+melanocytes+to+glia+through+a+neural+crest-derived+glial-melanocytic+progenitor.">Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor.</a> PNAS. 97, 7882-7887 (2000).</li><li>Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nacre+encodes+a+zebrafish+microphthalmia-related+protein+that+regulates+neural-crest-derived+pigment+cell+fate.">nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.</a> Development. 126, 3757-3767 (1999).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127, 5379-5389 (2000).</li><li>Levy, C., Khaled, M., Fisher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MITF:+master+regulator+of+melanocyte+development+and+melanoma+oncogene.">MITF: master regulator of melanocyte development and melanoma oncogene.</a> Trends mol med. 12, 406-414 (2006).</li><li>Phung, B., Sun, J., Schepsky, A., Steingrimsson, E., Ronnstrand, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-KIT+signaling+depends+on+microphthalmia-associated+transcription+factor+for+effects+on+cell+proliferation.">C-KIT signaling depends on microphthalmia-associated transcription factor for effects on cell proliferation.</a> PloS one. 6, e24064 (2011).</li><li>Grichnik, J. 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=KIT+expression+reveals+a+population+of+precursor+melanocytes+in+human+skin.">KIT expression reveals a population of precursor melanocytes in human skin.</a> J invest dermatol. 106, 967-971 (1996).</li><li>Li, L., 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=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J cell sci. 123, 853-860 (2010).</li><li>Nishimura, E. K., 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=Dominant+role+of+the+niche+in+melanocyte+stem-cell+fate+determination.">Dominant role of the niche in melanocyte stem-cell fate determination.</a> Nature. 416, 854-860 (2002).</li><li>Zur Nieden, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryonic stem cell therapy for osteo-degenerative diseases : methods and protocols. , (2011).</li><li>Lee, J. 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=high-content+screening+for+novel+pigmentation+regulators+using+a+keratinocyte/melanocyte+co-culture+system.">high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.</a> Exp dermatol. 23, 125-129 (2014).</li></ol>

著者らは、開示することは何の利害を持っていません。

hPSCsからメラノサイトの成功の分化のために以下の提案を考慮に入れなければなりません。何よりもまず、常に無菌培養条件下で動作することが不可欠です。さらに、多能性、完全未分化hPSCsで開始することが重要です。出発集団は、分化した細胞が含まれている場合、収率は常に汚染物質がメラノサイトに向けることができず、さらに適切に分化する細胞を破壊し得るよう低下します。 ...

ヒト多能性幹細胞（hPSCs）は、疾患モデル、薬物スクリーニング、および細胞置換療法1-6のためのスケーラブルな方法で正常な分化を模倣するためのプラットフォームを提供します。特に興味深いのは、hPSCsは、患者のサンプルが不足している単離することが困難またはまれ/一過性の細胞型を研究するための道を開きます。また、人工多能性幹細胞（性IPSC）のユニークなメカニズム1,2,7-11を解明するために、患者特異的に開発し、疾患モデルを研究する研究者を有効にします。 hPSCsからメラノサイトの分化のための以前に公開されたプロトコルは、分化の6週間まで必要とし、L-のWnt3a細胞12からの馴化培地で細胞を培養することを含みます。 1プロトコル第1雲母らによって提示。、ここでは説明は3週間で色素沈着細胞を生成し、馴化培地に関連した曖昧さや矛盾を削除します。 メラノサイトのareは神経堤、脊椎動物に特有の細胞の遊走集団から派生します。神経堤は、原腸陥入時に定義され、神経および非神経外胚葉の間で国境を接する、神経板のエッジで細胞の集団を表しています。神経胚形成の間に、神経組織が ​​神経管13,14の結果背側正中線に収束神経ひだを形成するために、神経板から進化します。 神経堤細胞は脊索の向かい、神経管の屋根板から出て、分化した細胞の多様な集団を生じさせるために離れて移行する前に、間葉への移行上皮を受けます。堤細胞の運命は、胚の体軸に沿って屋根板の解剖学的位置によって部分的に定義されています。神経堤細胞誘導体は、中胚葉（平滑筋細胞、骨芽細胞、脂肪細胞、軟骨細胞）および外胚葉細胞（メラノサイト、シュワンCの両方の特性系統を含みますells、ニューロン）14。神経堤幹細胞は、転写因子SOX10をアップレギュレートおよびp75とHNK1に対する抗体を用いて蛍光活性化セルソーティングにより単離することができます。 メラノサイトになることを運命づけられた神経堤細胞は、メラノブラストのステージを通過し、KITとMITF（小眼球症関連転写因子）をアップレギュレート6,21 MITFは、メラニン細胞の開発のマスター調節因子であり、メラノサイトの開発の多くを制御する役割を担う転写因子であります22- 24。人間melanoblastsは、彼らが毛膨らみまたは表皮中のケラチノサイトに囲まれた成熟した、色素性メラノサイトの前駆体として機能する（色素形成単位）のいずれかに存在し、表皮の基底層に移行します。色素性メラノサイトへのmelanoblastsの分化・成熟は、メラニン産生経路（TYRP1、TYR、OCA2の毛球と表現の植民地とそれに付随し発生し、PMEL）25,26。 患者からのヒトメラノサイトとmelanoblastsを分離すると、高価な困難や量に制限です。このプロトコルは、細胞選別せずに明確に定義され、迅速な、再現可能なスケーラブル、かつ安価な方法でメラノサイトまたはメラノサイト前駆体にhPSCsを（誘起または胚）を区別するために研究者を可能にします。プロトコルは、色素異常症を有する患者からのiPS細胞の分化時に疾患特異的な欠陥を識別するために以前に使用されました。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

feeder-free derivation of melanocytes from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

このプロトコルは、インビトロ無フィーダー、コスト効率の良い、そして再現可能な方法でhPSCsから完全に着色され、成熟したメラノサイトを導出するための方法を提供します。 HPSC由来のメラノサイトのために以前に確立された牙らプロトコルとは対照的に、概説したプロトコルは、馴化培地を必要とし、時間の要件を減少しません。牙らのプロ...

Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

この作品は、神経堤を介したヒト多能性幹細胞から色素沈着し、成熟したメラノサイトを生成し、無フィーダー、25日のプロトコルを使用して中間段階のメラノブラストするためのin vitroでの分化プロトコルを記述します。

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

feeder-free-derivation-melanocytes-from-human-pluripotent-stem

Research

JoVE Journal

Developmental Biology

10.2K ビュー.  Memorial Sloan-Kettering Cancer Center. この作品は、神経堤を介したヒト多能性幹細胞から色素沈着し、成熟したメラノサイトを生成し、無フィーダー、25日のプロトコルを使用して中間段階のメラノブラストするためのin vitroでの分化プロトコルを記述します。 

ビデオ: Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

発生生物学

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

ヒト人工多能性幹細胞由来の錐体ニューロンからの樹状突起棘の三次元定量

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

フィーダーフリー条件にセンダイウイルスを用いてヒト末梢T細胞から人工多能性幹細胞の生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

ヒト多能性幹細胞から肝細胞様細胞の定義され、スケーラブルな世代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

ヒト多能性幹細胞の肝細胞への効率的な分化

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...