著者らは、胎盤組織を提供するためにすぐに-チョルホン（医学博士、准教授、産婦人科、医科大学高麗大学）を感謝したいと思います。この作品は、韓国国立研究財団（NRF）、韓国からの助成金（R1211902）によって部分的にサポートされていました。

倫理文：ヒト胎盤研究は、高麗大学（AN09085-001）の人間の研究のための施設内倫理委員会の承認を得て、将来に向かって行きました。すべての実験は、高麗大学医療センターのクリーン無菌室の施設で行われました。 hPSCsを用いた実験の設計および手順は高麗大学医療センターの施設内倫理委員会（AN12277-003）によって承認されました。 楽器、文化メディア、および料理の作製<ol><li> 0.1％ゼラチンでコーティングすべての培養皿。オートクレーブリン酸緩衝生理食塩水（PBS）と鉗子。手術用はさみ、微量遠心管、および15分以上のための15ポンドのPSIの下で121℃でガーゼを滅菌します。 </li><li> 20％ウシ胎児血清（FBS）、100 U / mlペニシリン、100グラム/ mlを含む調製予め温めた0.25％トリプシン - エチレンアミン四酢酸（トリプシンEDTA）及び予熱濾過培地（DMEM） streptomyc手順を開始する前に。 </li></ol>ヒト胎盤から2細胞の単離<ol><li>書面によるインフォームドコンセントを得た後、妊娠7-32週で正常または治療的流産を受けた健康な妊婦から、帝王切開の出産後、胎盤組織を取得します。 </li><li>胎盤から産科専門外科の別々のヒト胎盤絨毛プレート（HPCの）を持っています。 37℃で20分間、0.25％トリプシン-EDTAでこれらをインキュベートし、次いで5 mlのPBSで洗浄します。 </li><li>はさみを使用してのHPCから絨毛を隔離。絨毛をミンチし、1ミリリットルPBSでそれらを3回洗浄します。ミンチした後、ピペットチップを使用して、マイクロチューブの中に絨毛を置きます。上下にピペッティングにより500μlのPBSでサンプルを洗浄し、3分間120×gでのためにチューブを遠心します。予め温めた培地500μlので二回組織を洗ってください。 </li><li> 37℃、5％CO 2でゼラチンコーティングしたプレート上で培地中の培養細胞</&gt;、および湿度95％の下のサブ。 37°Cで1時間0.1％ゼラチンでプレコートすべてのプレート。線維芽細胞様細胞のコロニーを形成し、約5〜7日、同じ培地で培養を開始した後、付着成長します。 </li><li>第3通路まで2〜3日ごとに培地を交換します。培養中、培養液中に浮遊する細胞の破片を除去します。成長胎盤線維芽細胞はプレートに取り付けます。文化その後、10 番目の通路にHPCまでに由来し、線維芽細胞様細胞は、トリプシン処理し、マイトマイシンC（10 / mlの）治療後のhESC培養物のためにそれらを収穫します。 </li><li>ストックサンプルとしてのHPCを凍結する前に、のHPCは、通常（M.·ファー、M.のヒオリニス、M.アルギニン、Mがマイコプラズマを含む胎盤、感染性病原体に汚染されていないことを確認するために逆転写ポリメラーゼ連鎖反応（RT-PCR）を使用し。orale、M.のsalivarium、M.ホミニス、M.プルモニス、M.のarthritidis、M.のneurolyticum、M。ハイオニューモニエ、とM.カプリコラム ）および細菌種、 ウレアプラズマリティクム 、マイコプラズマ検出キットを使用します。 <ol><li> RT-PCRを実行するための製造元の指示に従って、キットで提供されるすべてのプライマーとの混合物を使用しています。マイコプラズマ検出のための36日間培養された細胞の上清を使用してください。 PCR混合物にこれらのサンプルの3μlを添加して、35サイクルの1 回目のPCR手順を実行し、30秒間、94℃から成るそれぞれ、55℃で2分間、および72℃で1分間。 </li><li>慎重にPCR混合物に1 回目のPCR産物を0.5μlを添加し、前と同じ条件を用いて、30サイクルの2 回目のPCR手順を実行します。 1％アガロースゲル電気泳動により増幅産物を分析します。ゲルに第 1および第 2 回目のPCR産物の各10μlのを適用します。 </li></ol></li><li>のHPCは、病原体に汚染されることがわかっている場合は、これらを排除します製造者の指示に従って、抗生物質（BM-サイクリン）の組み合わせを使用して。 3日間BM-サイクリン110μg/ mlを含む新しい培地を追加し、さらに4日間5μg/ mlのBM-サイクリン2で細胞を処理します。 2回のサイクルを繰り返してから、再度マイコプラズマ汚染を確認してください。 </li></ol>ミディアム馴化3.収穫ヒト胎盤由来細胞（hPCCM） <ol><li>汚染を除外した後、付着した細胞（85〜90％コンフルエンス治療; 2時間30分間マイトマイシンC（10μg/ ml）で（1.5×10 7細胞/フラスコ）を、10 mlの細胞を3回洗浄しPBSを24時間マイトマイシンCなしで予め温めた培地で細胞をインキュベートします。 </li><li>処理の1日後に、10ミリリットル培地中の細胞をインキュベート（DMEM-F12が20％ノックアウト血清代替[KOSR]を補充し、0.1ミリモルのβメルカプトエタノール、1％NEAA、及び1％ペニシリン - ストレプトマイシン）。インキュベーションの24時間後、15 mlのコニカルチューブで収穫CMと0.22μmのシリンジフィルターを使用してCMをフィルタリングします。 </li><li>新しいメディアとのインキュベーションの別の10ミリリットルを追加します。 1週間毎日培養物から全ての上清を収集し、-80℃で回収培地を凍結します。凍結融解を繰り返さないでください。 </li></ol> 4.培養ヒトの胚性幹細胞（ヒトES細胞）および多能性幹細胞の誘導（性IPSC） <ol><li>ヒト胚性幹細胞株を取得し、（それぞれ、名前WA01とWA09の下でNIHのhESCレジストリに記載されている）H1およびH9細胞および誘導多能性幹細胞iPS細胞-1：のiPS（包皮）-1とiPS細胞-2（IISH1i- WiCell研究所（マディソン、WI、USA）からのBM）。細胞を、製造者の指示に従って解凍し、培養されるべきです。 </li><li>対照群では、37°C ​​で広く使用されているフィーダーを含まない無血清の規定された培地中で基底膜マトリックスでコーティングされた皿上で、5％CO 2で培養細胞。最初は、hPSCs私の80-100塊をシードnは35-mmディッシュ。機械的または酵素的方法（ディスパーゼ）を使用して、すべての5~6日に1回継代培養用の70〜80％コンフルエンスと日常的継代細胞まで、これらの皿で細胞を増殖させます。培地で細胞を2回洗浄し、1：1の割合でそれらをプレート：4。毎日新鮮な培地で培地を交換してください。 </li><li>実験群では、皿上hPCCMで培養細胞を0.1％ゼラチンで予めコーティングし、5日ごとに一度ルーチン継代を行い、機械的または酵素的に。 6-1：10 1の範囲の比率でプレート細胞。 hPSCsは、転送後2日以内にゼラチンに接続します。毎日新鮮な培地で培地を交換してください。 </li></ol>ヒト多能性幹細胞の5キャラクタリゼーション注：アルカリホスファターゼ染色、免疫組織化学、定量的リアルタイムPCR（QRT-PCR）、およびウェスタンブロッティングなどのいくつかの方法を用いてhPSCsを特徴付けます。 <ol><li>免疫蛍光染色<ol><li> immunofluorescenc用E染色、培養hPSCsと10分間、4％（w / v）のパラホルムアルデヒドを用いて8ウェルチャンバースライド中の細胞を固定します。次に、（v / v）のトリトンX100を15分間、0.1％を含有するPBS中の3％（v / v）の正常ウマ血清と1時間、細胞をブロック（v / v）の0.1％で細胞を透過処理Tween- 20。すべてのステップの間に5分間のPBSで二回洗浄します。 </li><li> 4℃、二次抗体を用いて、O / N：一次抗体（千OCT-4、SSEA4、TRA-1-81、TRA-1-60、1の希釈）で細胞をインキュベートします。 </li><li>取り付けの前に、暗所で5分間、4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）で細胞をインキュベートします。暗闇の中で、完全に5分間、細胞を厳密に3回ずつ、乾燥細胞を洗浄します。蛍光マウンティング培地中で細胞を維持し、蛍光顕微鏡下に観察します。 </li></ol></li><li>定量RT-PCR <ol><li>製造業者の仕様書に従ってRNeasyミニキットを用いて細胞から全RNAを分離し、ナノドロップ分光光度計を用いて抽出したRNAを定量化します。 </LI&gt; <li>オリゴ（dT）プライマーおよびSuperscript II逆転写酵素を含む20μlの反応混合液に2μgの全RNAを添加することによりcDNAを合成。この手順では、製造元の指示に従ってください。 </li><li>のiQ SYBRグリーン定量PCRマスターミックスと補足表S1に記載されたプライマー配列を用いて、Bio-Rad社のiCycler iQのシステムを用いて合成されたcDNAを増幅します。グリセルアルデヒド-3-リン酸デヒドロゲナーゼ（GAPDH）のものに目的の遺伝子のサイクル閾値の値を正規化します。 </li></ol></li><li>アルカリホスファターゼ（AP）染色<ol><li>製造業者のプロトコルに従って、ES細胞の特徴付けキットを使用して、AP活性を検出。 </li><li> 5日目に、培地を吸引し、12分間、PBS中の4％パラホルムアルデヒドでhPSCsを修正。 overfixないでください。より長い2分間細胞を固定すると、アルカリホスファターゼの不活性化をもたらします。固定液を吸引し、1×リンスバッファーですすいでください。しないウェルを工程間乾燥させます。 </li><li>各ウエル（35mm皿のウェルあたり1ml）にカバーするのに十分な染色液を加えます。室温で15分間、暗所でプレートをインキュベートします。 </li><li>インキュベーション後、1×リンスバッファーで皿を洗います。オリンパス顕微鏡を用いて染色された細胞の乾燥を防止し、検査し、画像ために、1×PBSで細胞を覆います。 </li></ol></li><li>ウエスタンブロット法<ol><li>以前に9で説明したようにウェスタンブロット分析を行います。簡潔には、細胞溶解物中のタンパク質濃度を決定します。ドデシル硫酸ナトリウム - ポリアクリルアミドゲル電気泳動（SDS-PAGE）ゲル上で等量のタンパク質を解決し、ポリフッ化ビニリデン（PVDF）膜上にタンパク質を移します。 </li><li>ブロットをブロックし、4℃で、異なる一次抗体/ O Nをそれを調べます。その後、1時間HRP結合二次抗体でブロットをインキュベートします。 10分のインキュベーションの間の各時間のために、膜を3回洗浄します。 ECL試薬を使用して信号を検出します。 </li&gt; </ol></li><li>短いタンデムリピート（STR）分析<ol><li> hPSCsは、同じ通路で対照群と実験群から採取しました。ゲノムDNAは、製造業者の説明書に従って、DNAマイクロキットを用いて抽出しました。 </li><li>メーカーの仕様およびキャピラリー電気泳動によると、AmpF / STR PCR増幅キットを使用して16個の異なる遺伝子座についてのゲノムDNAを増幅すると、遺伝子解析装置を用いて行きました。 </li></ol></li></ol>

<ol>
	<li>Xu, 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=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat Biotechnol. 19 (10), 971-974 (2001).</li><li>Amit, M. C., Shakiri, V., Margulets, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itskovitz-Eldor+Feeder+layer+and+serum-free+culture+of+human+embryonic+stem+cells.">Itskovitz-Eldor Feeder layer and serum-free culture of human embryonic stem cells.</a> Biol Reprod. 70 (3), 837-845 (2004).</li><li>Nagaoka, M., Si-Tayeb, K., Akaike, T., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+pluripotent+stem+cells+using+completely+defined+conditions+on+a+recombinant+E-cadherin+substratum.">Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.</a> BMC Dev Biol. 10, 60 (2010).</li><li>Jin, S., Yao, H., Weber, J. L., Melkoumian, Z. K., Ye, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic,+xeno-free+peptide+surface+for+expansion+and+directed+differentiation+of+human+induced+pluripotent+stem+cells.">A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells.</a> PLoS One. 7 (11), e50880 (2012).</li><li>Xu, R. 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=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Xu, 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=Basic+fibroblast+growth+factor+supports+undifferentiated+human+embryonic+stem+cell+growth+without+conditioned+medium.">Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.</a> Stem Cells. 23 (3), 315-323 (2005).</li><li>Levenstein, M. 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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, 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=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, 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=The+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, 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=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, 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=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. 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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. A., 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=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+differentiation+of+stem+cells+mesenchymal+progenitors+by+inhibition+of+TGF-beta/activin/nodal+using+SB-431542.">Enhanced differentiation of stem cells mesenchymal progenitors by inhibition of TGF-beta/activin/nodal using SB-431542.</a> J Bone Miner Res. 25 (6), 1216-1233 (2010).</li></ol>

著者らは、利害の衝突を示していません。

ヒト微小環境にhPSCsの操作を可能にする、などのbFGFまたはインスリンなどの外因性組換え増殖因子を添加することなくヒトフィーダーを含まない培養条件で、その特性を維持しながら、このモデルは、正常hPSCsを伝播するために開発されました。外因性のbFGF補充が一般的であり、そのようなマトリゲルまたはラミニンなどの基層の適用はhPSCs 14の無フィーダー培養のために不可欠です...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

このHPSC伝搬法の1つの利点は、ヒト細胞から分泌されたコンポーネントを使用することです。プロトコルの中で最もciriticalステップは、ヒト胎盤由来細胞の単離および培養です。これは、HPC板絨毛の正確な部分から必要とし、正確な解剖。 図1は、ヒト胎盤由来細胞を単離するための手順を示しています。このプロセスは、細胞の単離のための簡単​​で便利であり、かつ容易に1...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

ヒト多能性のための新しい文化モデルは、プラセンタ馴化培地中のゼラチンの細胞増殖幹

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

a-novel-culture-model-for-human-pluripotent-stem-cell-propagation-on

Research

JoVE Journal

Developmental Biology

7.8K ビュー.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

ビデオ: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

発生生物学

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Basicおよび前臨床研究のための密閉型細胞培養系でヒト多能性および神経幹細胞を培養します

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

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

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

マイクロ電極アレイ上のネットワーク・アクティビティを測定するための人工多能性幹細胞の急速な神経分化

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

創薬における多能性幹細胞由来肝細胞様細胞を誘導人間を使用して

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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.