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>

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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><tbody><tr><td>Mitomycin-C</td><td>Sigma-Aldrich Corporation</td><td>M4287</td><td>10 &amp;mu;g/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>1 mg/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>10 &amp;mu;g/ml</td></tr><tr><td>RNeasy mini kit&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>iNtRON Biotechnology</td><td>25011</td><td></td></tr><tr><td>QIAamp&amp;reg; DNA Micro kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>AmpF/STR&amp;reg; Identifiler&amp;reg; PCR Amplification kit</td><td>Applied Biosystems Inc.</td><td>4322288</td><td></td></tr><tr><td>Applied Biosystems&amp;reg; 3130xl Genetic Analyzer&amp;nbsp;</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

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

ロボット液体ハンドリングシステムを用いたiPS細胞培養および生産ベースのスケーラブルな96ウェルプレート

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

発生生物学

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

生物工学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神経科学

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

免疫・感染学

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Xeno 無料条件下におけるさまざまな剛性とポリビニル アルコール Co イタコン酸ゲルのひと多能性幹細胞文化

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

自動培養システムによる多能性幹細胞分化の誘導

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

著者スポットライト:iPS細胞培養の自動化による再現性の向上 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

iPS細胞からの効率的なヒト腸オルガノイド生成のためのマトリックス比較

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

著者スポットライト:iPS細胞を用いた創薬のためのオルガノイド生成の促進

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

ヒト幹細胞維持と腸管オルガノイド生成のための基底膜マトリックスの比較研究

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

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

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この動画の章

ロボット液体ハンドリングシステムを用いたiPS細胞培養および生産ベースのスケーラブルな96ウェルプレート

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

Xeno 無料条件下におけるさまざまな剛性とポリビニルアルコール Co イタコン酸ゲルのひと多能性幹細胞文化

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト幹細胞維持と腸管オルガノイド生成のための基底膜マトリックスの比較研究

ヒト幹細胞維持と腸管オルガノイド生成のための基底膜マトリックスの比較研究

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

要約

さらに動画を探す

この動画の章

ロボット液体ハンドリングシステムを用いたiPS細胞培養および生産ベースのスケーラブルな96ウェルプレート

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

Xeno 無料条件下におけるさまざまな剛性とポリビニル アルコール Co イタコン酸ゲルのひと多能性幹細胞文化

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト幹細胞維持と腸管オルガノイド生成のための基底膜マトリックスの比較研究

ヒト幹細胞維持と腸管オルガノイド生成のための基底膜マトリックスの比較研究

Xeno 無料条件下におけるさまざまな剛性とポリビニルアルコール Co イタコン酸ゲルのひと多能性幹細胞文化