Name: human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions
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この研究は科学技術、台湾省で許可番号 106-2119-M-008-003、105-2119-M-008-006 と 104-2221-E-008-107-MY3 の下で部分的に支持されました。本研究は、台湾 Landseed 病院プロジェクト (NCU LSH 105 A-001-) によっても支えられました。費補助金科学研究 (番号 15 K 06591) 教育省、文化、スポーツ、科学および技術の日本からも認められています。樋口は、大学院教育と研究、キング サウド大学 11451、サウジアラビア王国リヤドの国際科学のパートナーシップ プログラム (ISPP-0062) 副牧師の地位の確認したいと思います。

本研究では実験は、台湾 Landseed 病院 (IRB-13-05)、国立中央大学の倫理委員会によって承認されました。すべての実験は、この研究ででき、関連する政府や機関ガイドラインおよび規制に従って行われました。
1. ソリューションとメディアの準備
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ポリマーの精製
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<li>浄化の加水分解の程度とカルボン酸基を持つ P IA > 96.5% P IA をエタノールで洗浄し?...

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ヒト ES および iPS 細胞人間 AFS セル、広告細胞の培養のためだけでなく、無料の xeno の条件で 10 以上の通路を多能性を維持する長期的な拡大のため P IA oligoECM と可変剛性を有する P IA ECM ハイドロゲルを開発しました。そして造血幹細胞25,28,32。OligoECM で固定 P IA ヒドロゲルは分化の運命と様々 な種類の幹細胞として初代?...

阻害剤、成長因子によって調節される細胞、幹細胞、特にひと誘導多能性 (iPS) 細胞と人間の萌芽期の茎 (ES) 細胞の長期的な増殖の特定の血統に幹細胞の分化の運命は知られていると/または文化メディアの生理活性分子が小さく。最近では、生体材料、特に細胞文化生体材料の剛性の物理的な手がかりは、幹細胞の増殖と分化1,の2、運命を導く重要な要素に認識されています。 3,4,5,6。したがって、いくつかの研究は、主にさまざまな剛性を有するポリアクリルアミド ゲルを使用して分化、ゲル上培養した幹細胞の運命を調査し始めています。
焦点接着斑、細胞の形態、細胞表現型、および幹細胞接着、二次元 (2 D) 栽培1,2,3,5で特に生体材料の剛性を制御できます。幹細胞による生体微粒子のメカノ センサーは、焦点接着インテグリン受容体を介したシグナル伝達によって一般に制御されます。NMMIIA、非ミオシンのアクチン細胞骨格の IIA 依存性収縮は 2 D セル栽培システム3,4,5,における幹細胞の力覚機構プロセスで重要な役割を果たしている7,8,9,10,11。
エングラーおよび彼の同僚は骨髄幹 (BMS) 細胞特定のティッシュのような剛性を有する細胞培養材料の栽培など、成人の幹細胞に由来する細胞に分化する傾向にある興味深い概念を開発しました。特定のティッシュの5。BMS セル拡張メディア (脳組織に匹敵する剛性) と私は自発的に BMS 細胞に対し初期の神経系統に分化誘導された型コラーゲンでコーティングされた 2 D ソフト ポリアクリルアミド ゲル上培養筋肉や骨の膠原線維組織のような剛性を有するゲルは、2 D ポリアクリルアミド ゲル3、5、それぞれの筋細胞と骨芽細胞の初期に分化を誘導するために発見されました。多くの研究者は、分化の幹細胞の運命を調査したポリアクリルアミドの培養コラーゲン固定化ヒドロゲル I 型12,13,14,,1516,17,18,19,20,21しますただし、これは1、、いくつかの矛盾を報告に言及する必要があります18,22,23,24エングラーによって提案されたよく知られているアイデアの存在。ら5これは、エングラーのアイデア5ポリアクリルアミド ゲル上だけ開発された、生体材料 (ポリアクリルアミド) の特定の特性との物理的なキュー (剛性) からだけではなく、その結果が由来しています。生体材料。したがって、ハイドロゲルが剛性、ゲルの架橋によって制御することができます別の種類を開発することが重要です。この目的のため不活性ヒドロゲルは開発された異なる硬度の変化する架橋時間25、架橋度によって制御されたポリビニル アルコール co イタコン酸 (P IA) からを作製26,27,28,29,30,31,32. nonmodified P IA ゲルだけでなく、細胞外マトリックス (Ecm) とオリゴペプチド接木 P IA ヒドロゲル幹細胞を栽培できます。以前の研究25ひと造血幹細胞 (hHSCs) 臍帯血からが P IA ゲル上 3 kPa からフィブロネクチンやオリゴペプチドが由来 30 kPa 貯蔵弾性率の異なる剛性値で栽培しました。フィブロネクチン (CS1, EILDVPST) は P IA ヒドロゲルへ移植されました。HHSCs の倍拡大高前のヴィヴォは CS1 と 30 kPa2512 kPa に至る中間剛性を表示, フィブロネクチン, 接ぎ木 P IA ヒドロゲルで観察されました。
ヒト ES および iPS 細胞はビトロネクチンなどラミニンの多能性を維持するために、Ecm に特定のバインディングを必要とするため、ヒト iPS や ES 細胞を従来の培養ポリスチレン (TCP) 料理33,34を栽培することが長期間培養します。したがって、最適な剛性特性を持つ P IA ヒドロゲルをオリゴペプチド接木のいくつかの構造が設計され、単一のチェーン、ジョイント部分を持つ単一のチェーン、共同セグメントと分岐型デュアル チェーンの形成の準備チェーン32。オリゴペプチドのシーケンスは、Ecm のインテグリンとグリコサミノグリカン結合ドメインから選ばれました。約 25 kPa で貯蔵弾性率を持ち、デュアル チェーンまたは共同セグメント ビトロネクチン由来のオリゴペプチドをグラフトした P IA ヒドロゲルは xeno 無料と化学の下以上 12 通路をヒト ES および iPS 細胞の長期培養をサポート定義された条件32。共同セグメントとデュアル チェーン、ゲル上の細胞接着分子で容易に増殖し、ヒト ES および iPS の多能性細胞32。ここでは、(空気中の湿潤条件下で測定した 30 kpa の空気に 10 kPa からストレージ係数) と P IA ゲルの準備のプロトコル移植オリゴペプチドとまたは Ecm の説明。文化および (羊水幹細胞、脂肪由来幹細胞、ヒト ES 細胞、ヒト iPS 細胞を含む) いくつかの幹細胞を通過する方法が表示されます。

<table>
	<tbody>
		<tr>
			<td>GTPGPQGIAGQRGVV</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: Cyclic RGD, cRGD</td>
		</tr>
		<tr>
			<td>GACRGDCLGA</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: FN1</td>
		</tr>
		<tr>
			<td>KGGAVTGRGDSPASS</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: CS1</td>
		</tr>
		<tr>
			<td>EILDVPST</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1</td>
		</tr>
		<tr>
			<td>KGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP1</td>
		</tr>
		<tr>
			<td>GKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP2C</td>
		</tr>
		<tr>
			<td>CGGGKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1G</td>
		</tr>
		<tr>
			<td>GGGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN2C</td>
		</tr>
		<tr>
			<td>GCGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: rVN</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Thermo Fisher scientific</td>
			<td>A14700</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: FN</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Specification: Commercially available coating material 
			Abbreviation: Synthemax II</td>
		</tr>
		<tr>
			<td>Synthemax II</td>
			<td>Corning</td>
			<td>3535</td>
			<td>Specification: Polymer</td>
		</tr>
		<tr>
			<td>Polyvinylalcohol-co-itaconic acid</td>
			<td>Japan VAM &#38; Poval</td>
			<td>AF-17</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td>Specification: Chemical 
			Abbreviation: EDC</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E7750</td>
			<td>Specification: Chemical 
			Abbreviation: NHS</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D4693</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 6</td>
			<td>Thermo Fisher scientific</td>
			<td>A1516401</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 8</td>
			<td>Thermo Fisher scientific</td>
			<td>A1517001</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher scientific</td>
			<td>11330-032</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher scientific</td>
			<td>12800-017</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>MCDB 201</td>
			<td>Sigma-Aldrich</td>
			<td>M6770</td>
			<td>Specification: ES cell</td>
		</tr>
		<tr>
			<td>Human ES cell</td>
			<td>WiCell Research Institute, Inc..</td>
			<td>WA09</td>
			<td>Specification: iPS cell</td>
		</tr>
		<tr>
			<td>Human iPS cell</td>
			<td>Riken Cell Bank</td>
			<td>HS0077</td>
			<td>Specification: 35 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Specification: 60 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Specification: 24 well dish</td>
		</tr>
		<tr>
			<td>24 well dish</td>
			<td>Corning</td>
			<td>353047</td>
			<td>Specification: Blocking agent</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase live stain</td>
			<td>Thermo Fisher scientific</td>
			<td>A14353</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; eosin</td>
			<td>Sigma-Aldrich</td>
			<td>1.05175</td>
			<td>Specification: EB formation dish</td>
		</tr>
		<tr>
			<td>6-well ultralow attachment dish</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Specification: Mice</td>
		</tr>
		<tr>
			<td>NOD-SCID mice</td>
			<td>National Applied Research Laboratories</td>
			<td>None</td>
			<td>Specification: Serum</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td>Specification: Antibiotic</td>
		</tr>
		<tr>
			<td>antimycotic antibiotic</td>
			<td>Thermo Fisher scientific</td>
			<td>15240-062</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Nanog</td>
			<td>Invitrogen</td>
			<td>MA1-017</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for SSEA4</td>
			<td>Abcam</td>
			<td>ab16287</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for OCT3/4</td>
			<td>Invitrogen</td>
			<td>PA5-27438</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Sox2</td>
			<td>Invitrogen</td>
			<td>48-1400</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Smooth Muscle Actin</td>
			<td>Invitrogen</td>
			<td>PA5-19465</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for AFP</td>
			<td>Invitrogen</td>
			<td>PA5-21004</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody GFAP</td>
			<td>Invitrogen</td>
			<td>MA5-15086</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 &#8211; conjugated Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 &#8211; conjugated Goat anti-Rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td>Specification: Antibody</td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions

幹細胞の分化増殖に対する生体材料の剛性などの物理的な手がかりの効果は、いくつかの研究が検討されています。ただし、これらの調査のほとんどは、彼らの研究の幹細胞文化のポリアクリルアミドのゲルを使用しています。したがって、これらの結果かもしれないと生体材料の物理的なキュー (剛性) ではなく、ポリアクリルアミドの特定の特性から発生するため、その結果、物議を醸す。ここでは、ポリアクリルアミド、様々 な幹があり、人間の萌芽期の茎 (ES) の細胞を含む細胞、ひと誘導多能性幹 (iPS) 細胞が培養することができますに基づいていないゲルの準備のプロトコルについて述べる。架橋時間を変更することによって架橋度によって制御剛性不活性ポリビニル アルコール co イタコン酸 (P IA) から可変剛性を有するゲルを調製されました。幹細胞培養と分化のため将来のプラットフォームとして移植細胞外マトリックス由来オリゴペプチドと P IA ヒドロゲルを調べた。文化および羊水幹細胞、脂肪由来幹細胞、ヒト ES 細胞、ヒト iPS 細胞の通路は、ここで詳細に記述されます。オリゴペプチド P IA ヒドロゲルの剛性特性によって誘導された優れた性能を示した。このプロトコルは、生体材料、剛性特性と最後に、xeno フリー培養条件を用いた幹細胞の運命への影響を制御すると共に、彼らの表面操作の合成を報告します。最近の研究に基づく、そのような変更された基板として使用できます将来のプラットフォームをサポートし、異なるリンケージ; さまざまな幹細胞ラインの運命を指示さらに、再生成し、失われた臓器や組織の機能を回復します。

P IA ヒドロゲル グラフト ECM 由来ペプチド (oligoECM) または異なる弾力性と ECM で調製した反応スキームを以下図 1 aに見られるように oligoECM (図 1 b) の種類を使用しています。ゲルの弾力性は、応用架橋強度 (時間) (図 1) によって規制されました。25.3 kPa (24 h 架橋時間) の貯蔵弾性率を持つ、ビトロネク?...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions at JoVE.com

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

増殖と分化に生体材料の剛性の影響を調査するため、オリゴペプチド、接木されたさまざまな剛性とポリビニル アルコール co イタコン酸ゲルを準備するためのプロトコルを表示します。幹細胞。ゲルの剛性は、架橋時間によって制御されました。

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 ...

The overall goal of this experiment is to prepare either unmodified or oligopeptide or extra-cellular matrix-grafted polyvinyl alcohol-co-itaconic acid hydrogels with the optimal elasticity for culturing and passaging stem cells. This method can help answer key questions in the stem cell research field about long term content of stem cells under xeno-free conditions. The main advantage of this technique is that it provides a platform to investigate a different hydrogel elasticity on stem cell differentiation into specific lineages.Demonstrating the procedure will be me, Jia-Sin Yang and Yeh-Chia Tseng, graduate students from Professor Higuchi's laboratory. To begin the procedure, place 20 grams of P-IA and 200 milliliters of ethanol in a 500 milliliter conical flask. Stir the mixture for 24 to 30 hours, replacing the ethanol every eight to ten hours.Collect the purified P-IA on Buchner funnel. Dry the P-IA under vacuum at room temperature for 24 hours. Next, over the course of 15 minutes, slowly dissolve 15 milligrams of purified P-IA in 100 milliliters of ultra-pure water.Heat the solution to above 60 degrees Celsius and stir for one hour. Confirm that the solution has become clear before proceeding. Then, turn off the heat.Allow the P-IA solution to cool to room temperature on the cooling hot plate while stirring. Continue stirring the solution at room temperature for 48 hours. Then, leave the P-IA solution undisturbed at room temperature for 20 to 24 hours to ensure that visible air bubbles are not present in the solution.Next, dispense one milliliter of the P-IA solution into each of eight 35 milliliter tissue culture-treated dishes. Dry the dishes in an oven at 45 degrees Celsius for two days to produce P-IA films in the dishes. Next, add to 10 milliliters of ultra-pure water 100 microliters of a high-purity aqueous 25%glutaraldehyde solution, two grams of sodium sulfate and 100 microliters of sulfuric acid.Stir the mixture well to obtain the crosslinking solution. Gently add one milliliter of the crosslinking solution to each dish, being careful not to damage the fragile P-IA films. Leave each film immersed in the crosslinking solution for half, one, two, four, six, twelve, twenty four or forty eight hours to obtain the hydrogels.Rinse each hydrogel with one milliliter of ultra-pure water after its crosslinking period. Sterilize each hydrogel by immersion in an aqueous 75%ethanol solution for one minute. Rinse each sterilized hydrogel with ultrapure water six times.Cover the hydrogels in fresh ultra-pure water, place the lids on the dishes and store the hydrogel dishes in a clean area until ready for activation and grafting. Next, activate each sterilized hydrogel by immersion in one milliliter of an aqueous solution containing 10 milligrams per milliliter each of EDC and NHS, either for one hour at 37 degrees Celsius or four hours at four degrees Celsius. Then, rinse each activated hydrogel with one milliliter of Ph 7.2 phosphate-buffered saline three times.Immerse each hydrogel in one milliliter of a solution of either oligopeptides or ECMs in PBS for 24 hours at four degrees Celsius. Finally, wash each hydrogel with one milliliter of ultra-pure water three times after grafting is complete. Use standard method to culture human ES or IPS cells on a membrane matrix in six centimeter culture dishes until near confluent.Then, incubate the cells in DMMF-12 supplemented with two milligrams per milliliter of dispase-2 at 37 degrees Celsius for eight to ten minutes. Rinse the cells twice with DMMF-12. Add two milliliters of DMMF-12 to each culture dish and attach weekly adherent colonies with the pipette or cell scraper.Collect the cells in 15 milliliter centrifuge tubes and centrifuge the cells at 160 times G for five minutes at 37 degrees Celsius. Discard the supernatant and re-suspend the cells in one milliliter of xeno-free eight component medium. Count the cell density in the suspension.Then, obtain culture dishes or multi-well plates containing sterilized P-IA hydrogels grafted with either oligopeptides or ECMs. Load the appropriate volumes of the cell suspension onto the hydrogels. Passage the cells on grafted P-IA hydrogels until 80%confluent the desired number of times.To begin the immuno-staining process for a 24 well plate of passaged human ES or IPS cells on grafted hydrogels, add 0.5 milliliters of four per cent paraformaldahyde by volume to each well. Incubate the cells at four degrees Celsius for 15 minutes to fix the cells. Aspirate the paraformeldehyde solution from the wells.Add one milliliter of PBS to each well and aspirate the PBS. Perform this PBS rinse three times in total. Then, add 0.5 milliliters of 0.3%octoxynol 9 by volume in PBS to each well.Incubate for 30 minutes at room temperature. Aspirate the octoxynol 9 solution. Add 300 microliters of two per cent BSA and PBS to each well and incubate for another 30 minutes at room temperature.Aspirate the two per cent BSA solution from the cells and add the primary antibodies. Incubate the cells for one day at four degrees Celsius. Then, aspirate the primary antibody solutions and wash the cells with 300 microliters of 05 per cent polysorbate 20 in PBS three times.Add 300 microliters of secondary antibodies specific to the primary IDG subtype in two per cent BSA to each well. Incubate the cells for one hour at room temperature in the absence of light. Finally, wash the cells with 300 microliters of 05 per cent polysorbate 20 and PBS three times in the dark.Image the stained cells with fluorescence microscopy. P-IA hydrogels crosslinked for 24 hours and grafted with vitronectin-derived oligopeptides, supported cultures of human ES and IPS cells for at least 10 to 20 passages. Human ESLs cultured on commercially available coated dishes were found to be more easily differentiated at the edges of the colonies.Differentiated cells were not observed on the hydrogels, indicating that human ESLs maintained their pluripotency. Pluripotency proteins were satisfactorily expressed on human ES cells cultured on hydrogels grafted with three different Vitronectin-derived oliogopeptide designs for ten passages. Satisfactory expression was also observed on recomibinent Vitronectin-coated dishes.Similar results were observed for human IPS cells. Both human ES and IPS cells differentiated into cells derived from all three germ layers after cultivation on P-IA hydrogels and on recombinant Vitronectin-coated dishes for at least ten passages. Human ES cells cultivated on hydrogels grafted with Vitronectin-derived oligopeptides were injected into NOD mice.The harvested teratomas exhibited cells originating from all three germ layers indicating that pluripotency is maintained in vivo. Following this procedure, methods like media screening can be performed to answer additional questions like the effect of calcium arterial and stem cell differentiation and proliferation. After this development, this technique paved the way for researchers in the field of stem cell co-division to explore tissue or organ regeneration in neurodegenerative medicine.Once mastered, this technique can be done in four days if it's performed properly. After watching this video, you should have a good understanding of how to prepare P-IA hydrogel dishes, both with and without ECM. While attempting this procedure, remember to set up a clean environment for preparing P-IA hydrogel dishes.Don't forget that working with ethanol, hot water and the sulfuric acid can be extremely hazardous. The appropriate lab coat, gloves and the protective glasses should always be worn while performing this procedure.

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

開発、展開、および<em生体内で&gt;</em&gt;ヒト胚性幹細胞（ヒトES細胞）からヒトNK細胞のモニタリングとや人工多能性幹細胞（性IPSC）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

生物工学

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

ヒアルロン酸を用いた神経膠芽腫患者由来細胞の 3次元培養ヒドロゲル

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

化学的に定義された条件下でのヒト人工多能性幹細胞からの成熟した腎臓ポドサイトの誘導分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

静止ヒト造血幹細胞を維持する培養法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

ヒト多能性幹細胞スフェロイドを担持したコアシェル型マイクロカプセルのマイクロ流体作製

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

ヒト人工多能性幹細胞から改変された同質遺伝子腎臓糸球体チップ

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

多能性幹細胞からのヒト血管オルガノイドの作製

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

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

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