著者らは次のスタッフ メンバーに感謝し、彼らの貢献のための人員を研究: マシュー Librach、レイラ ・ Maghen、ターニャ A. Baretto、Shlomit Kenigsberg Andrée ゴーティエ ・ フィッシャー。この作品は、オンタリオ州研究基金 - 研究の卓越性 (ORF 日時、ラウンド #7) と作成プログラム株式会社によって支えられました。

All studies involving animals were conducted and reported according to ARRIVE guidelines48. All studies were performed with institutional research ethics board approval (REB number 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Institutes of Health 2011).
1. Tissue Culture
<ol>
	<li>Culture FTM HUCPVCs, term HUCPVCs (previously established, n &#8805; 3 independent lines for each)42 and commercially available BMSCs in alpha-minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and a 1% penicillin/streptomycin (P/S) cocktail. Culture rat primary cardiomyocytes and MSC-cardiomyocyte co-cultures in Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) containing 10% FBS and 1% P/S. 
	NOTE: Sterilize the medium using a 0.2-&#181;m filter. Store prepared medium solutions at 4 &#176;C for up to 3 weeks.</li>
	<li>Maintain cell cultures in humidified incubators (95% relative humidity, 37 &#176;C, and 5% CO2) and passage at 70-80% confluency, determined by phase-contrast microscopy. Use appropriate volumes of medium for the size of tissue culture dish used (e.g., 10 mL in a 10-cm dish and 2 mL per well in 6-well tissue culture plate). Use these culture conditions for the duration of the protocol.</li>
	<li>Dissociate MSC monolayers for passaging or MSC-cardiomyocyte co-culture establishment using a dissociation enzyme solution (2 mL/well in a 6-well plate) and incubate at 37 &#176;C for 4 min.</li>
	<li>Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.</li>
	<li>Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of a culture medium appropriate for counting using an automated cell counter. Seed the cells as described in the following protocol sections.</li>
</ol>
2. Preparation of Primary Rat Cardiomyocyte-MSC Co-cultures
<ol>
	<li>Obtain heart tissue for primary cardiomyocyte isolation.
	<ol>
		<li>Euthanize rat pups (5-6 days postnatal) using CO2 asphyxiation. Set CO2 chambers to 20% gas replacement (flow rate = 0.2 x chamber volume per min). Confirm exitus by the absence of the pinch reflex.</li>
		<li>Remove the atria with the connecting major blood vessels using sterilized instruments (i.e., forceps and curved scissors)41. Transfer the hearts to 50-mL tubes containing sterile PBS with1% P/S (PBS-P/S) on ice.</li>
		<li>Cut the ventriculi in half and let the blood wash out in a 10-cm dish with 10 mL of PBS-P/S on ice. Cut the ventricular walls into small pieces (diameter = 2-3 mm) using curved scissors.</li>
		<li>Transfer the heart pieces from 10-12 animals to a 50-mL tube using a serological pipette and let them settle.</li>
		<li>Remove as much PBS-P/S as possible without removing any heart pieces. Add 10 mL of new PBS-P/S.</li>
	</ol>
	</li>
	<li>Digest the heart tissue to isolate the cardiomyocytes.
	<ol>
		<li>Allow the heart pieces to settle. Replace the PBS-P/S with 10 mL of 0.15% trypsin in PBS and shake at 37 &#176;C for 10 min.</li>
		<li>Discard the supernatant. Repeat the digestion described in step 2.2.1 three more times, but decant the supernatants into 50-mL collection tubes containing 10 mL of 100% FBS.</li>
	</ol>
	</li>
	<li>Centrifuge the cells (400 x g, 5 min) and aspirate the supernatant. Resuspend the cells in DMEM-F12 containing 10% FBS and 1% P/S and seed onto a 6-well plate (1 x 105 cells/cm2, 2 mL of medium per well).</li>
	<li>After 1 h, transfer the medium containing non-attached cells to a 50-mL tube and discard the attached cells. Count the cells in suspension and re-plate them into new 6-well plates (1 x 105 cells/cm2, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well).</li>
	<li>Inhibit cell proliferation with bromodeoxyuridine (BrdU). 
	Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training is provided and refer to the safety data sheet before use.
	<ol>
		<li>Once cells have attached, replace the medium in the 6-well plate with DMEM-F12 containing 10% FBS, 1% P/S (2 mL of medium per well), and 5 &#181;M BrdU. Incubate for 16 h (37 &#176;C, 5% CO2).</li>
		<li>Remove the BrdU-containing medium and replace with DMEM-F12 containing 10% FBS and 1% P/S (2 mL of medium per well).</li>
	</ol>
	</li>
	<li>Prepare pre-stained MSCs.
	<ol>
		<li>Once MSC cultures are at 70-80% confluency in 10-cm dishes, remove the culture medium and add 3 mL of cell dissociation solution. Incubate the dish at 37 &#176;C and 5% CO2 for 5 min.</li>
		<li>Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.</li>
		<li>Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of DMEM-F12 containing 10% FBS and 1% P/S for counting using an automated cell counter.</li>
		<li>Dilute the cells to a concentration of 1 x 106 MSC/mL of DMEM-F12 containing 10% FBS and 1% P/S.</li>
		<li>Incubate the MSCs with viable, non-transferable fluorescent dye (5 &#181;M, 30 min, 37 &#176;C, 5% CO2) in 1.5-mL centrifuge tubes for 1 h.</li>
		<li>Centrifuge the tubes at 400 x g for 5 min. Aspirate the supernatant and resuspend the pellet in DMEM-F12 containing 10% FBS and 1% P/S for a cell concentration of 1 x 106 MSC/mL. Repeat this a total of 3 times.</li>
	</ol>
	</li>
	<li>Transfer the MSCs onto cardiomyocytes (step 2.5.2) at a concentration of 10 x 104 cells per well of the 6-well plate.</li>
</ol>
3. Preparation of Aggregate Co-cultures
<ol>
	<li>Prepare a single-cell suspension of MSCs (2 x 104 cells/mL of medium, passage # &#8804; 6) in alpha-MEM supplemented with 10% FBS and 1% P/S (see step 2.6). 
	NOTE: Refer to section 1 of the protocol for the passaging of cells. Alternatively, pre-stain MSCs as per step 2.6.</li>
	<li>Initiate aggregate formation by placing 25-&#181;L drops of cell suspension (500 cells) on the inner surface of the lids of 10-cm tissue culture dishes (up to 50 drops per lid). Place the lids on their bottom counterparts containing PBS-P/S. Incubate at 37 &#176;C and 5% CO2. 
	NOTE: Place 5-7 mL of PBS-P/S into the culture dish below the hanging drops to avoid drop evaporation.</li>
	<li>Observe aggregate formation in the drops after 3 days using a stereomicroscope. If over 40 out of 50 drops contain formed aggregates, collect the drops from the lids using a 1-mL micropipette and transfer the aggregates directly onto primary rat cardiomyocyte monolayers (prepared in steps 2.1-2.7; 10 drops/well). Avoid vigorous pipetting to preserve aggregate integrity.</li>
	<li>Keep aggregate co-cultures in the incubators for up to 2 weeks, changing the full volume of medium (2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well) every 72 h.
	<ol>
		<li>Daily observe aggregates attaching on feeder cell layers using bright-field microscopy. Record contracting aggregates when observed.</li>
	</ol>
	</li>
	<li>Prepare aggregates for analysis.
	<ol>
		<li>Remove the medium and add 2 mL of PBS per well of a 6-well tissue culture dish. Remove the PBS and add 2 mL of dissociation solution per well. Incubate for 3 min at 37 &#176;C and 5% CO2.</li>
		<li>Centrifuge at 400 x g for 5 min to obtain a cell pellet. Resuspend in medium, as specified for the applications described in the subsequent steps (see steps 4.1, 5.1, and 6.1) and pass through a 70-&#181;m cell strainer.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry (FC) and Fluorescence-activated Cell Sorting (FACS)
<ol>
	<li>Incubate cell suspensions (1 x 105 cells in 200 &#181;L of PBS containing 3% FBS) with fluorophore-conjugated (FITC or APC) primary antibodies (i.e., CD49f, Cx43, TRA-1-85, HLA-A, HLA-G, and SIRPA for FC or TRA-1-85 for FACS; 1:40) at 4 &#176;C for 30 min, protected from light.</li>
	<li>Centrifuge (400 x g, 5 min) and resuspend the cells in 1 mL of PBS with 3% FBS for FC or PBS with 0.5% FBS for FACS. 
	NOTE: The FC of MSCs was optimized by Hong et al.41.</li>
	<li>Maintain the cells at 4 &#176;C in the dark until they are ready to be analyzed by FC (at least 1 x 104 events) or FACS. Sort the cells as described41. Re-plate TRA-1-85 high-positive sorted cells in 6-well plates (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S) within 1 h. 
	NOTE: For the gating strategy of the TRA-1-85 human cell surface antigen, see the Supplementary Figure.</li>
</ol>
5. Immunocytochemistry (ICC) and Microscopy
<ol>
	<li>Re-plate the cell suspensions obtained from the co-cultures (step 3.5.2) or FACS (section 4) onto chamber slides (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well). Let the cells attach overnight in a tissue culture incubator (see section 1 for the conditions).</li>
	<li>Fix the cells using 3 mL of 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Wash 3 times with 3 mL of PBS containing 1% bovine serum albumin (BSA; PBS-BSA) for 5 min per wash. 
	Caution: Wear appropriate personal protective equipment when handling PFA.</li>
	<li>Permeabilize the cells in 3 mL of PBS-BSA with 0.1% Triton X-100. Incubate at room temperature for 10 min for intracellular antigens (i.e., alpha sarcomeric actinin (aSarc) and Cx43), or 25 min for intra-nuclear antigens (i.e., Mef2c and human nuclear antigen (HuNu)). Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.</li>
	<li>Block the samples against non-specific antibody reactions with 3 mL of PBS containing 5% normal goat serum (NGS) and 1% BSA for 15 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.</li>
	<li>Incubate the cells in the primary antibodies (i.e., Mef2c, aSarc, Cx43, and HuNu) diluted 1:200 in 3 mL of PBS-BSA at 4 &#176;C overnight.</li>
	<li>Wash 3 times with 3 mL of PBS-BSA for 5 min per wash and incubate with secondary antibodies for 30 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.</li>
	<li>Store the stained specimens in 3 mL of of mounting medium.</li>
	<li>Acquire images using a fluorescence microscope. Use a 10X objective (NA = 0.3), and a 20X objective (NA = 0.45) for lower-magnification imaging. Use fluorescence filter cubes and wavelengths for GFP (ex = 470/22 nm, em = 525/50 nm) and RFP (ex = 531/40 nm, em = 593/40 nm) for the secondary antibodies used (see the Materials and Equipment Table).</li>
	<li>Quantify images using imaging software (see the Materials and Equipment Table for the recommended software). Normalize the fluorescence intensity readings to the secondary control acquisitions.</li>
</ol>
6. RNA Isolation and Quantitative RT-PCR
<ol>
	<li>Prepare RNA samples from undifferentiated MSC cultures or MSCs sorted from co-cultures using column-based RNA isolation, according to the manufacturer&#39;s instructions. Prepare 1 x 104 to 1 x 106 cells in 0.7 mL of cell lysis buffer (provided with the RNA isolation kit) per sample.</li>
	<li>Prepare cDNA from up to 2 &#181;g of RNA per 100-&#181;L RT reaction.</li>
	<li>Perform qPCR using 10 ng of cDNA per reaction (40 cycles, 60 &#176;C annealing/extending temperature).
	<ol>
		<li>Use primers for human MY6H and cTnT in a 500-nM concentration and 1-100 ng of cDNA per reaction (see the Materials and Equipment Table). Use GAPDH, ACTB, and HPRT as internal housekeeping normalizers. Use commercially available human-induced pluripotent stem cell-derived cardiomyocytes as a positive control. 
		NOTE: Express the fold-change of expression compared to undifferentiated MSC-derived cDNA samples.</li>
	</ol>
	</li>
</ol>

博士クリフォード ・ l ・ Librach は、特許の共同保有者:最初の学期臍帯組織由来細胞の分離とファージのメソッドカナダ、オーストラリアで。

幹細胞の心筋分化は、MSC のソースから心筋細胞のような細胞の生成に使用されているいくつかの異なる戦略と 2 年以上にわたって開発を進めています。これらの戦略の多くはただし、効率的ではありません、使用条件、環境移植細胞の出会い、生体内の代表的なよくないです。
既存のメソッドとは対照的は、ここで提示されたプロトコルはプライマリ心臓フィーダー層と MSC 形成の組み合わせを利用しています。主な心臓フィーダー層と同様に、hMSCs が移植時にさらされている、差別化環境を提供します。凝集体は、酸素と栄養のグラデーションを分化系列決定を開始する誘導効果を持つことができる環境を生成します。低酸素向き前処理を用いた PSC と ESC に基づく心臓差別化戦略と会合体形成幹細胞5,52,53この誘導効果を設けてください。さらに、3 D 構造より提供される細胞間の接続の体内に似ています。このメソッドを使用して、我々 は正常に同期的に契約立体的集計の最初時間立体的型は私達の知識にこれを達成することができたを作り出した。重要なは、最年少立体的細胞ソースのみ適用、FTM の HUCPVCs は、この能力を展示しました。
心筋再生の Esc または Psc ナイーブまたは事前分化 MSCs を使用しての重要な利点は、免疫調節または immunoprivileged プロパティであります。ただし、MSCs の場合でも免疫調節特性は年齢とソース54によって異なります。BMSCs がしばしば注入55に彼らの拒絶につながるその免疫原性のプロパティの後分化を変更する示されています。前述したように、臍帯から MSCs 起源の子宮の起源のための特別な免疫特権56を所有しています。プロトコルを使用して、高レベル (つまり、生得の免疫反応を積極的に減衰) HLA G の用語と FTM HUCPVCs 心筋細胞のような細胞への分化後も、上記し、(T 細胞による細胞毒性を決定) する HLA A の低レベルを維持します。
この分化のプロトコルには、便利な複数の分析レベルを可能にするという利点があります。まず、契約の集計機能、ライブ イメージングは送り装置の層に統合された単一のセルを区別するよりもより現実的です。QPCR 解析するプロトコルは、混合種共培養を利用するとひと特異プライマーが適用されます、FACS や ICC-具体的には、表現型の迅速な同定心筋マーカー式 (図 2)。前述のプロトコルを使用して、SIRPA、cTnT、MYH6、aSarc、Mef2c、配列とコネキシン 43 の高値を認めた.HuNu と連携して、Mef2c と差別化された hMSCs の aSarc の式を識別するために、ラットの心筋細胞由来の送り装置の層または融合細胞を区別することが可能です。送り装置の層の租税の集計の識別は比較的簡単、収集またはそれらを収穫制限を提起できます。このような課題を処理するマイクロマニピュレーターを搭載顕微鏡を用いることができます。また、人間の細胞はひと特異抗-トラ-1-85 抗体を用いた共培養から効率的にソートできます。共培養は、潜在的な細胞融合への挑戦を提示するも、これも克服できる人間核と細胞表面のマーカーのトレーサー (補足図 1) の組み合わせを使用しています。
私たちの微分法の改造が可能は、凝集体の形成を調整することです。骨材粒径が最適な分化の重要なパラメーターをすることができますが示唆されました。集計がより接着性細胞の選択的な、CD49f 陽性 hMSCs の事前選択ステップとして機能できます。強化された MSC 球形成が CD49f 式49の増加にリンクされている、CD49f またと関連付けられる高い未分化 OCT4、SOX2 の49に直接接続されているため。CD49f 陽性細胞の観察された減少の合計サイズを説明することができる FTM HUCPVCs に比べて hMSCs 古いソースの低い数字を見ました。我々 は高い総計のサイズを達成するために集計ごと hMSCs 数の増加によってこの選択を補償、従っておそらく凝集誘導される分化の効率を高める理論化します。
将来のアプリケーションは、同様のプロトコルに変更を必要があります。事前分化 hMSCs の臨床応用が xeno 無料条件の場所をとることが不可欠です。人工心臓筋肉ティッシュの作成のためのこのプロトコルを使用して生成される契約の集計も使えます。臨床応用可能な製品を生産するには、cGMP 準拠のワークフローが必要です。Xeno 自由な文化媒体は hMSCs、動物無料フィーダー層を提供することに挑戦する可能性があります。ただし、Ips などの他のソースの幹細胞の分化済みフィーダー層を加えることは、可能なソリューションです。
結論としては、便利で再現可能な方法で hMSCs 若い源から心筋細胞のような細胞を生成する説明した微分法を適用できます。

うっ血性心不全 (CHF) は、罹患率と死亡率の世界の一流の原因として保持します。スイスフランは、心筋細胞の大規模な損失と心筋梗塞 (MI)1の病理の結果として瘢痕組織の細胞の開発を多く発生します。中心部は部分的に自己更新器官が、豊かさと多くの場合受傷後適切に回復するが不十分となった高齢者の患者の機能で常駐幹・前駆細胞プール大幅組織再生を実行するための責任が減少します。従って、健康なドナー細胞の損傷を受けた心筋への移植を含む実験的治療法の開発に大きな関心があります。ドナー細胞だけでなく、組織の構造を復元、また影響を受けた心筋の機能回復を達成するために不可欠です。
ネイティブの心は心臓組織に常駐を採用し、受傷後の内因性骨髄由来幹細胞修復2,3,4。再生細胞ホストとドナー由来もありますは、適切な表現と効率的にかつ安全に失われた細胞を置換する機能と共に、改造の心筋の微小環境で関数を取得する能力を持っています。In vitro分化の方法は、高効率、幹細胞を用いた心筋細胞生産5,6を達成するために広く使用されています。心臓系統マーカーの発現プロファイルを使用して、心臓系統7に向かって幹細胞分化のプロセスを定義します。初期分化マーカー、NKX2.5、心筋増強因子 2 C など (Mef2c) および GATA48,9、cardiomyogenic プロセスの開始を示すことができます。差別化の有効性を評価するために一般的に使用される成熟心筋細胞マーカーが信号調節タンパク質 α (SIRPA)10, 心筋トロポニン T (cTnT)11、重鎖心筋ミオシン (MYH6)8,12,13, とコネキシン 43 (cx 43)14,,1516。胚性幹細胞 (Esc) と多能性幹細胞 (Psc) を使用してメソッドは徹底的に最適し、誘導要因、酸素および栄養素のグラデーションの詳細とアクション5,6,7,17,18の正確なタイミングについて説明しました。それにもかかわらず、ESC と PSC ベースの技術はまだ次善の電気生理学的および免疫学的機能19,20と共に複数の倫理と安全性懸念を提示します。多くの場合これらの細胞移植ホストは、immunorejection を体験し、永久的な免疫抑制を必要とします。これは、主に主要組織適合性の複雑な (MHC) 分子ホストとドナーおよび結果 T 細胞応答21までの不一致によるものです。ながら個々 の MHC クラス I 可能な解決策は、マッチング、やすく実習が拒絶反応の懸念を克服するために immunoprivileged では普遍的に細胞ソースが必要があります。
特に、BMSCs、臨床では、MSCs 用代替細胞ソースとして 1995年22で彼らの初期の説明から歯周組織再生用検討しました。MSCs は、ほぼすべての血管柄付き組織23で見つけることができる居住者の再生細胞であると考えられています。目的のソースからの分離、MSCs 文化で容易に拡張することができます、広範な傍分泌能力を持っている、しばしば immunoprivileged または免疫調節特性24,25を持っています。その安全性と有効性既に示されているいくつかの臨床研究で特に心筋再生3,26。
多くの MSC 差別化戦略は、変数効率 5-アザシチジン22や DMSO27、および成長のような Bmp5,7,28,29またはアンジオテンシン II30、形態形成の要因などの薬理学的エージェントを利用します。これらの戦略は、素朴な再生細胞はホーミングまたは傷害のサイトに配信されている後に発生する可能性が障害物に基づいていないただし、体内。もっと生理学的に関連する戦略は、一方が難しく定義し操作、組織微小環境自体からの信号を通して MSC 分化を誘起することを前提に基づいています。以前の研究は心臓セル lysates31または心室心筋32,33, への曝露が示されているまたは直接プライマリ心筋細胞の in vitro15,34, との接触が MSCs で心臓マーカーの発現を高めることができます。他の人は一部、BMSCs と心筋細胞39,40の融合生成初期の心筋が MSCs35,36,37,38, 心臓傷害の治療後特発性心筋を実証しています。私たちの知る限り、ヒト MSCs (hMSCs) の組織の任意のソースから機能、自発的に引き締まる心筋細胞がまだ報告されていません。
現在のコンセンサスは、MSCs のすべてが血管周囲細胞23から発生することです。周皮細胞プロパティを持つ若い MSCs は、ひと臍帯組織41,42,43の血管領域から分離できます。BMSCs と比較して HUCPVCs は、潜在的な増加の分化と他のいくつかの再生の利点、両方生体外で41,44とin vivo45,46,47を所有しています。特に、妊婦の胎児のインターフェイス ソース、HUCPVCs MSCs の大人の情報源と比較して有意に低い免疫原性があります。我々 の研究に焦点を当てて評価と FTM HUCPVCs、調査、MSCs の最年少のソースの前臨床応用増殖性と高い multilineage を増加するいると我々 は以前に、分化能力、cardiomyogenic 系統41を含みます。
ここでは、MSCs。 集計の完了 cardiomyogenic 差別化を達成するために誘導の力より良い条件モデル生体内で2D 付着性文化と比較して 3 D 環境を提供するように、粒の形成と心筋細胞の主要な送り装置の層を組み合わせたプロトコルを提案する.心臓フィーダー層を利用した MSCs の究極移植サイトの代表である環境を提供します。集合体を形成し、彼らの免疫特権を維持しながらアダルト BMSCs に比べて心臓表現型に到達する能力が高いを前または出生後のへその緒から分離された MSCs の若いソースにことを示します。心臓系統マーカー遺伝子の誘導発現細胞内急昇格以外 (すなわち、 cTnT および MYH6) および細胞表面蛋白質 (すなわちSIRPA と配列とコネキシン 43) 心筋細胞の特定、紹介は FTM HUCPVCs の微分の潜在性ことができますこの方法で活かされることや心筋細胞のような細胞を自発的に契約に上昇を与えることができます。

<table><tbody><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200056</td><td>For cell dissociation</td></tr><tr><td>Alpha-MEM</td><td>Gibco</td><td>12571071</td><td>For HUCPVC and BMSC culture media.</td></tr><tr><td>PE-conjugated anti-human/mouse CD49f antibody</td><td>Biolegend</td><td>313612</td><td>Integrin marker for FC</td></tr><tr><td>APC-conjugated human Cx43/GJA1 antibody</td><td>R&amp;amp;D Systems</td><td>FAB7737A</td><td>Connexin 43 marker for FC</td></tr><tr><td>FITC-conjugated HLA-A2 antibody</td><td>Genway Biotech Inc.</td><td>GWB-66FBD2</td><td>Immunogenicity marker for FC</td></tr><tr><td>FITC-conjugated anti-HLA-G [MEM-G/9] antibody</td><td>Abcam</td><td>ab7904</td><td>Immunogenicity marker for FC</td></tr><tr><td>FITC-conjugated mouse anti-human SIRPA/CD172a antibody</td><td>AbD Serotec/Bio-Rad</td><td>MCA2518F</td><td>Cardiac marker for FC</td></tr><tr><td>APC-conjugated human TRA-1-85/CD147 antibody</td><td>R&amp;amp;D Systems</td><td>FAB3195A</td><td>Human cell marker for FC and FACS</td></tr><tr><td>FITC-conjugated human TRA-1-85/CD147 antibody</td><td>R&amp;amp;D Systems</td><td>FAB3195F</td><td>Human cell marker for FC and FACS</td></tr><tr><td>Anti-connexin 43/GJA1 antibody</td><td>Abcam</td><td>ab11370</td><td>Cx43. For ICC</td></tr><tr><td>Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555</td><td>Life Technologies</td><td>A-21428</td><td>For ICC</td></tr><tr><td>Anti-sarcomeric alpha actinin [EA-53] antibody</td><td>Abcam</td><td>ab9465</td><td>aSARC. For ICC</td></tr><tr><td>Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555</td><td>Life Technologies</td><td>A-21426</td><td>For ICC</td></tr><tr><td>Mef2C (D80C1) XP rabbit antibody</td><td>New England BioLabs Ltd.</td><td>5030S</td><td>For ICC</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488</td><td>Life Technologies</td><td>A-21206</td><td>For ICC</td></tr><tr><td>Anti-nuclei (HuNu) (clone 235-1) antibody</td><td>EMD Millipore</td><td>MAB1281</td><td>For ICC</td></tr><tr><td>MZ9.5 Stereomicroscope</td><td>Leica</td><td></td><td>For imaging aggregates.</td></tr><tr><td>1.5 ml centrifuge microtubes</td><td>Axygen</td><td>MCT-150-C</td><td>For staining MSCs with fluorescent dye.</td></tr><tr><td>ImageJ</td><td></td><td></td><td>Open source image processing software.</td></tr><tr><td>Aria II&amp;nbsp;</td><td>BD</td><td></td><td>UHN SickKids FC Facility. For cell sorting.</td></tr><tr><td>Bone marrow mesechymal stromal cells</td><td>Lonza</td><td>PT-2501</td><td>BMSCs</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030-100G</td><td>BSA. To prepare solutions for ICC</td></tr><tr><td>BrdU</td><td>EMD Millipore</td><td>MAB3424</td><td>Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.</td></tr><tr><td>Canto II</td><td>BD</td><td></td><td>UHN SickKids FC Facility. For flow cytometry.</td></tr><tr><td>cDNA EcoDry Premix</td><td>Clontech/Takara</td><td>639570</td><td>For preparation of cDNA for qPCR</td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Life Technologies</td><td>C7025</td><td>Fluorescent imaging of cell cytoplasm</td></tr><tr><td>Countess automated cell counter</td><td>Invitrogen Inc.</td><td>C10227</td><td>For cell counting</td></tr><tr><td>DMEM-F12</td><td>Sigma-Aldrich</td><td>D6421</td><td>For rat primary cardiomyocyte culture medium.</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Gibco</td><td>10010023</td><td>D-PBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>EVOS</td><td>Life Technologies</td><td></td><td>In-house fluorescent microscope</td></tr><tr><td>FACSCalibur</td><td>BD</td><td></td><td>In-house. For flow cytometry.</td></tr><tr><td>Fetal bovine serum (Hyclone)</td><td>GE Healthcare</td><td>SH3039603</td><td>FBS. Component of cell culture medium.</td></tr><tr><td>IDT Prime Time qPCR probes</td><td>Integrated Data Technologies</td><td>FAM fluorophore</td><td>http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers</td></tr><tr><td>Lab Vision PermaFluor Aqueous Mounting Medium</td><td>ThermoScientific</td><td>TA-030-FM</td><td>For storage of cells to undergo ICC</td></tr><tr><td>LSR II&amp;nbsp;</td><td>BD</td><td></td><td>UHN SickKids FC Facility. For flow cytometry.</td></tr><tr><td>MoFlo Astrios</td><td>Beckman Coulter</td><td></td><td>UHN SickKids FC Facility. For cell sorting.</td></tr><tr><td>Normal goat serum</td><td>Cell Signaling Technology</td><td>5425S</td><td>NGS. Used in blocking solution for ICC</td></tr><tr><td>Nunc Lab-Tek II Chamber Coverglass, 8-wells</td><td>Thermo Scientific Nunc</td><td>155409</td><td>To prepare samples for ICC</td></tr><tr><td>OmniPur Triton X-100 Surfactant</td><td>EMD Millipore</td><td>9410-OP</td><td>As a component of permeabilizing solution when preparing cells for ICC</td></tr><tr><td>Paraformaldehyde, 16% Solution, EM Grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>For fixing cells for ICC.</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td>Component of cell culture medium.</td></tr><tr><td>Primers</td><td>Sigma</td><td>Custom Standard DNA Oligos, Desalted, 0.2 &amp;mu;mol</td><td>&lt;strong&gt;CTnT_F:&lt;/strong&gt; GGC AGC GGA AGA GGA TGC TGA A; &lt;strong&gt;CTnT_R:&lt;/strong&gt; GAG GCA CCA AGT TGG GCA TGA ACG A; &lt;strong&gt;MYH6 F:&lt;/strong&gt; GCA AAG TAC TGG ATG ACA CGC T; &lt;strong&gt;MYH6 R:&lt;/strong&gt; GTC ATT GCT GAA ACC GAG AAT G</td></tr><tr><td>Quorum Spinning Disk Confocal</td><td>Zeiss</td><td></td><td>SickKids Imaging Facility</td></tr><tr><td>ReproCardio hiPS cell derived cardiomyocytes</td><td>ReproCell</td><td>RCD001N</td><td>Positive control for qPCR</td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74106</td><td>To isolate RNA for qPCR</td></tr><tr><td>Rotor-Gene SYBR Green PCR Kit</td><td>Qiagen</td><td>204074</td><td>For qPCR with master mix</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>A1049101</td><td>For MSC, monocyte coculture medium.</td></tr><tr><td>TaqMan qPCR primer assays</td><td>Thermo Fisher Scientific</td><td>4444556</td><td>For qPCR</td></tr><tr><td>Trypan Blue</td><td>Life Technologies</td><td>T10282</td><td>Staining of cells for viability and counting</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>272500108</td><td>For cell dissociation</td></tr><tr><td>Volocity</td><td>Perkin-Elmer</td><td>Volocity 6.3</td><td>Imaging software</td></tr><tr><td>0.2 &amp;mu;m pore filter</td><td>Thermo Fisher Scientific</td><td>566-0020</td><td>For sterilizing tissue culture media</td></tr><tr><td>HERAcell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific</td><td>51026410</td><td>For incubating cells</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>PBS. 1X, Without calcium chloride and magnesium chloride</td></tr><tr><td>Forceps</td><td>Almedic</td><td>7727-A10-704</td><td>For handing rat heart. Can use any similar forceps.</td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14059-11</td><td>For mincing rat heart. Curved scissors recommended.</td></tr><tr><td>50 mL tube</td><td>BD Falcon</td><td>352070</td><td>For collection during cardiomyocyte collection and general tissue culture procedures</td></tr><tr><td>15 mL tube</td><td>BD Falcon</td><td>352096</td><td>For general tissue culture procedures</td></tr><tr><td>6-well plates</td><td>Thermo Scientific Nunc</td><td>CA73520-906</td><td>For tissue culture</td></tr><tr><td>10 cm tissue culture dishes</td><td>Corning</td><td>25382-428</td><td>For aggregate formation</td></tr><tr><td>Axiovert 40C Microscope</td><td>Zeiss</td><td></td><td>For bright-field imaging through out tissue culture and the rest of the protocol</td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Fisherbrand</td><td>22363548</td><td>To ensure a single cell suspension before flow cytometry or sorting</td></tr><tr><td>Triton X-100</td><td>EMD Millipore</td><td>9410-1L</td><td>Used in permeabilization solution for ICC</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H1399</td><td>Stain used during visualization of Cx43 localization</td></tr></tbody></table>

in vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells

心筋梗塞とその後虚血性連鎖結果心筋細胞、うっ血性心不全、世界の死亡率の一流の原因につながるの広汎な損失。間葉系幹細胞 (Msc) は、現在、侵襲的な技術を交換する細胞ベースの治療に有望なオプションです。MSCs は、心筋細胞の種類を含む、間葉系の系統に区別できるが、完全な機能的な細胞分化がまだ達成されていません。以前の差別化の方法は、薬理学的エージェントまたは成長の要因に基づいていた。しかし、もっと生理学的に関連する戦略には MSCs cardiomyogenic 変換を受けることも可能です。心筋細胞のような収縮細胞を生産する心筋細胞の送り装置の層に MSC 骨材を用いた微分法を紹介します。
ひと臍帯血管周囲細胞 (HUCPVCs) がより大きい微分よりも潜在的なを持っている示されている一般的骨髄 MSCs (BMSCs) など、MSC の種類を調査しました。ひきつづいて若いソースとして古いソースと比較して妊娠 (FTM) の HUCPVCs の cardiomyogenic の可能性を検討した.FTM の HUCPVCs プロパティを保持、子宮内immunoprivileged 培養 MSCs の小説、豊かな源である生体外で。HUCPVCs この分化プロトコル、FTM の用語を使用して (すなわち、心筋増強物の要因 2 C、心筋トロポニン T、心筋ミオシンの重鎖、信号規定する蛋白質の α とコネキシン 43) 心筋細胞マーカーの発現の増加によって示されるように、BMSCs と比較して大幅に増加した cardiomyogenic 分化を達成しました。彼らはまた彼らの低い HLA A 式と高い HLA G 式で実証としての有意に低い免疫原性を維持しました。分化の集計ベースを適用すると、FTM HUCPVCs は、潜在的なと心臓フィーダー層を共培養後 1 週間以内のセルのクラスターを契約、そう最初の MSC 型になって生成された高められた形成を示した。
我々 の結果はこの差別化戦略 FTM HUCPVCs などの若い MSCs の cardiomyogenic の可能性を効果的に活用することができることを実証してから、その再生効果で体内を向上させる潜在的な戦略ができるその体外前分化を示唆しています。

Watch this Scientific Journal Video about In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells at JoVE.com

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

ここでは、機能、契約、心筋細胞のような細胞を生成するために若いヒト間葉系幹細胞源の心臓微分の潜在性を効率的に活用する方法を提案するの in vitro 。

体外ヒト間葉系幹細胞の心筋細胞のような細胞への分化

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

in-vitro-differentiation-human-mesenchymal-stem-cells-into-functional

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

10.8K ビュー.  Create Fertility Centre. ここでは、機能、契約、心筋細胞のような細胞を生成するために若いヒト間葉系幹細胞源の心臓微分の潜在性を効率的に活用する方法を提案するの in vitro 。

ビデオ: In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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

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

胚性幹細胞から心臓前駆細胞の誘導

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

発生生物学

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

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

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

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

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

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

BMPアンタゴニストGrem2を使用した多能性幹細胞から心房心筋細胞の分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

ハンギングドロップ法を用いたマウス胚性幹（MES）細胞のin vitroにおける分化

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

生物学

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

多電極アレイ（MEA）を用いたヒト多能性幹細胞由来心筋細胞（hPSC-CM）の電気生理学的解析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

生物工学

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者末梢血単核細胞からのヒト心筋細胞の発生と拡大

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. 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 tumorigenicity6-8 (see a schematic in Fig. 1A). 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 problematic9-11. 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 molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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

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

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

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

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

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

カルシウム処理特性評価のための心室様HiPSC由来心筋細胞と高品質細胞製剤の生成

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...