Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

全ての実験手順は、アラバマ大学バーミンガム校の動物ガイドラインに従って行われています。 1. hiPSC-のCMにhiPSCsの区別<ol><li>一晩4℃で予め冷却した成長因子低減ゼラチン状タンパク質混合物でコート6ウェルプレートのウェル。使用前にゲル状のタンパク質混合物を吸引。 10μMのROCK阻害剤とのmTeSR1培地補充で5％CO 2、37℃で細胞（ウェル当たり1×10 5細?...

<ol>
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hiPSC-のCMの歩留まりの向上/純度 CMSにヒト幹細胞を分化するための従来のプロトコルは、多くの場合、低い収率および純度によって制限されます。例えば、ヒトES細胞-CMSのわずか35から66まで%は遅いミオシン重鎖またはcTnTの6を表明パーコール分離および心臓体形成を介して得られます。分化hiPSC-CM集団の純度は、実質的にCM特異的タンパク質<s...

ヒトの人工多能性幹細胞（hiPSCs）は、患者の免疫系によって拒絶されていない細胞の潜在的に無限の範囲と量に分化させることができるので、再生細胞療法のための最も有望な薬剤の一つです。 （平滑筋細胞を但し、自己複製および分化のための能力はまた、腫瘍形成につながる可能性があり、その結果、hiPSCsは、完全にこのような心筋細胞（CMS）、内皮細胞（EC）、および平滑筋細胞などの特定の細胞型に分化することが必要）、投与前。細胞投与の最も簡単で最も一般的な方法の1つは、直接心筋内注射であるが、本来の心筋組織により生着された移植細胞の数が非常に低いです。このスレの多くは、虚血組織の細胞毒性環境に起因することができます。しかし、マウスの胚性幹細胞（ESC）は、O、無傷心臓の心筋に直接注入しました。NLY〜配信500万細胞の40％は、それらが中に製造された高圧力によって針トラックを通って絞り出されたせいか、投与された細胞のかなりの割合は、投与部位を終了したことを示唆している3-5時間1、のために保持しました心筋収縮。 ここでは、（SMCの）4 hiPSC由来の心筋細胞（hiPSC-CMS）2、内皮細胞（hiPSC-ECS）3、および平滑筋細胞を生成するための新規かつ、実質的に、より効率的な方法を提示します。注目すべきは、このhiPSC-SMCプロトコルは、主に合成または収縮SMC表現型に向かって細胞を向けることによって、体細胞のSMC 5で観察された形態学的および機能的特性の広い範囲を模倣する最初のものです。我々はまた、サイトカインを含むフィブリンPを作成することにより、注入された細胞の生着率を改善細胞送達の方法を提供します注射部位の上にATCH。パッチは、少なくとも3日間の期間にわたってインスリン様成長因子（IGF）を放出することにより、心筋から出るセルを防止する針トラック、及び細胞生存を封止することにより、両方の細胞保持を改善するように見えます。

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 monofilament suture</td>
			<td>Ethilon</td>
			<td>627H</td>
			<td></td>
		</tr>
	</tbody>
</table>

pluripotent stem cell derived cardiac cells for myocardial repair

ヒト人工多能性幹細胞（hiPSCs）が完全に投与する前に、特定の細胞型に分化されなければならないが、心筋細胞（hiPSC-CMS）にhiPSCsを区別するための従来のプロトコル、内皮細胞（hiPSC-ECS）、および平滑筋細胞（平滑筋細胞）は、しばしば低収量、純度、および/または貧弱な表現型の安定性によって制限されます。ここで、我々は、新規hiPSC-のCMを生成するためのプロトコル、-ECs、実質的に従来の方法よりも効率的です-SMCs、ならびに投与のサイト上で作成されたサイトカインを含むパッチで細胞注入を組み合わせるための方法を提示します。パッチは、長期間にわたってインスリン様成長因子（IGF）を放出することにより、心筋から絞り出されてから細胞を防ぐために、針の軌道、及び細胞生存を封止することにより、注入された細胞の保持の両方を改善します。心筋虚血再灌流傷害のブタモデルにおいて、生着率が時より2倍大きかったです細胞は、細胞とパッチの両方を有するサイトカイン含有貼付せずに細胞に比べ、パッチ、および治療を投与したが、一人ではない細胞を用いて、心機能および梗塞サイズの大幅な改善と関連していました。

差別hiPSC-CMを、-ECs、および-SMCsのキャラクタリゼーション hiPSCsの微分容量は2、3、4を評価しました。フローサイトメトリー心臓トロポニンT（cTnTの）の発現の分析は、最終的なhiPSC-CM集団の純度は90％（ 図1A、1B、パネルB1）を超え...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

我々は、心筋細胞、内皮細胞、および平滑筋細胞およびパッチ媒介性サイトカインの送達と細胞注射を組み合わせることにより、移植細胞の生着を改善する配送方法に、ヒト人工多能性幹細胞を分化するための3つの新規かつより効率的なプロトコルを提示します。

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

The overall goal of this video is to introduce a technique for differentiating human pluripotent stem cells into cardiomyocytes, and to demonstrate the use of these cells for studying myocardial repair after an ischemia reperfusion injury. This method can help answer key questions about engineering heart tissue with human iPS cell derived from cardiac cells for myocardial repair. The main advantage of this procedure is that it allows scientists to collect purified cardiomyocytes, smooth muscle cells, and endothelial cells, and generate heart tissue with these progenitor cells.Ling Gao is a post-doc in Doctor Zhang's own lab in UAB and Greg Walcott is a cardiologist in UAB hospital. Trina is a MRI specialist in UAB hospital. To induce pluripotent stem cells into cardiomyocytes, first, coat the wells of a six-well plate with growth-factor reduced gelatinous protein mixture cooled to 4 degrees Celsius.Then, refrigerate the plate overnight at 4 degrees Celsius. The next day, aspirate the gelatinous protein mixture and seed the plates with 100 million stem cells per well. Never keep the stem cells out of the incubator for more than 15 minutes.Also, always use fresh media. In this case, use mTeSR1 medium, supplemented with 10 micromolar of ROCK inhibitor. Then, culture the cells with 5%carbon dioxide at 37 degrees Celsius, and refresh the medium daily by gently aspirating away the old medium without touching the cells, and using a transfer pipette to apply fresh medium.Grow the cells until they are 90%confluent. Three days into the differentiation process, start checking for clusters of contracting cells. As soon as contractions are noted, which can take a week or so, collect the cells.Count the contracting cells using a hemocytometer, and then culture the cells on 10 centimeter plates at a concentration of one million cells per milliliter. It is also possible to make endothelial cells from stem cells, as well as smooth muscle cells. The processes are different, but are carefully detailed in the text protocol.First, surgically induce a myocardial infarction in a swine heart. Briefly perform a fourth intercostal thoracotomy to expose the left anterior descending coronary artery. Then, occlude the coronary artery using a ligature for 60 minutes.Defibrillate the heart as necessary. Suspend five milligrams of microspheres in one milliliter of fibrinogen solution, and load this into a syringe. Third, fill another syringe with one milliliter of thrombin solution.After an hour has passed, remove the ligature used to create the myocardial infarction. Then, position a plastic ring on the epicardium of the infarcted region, and simultaneously inject the microspheres and fibrinogen and the thrombin solutions into the ring. Allow the mixture of solutions to solidify, which takes about 30 seconds.This makes the patch. Then, insert the needle of the cell-containing syringe through the patch into the infarcted myocardium and inject the cells. Now, close the muscle layers, subcutaneous tissue, and the chest wall with 30 or 20 Monocryl suture, depending on the animal's size.One week and four weeks after the procedure, evaluate the animal's cardiac function using a cardiac MRI. As described by the first presented protocol, human pluripotent stem cells were differentiated into cardiomyocytes. To assess their purity, flow cytometry for cardiac troponin T showed it was present in excess of 90%of cells.Other markers were also observed. Nearly all of the cells expressed slow myocin heavy chain, and nearly all the cells expressed alpha sarcomeric actin. About one quarter of the cells expressed 2v isoform of myosin light chain, a marker for ventricular cardiomyocytes.In comparing stem cell lines, it was found that the efficiency of the differentiation was substantially higher with cells from the GRiPS line than from the PCBC16iPS line. Using the patch-mediated cell transplantation protocol, a mixture of cells, made up of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells was injected into the injured tissue. Four weeks later, more cells were retained with the patch than without it.Within one week, treatment using the cell injection with the patch resulted in the most improvement of the infarct size. Also, there was no appreciable difference in injecting with just cardiomyocytes compared to injecting with three cell types. After watching this video, you should have a good understanding on how to differentiate human iPS cells into three different cardiac ligature cells, including cardiomyocytes, smooth muscle cells, and endothelial cells.You should also have a good understanding of how to generate human heart tissue with the progenitor cells for myocardial repair. Since the development, this technique has advanced investigations on myocardial repair and stem cell tissue engineering. It should be noted that when attempting this procedure, it is very important to use high quantity of iPS cells and their cardiac derivatives.

Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

MYH6プロモーター - 選択と組み合わせたES細胞のプログラミングに基づいて、マウス心臓ペースメーカー細胞凝集体の生成

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

発生生物学

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

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

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

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

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

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

末梢血由来ヒト誘導多能性幹細胞からの軟骨細胞の分化

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

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

Single-cell Photoconversion in Living Intact Zebrafish

生活そのままゼブラフィッシュの単一セルの光電

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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

創薬における多能性幹細胞由来肝細胞様細胞を誘導人間を使用して

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

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

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

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