Name: in vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells
Uploaded: 2017-08-09T12:30:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells at JoVE.com

作者感谢以下人员和研究人员所作的贡献: 马修 Librach、 莱拉 Maghen、 坦尼娅 A.Baretto，Shlomit Kenigsberg 和安德鲁 Gauthier 费舍尔。这项工作被支持由安大略省研究基金-研究卓越 （ORF RE，圆 #7） 和创建程序公司。

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>

克利福德 · L.Librach 博士是联合专利持有人:隔离和使用来自第一孕期胎儿脐带组织细胞的方法，授予在加拿大和澳大利亚。

2 年来，发展与几个不同的策略被用来从 MSC 来源生成心肌样细胞受到了心脏干细胞分化。许多这些策略，然而，是低效的而且使用的条件往往不代表环境移植细胞遇到的体内。
与现有的方法，相比议定书 》 在这里提出了一种利用原发性心脏饲养层和 MSC 聚合形成的组合。原发性心脏饲养层提供类似于间充质干细胞移植后暴露的分化环境。聚合生成环境与氧气和养分的...

充血性心力衰竭 (CHF) 仍是全球发病率和死亡率的主要原因。CHF 经常发生后心肌细胞的巨大损失和心肌梗死 (MI)1的病理结果无细胞瘢痕组织的发展。虽然心脏是部分自我更新的器官，驻地干、 祖细胞池负责执行组织再生显著降低中丰度和老年患者，往往变得不足以最佳恢复的损伤后的功能。因此，是极大的兴趣发展涉及的健康供者细胞移植到受损心肌的实验性治疗。势在必行的是供者细胞不仅恢复结构的组织，但也达到心肌功能恢复的影响。
本机的心员工心组织居民和损伤后内源性的骨髓源性干细胞修复2，，34。蓄热式导出细胞宿主和捐助者都必须有获得适当的表型和功能的重塑心肌，并有能力以高效、 安全地更换丢失的细胞微环境的能力。在体外分化方法曾经广泛实现高效率、 基于干细胞的心肌细胞生产5，6。表达谱的心脏沿袭标记用于定义向心脏沿袭7干细胞分化的过程。早期分化标记，如 NKX2.5，肌细胞增强因子 2 C (Mef2c) 和 GATA48，9，可以指示开始向心肌细胞进程。成熟的心肌细胞标记通常用于评估分化效果是信号调节蛋白 α （关于）10、 心肌肌钙蛋白 T （肌钙）11、 重链心肌肌球蛋白 (MYH6)8，，1213和缝隙连接蛋白 43 (Cx43)14，，1516。利用胚胎干细胞 (Esc) 和多能性干细胞 (Psc) 的方法彻底优化并讨论了有关的诱导因素，氧气和养分的渐变，细节和行动5，，67，，1718的确切时间。尽管如此，基于 ESC 和 PSC 的技术仍然存在多个的伦理和安全问题，和次优的电生理及免疫学特征19，20。主机经常用这些细胞移植免疫排斥的经验和需要永久免疫抑制。这主要是由于失主要组织相容性复合体 (MHC) 分子在东道国和捐助和由此产生的 T 细胞反应21。同时个别 MHC I 类匹配是一个可能的解决方案，更易于访问的临床实践需要普遍是理想来克服排斥反应的关切的细胞来源。
作为替代细胞来源，用于在临床应用中，骨髓间充质干细胞和骨髓间充质干细胞，特别被进行了自其最初被描述在 1995年22组织再生使用。骨髓间充质干细胞据说是驻地的再生细胞在几乎任何带血管蒂的组织23中可以发现。骨髓间充质干细胞后从所需的源的隔离，很容易可以扩大在文化，有广泛的旁分泌能力，经常拥有理想或免疫调节属性24，25。其安全性和有效性已经进行了几个临床前研究，特别是心脏再生3，，26。
许多 MSC 分化策略利用药理剂 5-氮杂胞苷22和二甲基亚砜27，和增长或人骨的因素，像 BMPs5，7，，2829或血管紧张素 II30，与变量效率。这些策略，不过，不基于天真再生细胞有望后归巢或传递到损伤的部位，遇到的障碍体内。更多生理有关的战略，同时更难定义和操纵，都基于的前提下，可以通过从组织微环境本身的信号诱导 MSC 分化。先前的研究显示，暴露于心肌细胞裂解物31或左室心肌32，33，或直接与原发性心肌细胞体外15，34，接触可以增加心肌标志物检测在骨髓间充质干细胞中表达。别人有后，表现出自发再生治疗心肌损伤与骨髓间充质干细胞35，36，，3738，尽管部分，骨髓间充质干细胞和心肌细胞39，40融合生成新生心肌。我们所知，从人骨髓间充质干细胞 (hMSCs) 的任何组织源的功能、 自发订约心肌细胞尚未报告。
目前的共识是所有骨髓间充质干细胞产生的血管周围细胞23。年轻骨髓间充质干细胞与周细胞属性可以从人脐带组织41，，4243的血管周围地区隔离。与骨髓间充质干细胞，HUCPVCs 拥有更多的分化潜能和几个其他再生的优势，两个体外41，44和体内45，，4647。值得注意的是，被母胎界面的来源，HUCPVCs 有显著较低免疫原性相比成人骨髓间充质干细胞的来源。我们的研究重点的表征和 FTM HUCPVCs，年轻源间充质干细胞研究，临床前应用，我们以前已经增加了增殖能力和较高的 multilineage 分化的能力，包括在向心肌细胞谱系41。
在这里，我们提出一种协议，结合聚合形成和原发性心脏细胞饲养层，归纳部队达到完成向心肌细胞分化的骨料骨髓间充质干细胞。 提供 3D 的环境，更好地模型条件体内相比 2D 遗民文化。利用心脏饲养层提供一种环境，是对骨髓间充质干细胞的最终移植网站的代表。我们证明年轻来源的间充质干细胞分离产前或产后的脐带有较高的能力，与窗体集料，达到相比成人骨髓间充质干细胞，同时仍然保持其免疫特权心肌表型。除了陡海拔及心脏沿袭标记基因诱导的表达的细胞内 （即， cTnT 与 MYH6） 和细胞表面蛋白 (即，关于和 Cx43) 具体为心肌细胞，我们表明可以用这种方法利用 FTM HUCPVCs 分化潜力，它们可以导致自发性收缩心肌样细胞。

<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

心肌梗死和随后的缺血级联导致大量伤亡，心肌细胞，导致充血性心衰，全世界死亡的主要原因。骨髓间充质干细胞 (MSCs) 是一种有前景的细胞为基础的疗法，以取代当前的侵入性技术。骨髓间充质干细胞可以分化成间充质血统，其中包括心脏的细胞类型，但尚未实现完整功能的细胞分化。以前的分化的方法基于药物制剂或生长因子。然而，更多生理有关的战略也可以启用骨髓间充质干细胞向心肌细胞转化。在这里，我们提出一种利用心肌细胞饲养层上 MSC 聚合产生订约心肌样细胞的分化方法。
人脐带血管周围细胞 (HUCPVCs) 已被证明有更大的分化潜能比一般调查 MSC 类型，如骨髓间充质干细胞 (BMSCs)。作为一个 ontogenetically 年轻的来源，我们调查了早孕 (FTM) HUCPVCs 相比更旧的来源的心肌样潜力。FTM HUCPVCs 是新颖、 丰富的来源，保留其在子宫内理想属性时培养的间充质干细胞在体外。HUCPVCs 使用此分化协议、 FTM 和期限取得显著增加向心肌细胞分化与骨髓间充质干细胞，相比，增加表达的心肌细胞标记 （即，肌细胞增强因子 2 C、 心肌肌钙蛋白 T、 重链心肌肌球蛋白、 信号调节蛋白 α 和缝隙连接蛋白 43） 所示。他们还保持显著低免疫原性，证明了他们低 HLA A 表达和更高的 HLA — G 表达这一点。应用基于聚合的分化，FTM HUCPVCs 表明增加聚合形成潜在和生成的共培养对心脏饲养层 1 星期内收缩细胞集群，成为第一的 MSC 类型，这样做。
我们的结果表明，这种分化策略可以有效利用向心肌细胞的潜力，年轻的骨髓间充质干细胞，如 FTM HUCPVCs，表明那种体外预分化可能是一个潜在的战略，以增加其再生效果的体内。

HUCPVCs Display Higher Aggregate-formation Potential and CD49f Expression Levels Compared to BMSCs:
To induce the differentiation of hMSCs (i.e., FTM HUCPVCs, term HUCPVCs, and BMSCs), single-cell suspensions of undifferentiated MSCs or MSC-containing hanging drops (Table 1) were transferred onto rat primary cardiomyocyte monolayers to establish direct co-cultures or aggregate co-cultur...

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

在这里，我们提出一种方法，有效地利用的心肌细胞分化潜力，年轻的人骨髓间充质干细胞的来源以生成功能、 订约、 心肌样细胞体外。

在体外人骨髓间充质干细胞分化为心肌样细胞功能

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

Research

JoVE Journal

Developmental Biology

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

10.8K 次观看.  Create Fertility Centre. 在这里，我们提出一种方法，有效地利用的心肌细胞分化潜力，年轻的人骨髓间充质干细胞的来源以生成功能、 订约、 心肌样细胞体外。

视频: 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.

从胚胎干细胞衍生心脏祖细胞

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

科研

发育生物学

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.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

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.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

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.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

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.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

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.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

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

生物工程

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.

患者外周血单核细胞中人心肌细胞的生成和扩增

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.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

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.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

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.

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

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