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 border="0" cellpadding="0" cellspacing="0" width="917">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">0.25% Trypsin/EDTA</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:119px;">25200056</td>
			<td style="width:299px;">For cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Alpha-MEM</td>
			<td style="width:203px;">Gibco</td>
			<td>12571071</td>
			<td style="width:299px;">For HUCPVC and BMSC culture media.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">PE-conjugated anti-human/mouse CD49f antibody</td>
			<td style="width:203px;">Biolegend</td>
			<td style="width:119px;">313612</td>
			<td style="width:299px;">Integrin marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">APC-conjugated human Cx43/GJA1 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB7737A</td>
			<td style="width:299px;">Connexin 43 marker for FC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">FITC-conjugated HLA-A2 antibody</td>
			<td style="width:203px;">Genway Biotech Inc.</td>
			<td style="width:119px;">GWB-66FBD2</td>
			<td style="width:299px;">Immunogenicity marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated anti-HLA-G [MEM-G/9] antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab7904</td>
			<td style="width:299px;">Immunogenicity marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated mouse anti-human SIRPA/CD172a antibody</td>
			<td style="width:203px;">AbD Serotec/Bio-Rad</td>
			<td style="width:119px;">MCA2518F</td>
			<td style="width:299px;">Cardiac marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">APC-conjugated human TRA-1-85/CD147 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB3195A</td>
			<td style="width:299px;">Human cell marker for FC and FACS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated human TRA-1-85/CD147 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB3195F</td>
			<td style="width:299px;">Human cell marker for FC and FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Anti-connexin 43/GJA1 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab11370</td>
			<td style="width:299px;">Cx43. For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">A-21428</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Anti-sarcomeric alpha actinin [EA-53] antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:299px;">aSARC. For ICC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td style="width:203px;">Life Technologies</td>
			<td>A-21426</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Mef2C (D80C1) XP rabbit antibody</td>
			<td style="width:203px;">New England BioLabs Ltd.</td>
			<td style="width:119px;">5030S</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">A-21206</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Anti-nuclei (HuNu) (clone 235-1) antibody</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">MAB1281</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MZ9.5 Stereomicroscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">For imaging aggregates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">1.5 ml centrifuge microtubes</td>
			<td style="width:203px;">Axygen</td>
			<td style="width:119px;">MCT-150-C</td>
			<td style="width:299px;">For staining MSCs with fluorescent dye.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ImageJ</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Open source image processing software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aria II&#160;</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bone marrow mesechymal stromal cells</td>
			<td style="width:203px;">Lonza</td>
			<td style="width:119px;">PT-2501</td>
			<td style="width:299px;">BMSCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7030-100G</td>
			<td style="width:299px;">BSA. To prepare solutions for ICC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">BrdU</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">MAB3424</td>
			<td style="width:299px;">Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Canto II</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">cDNA EcoDry Premix</td>
			<td style="width:203px;">Clontech/Takara</td>
			<td style="width:119px;">639570</td>
			<td style="width:299px;">For preparation of cDNA for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">CellTracker Green CMFDA Dye</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:299px;">Fluorescent imaging of cell cytoplasm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Countess automated cell counter</td>
			<td style="width:203px;">Invitrogen Inc.</td>
			<td style="width:119px;">C10227</td>
			<td style="width:299px;">For cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM-F12</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6421</td>
			<td style="width:299px;">For rat primary cardiomyocyte culture medium.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:203px;">Gibco</td>
			<td>10010023</td>
			<td style="width:299px;">D-PBS, without Ca2+, Mg2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">EVOS</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">In-house fluorescent microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">FACSCalibur</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">In-house. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Fetal bovine serum (Hyclone)</td>
			<td style="width:203px;">GE Healthcare</td>
			<td style="width:119px;">SH3039603</td>
			<td style="width:299px;">FBS. Component of cell culture medium.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">IDT Prime Time qPCR probes</td>
			<td style="width:203px;">Integrated Data Technologies</td>
			<td style="width:119px;">FAM fluorophore</td>
			<td style="width:299px;">http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Lab Vision PermaFluor Aqueous Mounting Medium</td>
			<td style="width:203px;">ThermoScientific</td>
			<td style="width:119px;">TA-030-FM</td>
			<td style="width:299px;">For storage of cells to undergo ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">LSR II&#160;</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MoFlo Astrios</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Normal goat serum</td>
			<td style="width:203px;">Cell Signaling Technology</td>
			<td style="width:119px;">5425S</td>
			<td style="width:299px;">NGS. Used in blocking solution for ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Nunc Lab-Tek II Chamber Coverglass, 8-wells</td>
			<td style="width:203px;">Thermo Scientific Nunc</td>
			<td style="width:119px;">155409</td>
			<td style="width:299px;">To prepare samples for ICC</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:297px;">OmniPur Triton X-100 Surfactant</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">9410-OP</td>
			<td style="width:299px;">As a component of permeabilizing solution when preparing cells for ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:203px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td style="width:299px;">For fixing cells for ICC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Penicillin/streptomycin</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td style="width:299px;">Component of cell culture medium.</td>
		</tr>
		<tr height="110">
			<td height="110" style="height:110px;width:297px;">Primers</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:119px;">Custom Standard DNA Oligos, Desalted, 0.2 &#956;mol</td>
			<td style="width:299px;">CTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Quorum Spinning Disk Confocal</td>
			<td style="width:203px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">SickKids Imaging Facility</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">ReproCardio hiPS cell derived cardiomyocytes</td>
			<td style="width:203px;">ReproCell</td>
			<td style="width:119px;">RCD001N</td>
			<td style="width:299px;">Positive control for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">RNeasy mini kit</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td style="width:299px;">To isolate RNA for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rotor-Gene SYBR Green PCR Kit</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:119px;">204074</td>
			<td style="width:299px;">For qPCR with master mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">RPMI 1640</td>
			<td style="width:203px;">Gibco</td>
			<td>A1049101</td>
			<td style="width:299px;">For MSC, monocyte coculture medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">TaqMan qPCR primer assays</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4444556</td>
			<td style="width:299px;">For qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypan Blue</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">T10282</td>
			<td style="width:299px;">Staining of cells for viability and counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypsin</td>
			<td style="width:203px;">Gibco</td>
			<td>272500108</td>
			<td style="width:299px;">For cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Volocity</td>
			<td style="width:203px;">Perkin-Elmer</td>
			<td style="width:119px;">Volocity 6.3</td>
			<td style="width:299px;">Imaging software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">0.2 &#956;m pore filter</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">566-0020</td>
			<td style="width:299px;">For sterilizing tissue culture media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:297px;">HERAcell 150i CO2 Incubator</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">51026410</td>
			<td style="width:299px;">For incubating cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Dulbecco&#39;s phosphate buffered saline</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td style="width:299px;">PBS. 1X, Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Forceps</td>
			<td style="width:203px;">Almedic</td>
			<td style="width:119px;">7727-A10-704</td>
			<td style="width:299px;">For handing rat heart. Can use any similar forceps.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Scissors</td>
			<td style="width:203px;">Fine Science Tools</td>
			<td style="width:119px;">14059-11</td>
			<td style="width:299px;">For mincing rat heart. Curved scissors recommended.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">50 mL tube</td>
			<td style="width:203px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:299px;">For collection during cardiomyocyte collection and general tissue culture procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">15 mL tube</td>
			<td style="width:203px;">BD Falcon</td>
			<td align="right" style="width:119px;">352096</td>
			<td style="width:299px;">For general tissue culture procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">6-well plates</td>
			<td style="width:203px;">Thermo Scientific Nunc</td>
			<td style="width:119px;">CA73520-906</td>
			<td style="width:299px;">For tissue culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">10 cm tissue culture dishes</td>
			<td style="width:203px;">Corning</td>
			<td style="width:119px;">25382-428</td>
			<td style="width:299px;">For aggregate formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Axiovert 40C Microscope</td>
			<td style="width:203px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">For bright-field imaging through out tissue culture and the rest of the protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">70 &#956;m cell strainer</td>
			<td style="width:203px;">Fisherbrand</td>
			<td align="right" style="width:119px;">22363548</td>
			<td style="width:299px;">To ensure a single cell suspension before flow cytometry or sorting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">9410-1L</td>
			<td style="width:299px;">Used in permeabilization solution for ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Hoechst 33342</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">H1399</td>
			<td style="width:299px;">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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

科研

发育生物学

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

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 ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

在体外人骨髓间充质干细胞分化为心肌样细胞功能

摘要

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此视频中的章节

人多能干细胞对原始和最终造血祖细胞定向分化的研究

在人多能的体外干细胞向滋养层细胞

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

活完好斑马鱼的单细胞 Photoconversion

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

犬卵巢间充质干细胞的提取与分化

人类多能干细胞有效分化为肝细胞

人诱导多能干细胞体外生成体细胞衍生物的研究

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

在体外人骨髓间充质干细胞分化为心肌样细胞功能

摘要

探索更多视频

此视频中的章节

人多能干细胞对原始和最终造血祖细胞定向分化的研究

在人多能的体外干细胞向滋养层细胞

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

活完好斑马鱼的单细胞 Photoconversion

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

犬卵巢间充质干细胞的提取与分化

人类多能干细胞有效分化为肝细胞

人诱导多能干细胞体外生成体细胞衍生物的研究

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应