作者感谢朱利奥·科苏、玛蒂娜·拉加齐和整个实验室的有益讨论和支持，感谢杰夫·张伯伦亲切地提供MyoD-ER载体。所有涉及活体动物的实验都是按照所有相关的监管和体制机构、条例和准则完成的。作者实验室的工作得到了英国医学研究理事会、欧洲共同体第七框架项目Optisem和Biodesign以及意大利杜尚家长项目的支持。

1. 对肌原和增生潜力的评估
<ol><li>
体外： 肌电图诱导分化<ol>
<li>生成稳定的IDEM细胞系，该细胞系与塔莫西芬-可致病肌-ER扁桃体载体一起转化，对感染的多重性（MOI： 例如，1、5 和 50）使用 1.1.9 （MyHC） 中描述的染色作为程序效率的结果。</li>
		<li>涂上3.5厘米的菜，1毫升1%的甲基凝乳，在37°C孵育30分钟。</li>
		<li>用介质（种子 1 x 105 MyoD-ER）在 3.5 厘米组织培养盘中两次清洗盘子，并在生长介质中以 37 °C 孵育。</li>
		<li>预计细胞在一两天内达到汇合，然后在生长介质（1st 剂量）中加入 1 μM 4OH-tamoxifen。</li>
		<li>24小时后，用分化介质替换生长介质，辅以1μM 4OH-tamoxifen（第2次和最后一 剂）。</li>
		<li>每隔一天用新鲜分化介质替换一半的介质。</li>
		<li>每天检查肌管形成的文化。</li>
		<li>一周后（分化介质为5天），用PBS轻轻清洗板，并在RT用4%的副甲醛固定5分钟。</li>
		<li>对肌素重链 （MyHC） 进行免疫荧光染色，以确认肌管的存在。用核染料（如霍奇斯特）进行计数器。</li>
	</ol>
</li>
</ol>区分效率被评估为MyHC阳性细胞内核的百分比：如果效率为50%>，则进入下一步。
<ol start="2"><li>
体内：急性肌肉再生模型的灌动 为了评估MyoD-ER IDEM对肌肉再生的 体内 贡献，这些细胞以前被标记为绿色荧光蛋白（GFP）的载体编码，该载体能够在组织内追踪它们。然后将MyoD-ER GFP IDEM注射到成人肌肉中，以前曾因肌毒素（如 心毒素）而受伤。为了避免对异种基因（如HIDEM）或基因操纵/矫正细胞的免疫排斥，需要使用免疫缺陷或免疫抑制小鼠。<ol>
<li>移植前24小时，在移植和预处理细胞前24小时，在生长介质中加入1微米4OH-tamoxifen（血浆形式），在移植日前将3.5微克/克的10毫克/毫升塔莫西芬（脂溶性形式）注射到动物身上。</li>
		<li>按照规范动物设施外科手术的具体指南，对小鼠进行麻醉和镇痛。</li>
		<li>注射25微克100μM心毒素（CTX：来自 纳贾莫桑比卡莫桑比卡;注意：头 肌前部 （TA）肌肉中潜在的有害物质）</li>
		<li>用塔莫西芬和CTX治疗动物24小时后，通过尝试去皮素化和计数分离细胞。</li>
		<li>将细胞以232 x g分5分钟进行离心。</li>
		<li>在 Ca2+和 Mg2 +无 PBS、离心机中清洗细胞颗粒，然后轻轻地将 Ca2+中的细胞颗粒和 Mg2+无 PBS 中恢复到 106 个细胞/30 μl 的最终浓度，这将是每次注射的最终体积。</li>
		<li>使用29或30 G针的注射器将30μl的细胞悬浮注射到先前受伤的肌肉中。在从肌肉中取出针头时要特别注意。慢慢做，避免将细胞悬架溢出到针头的轨道上。不要移植反向 TA 并将其用作控制，注射 30 μl 的 Ca2+- 和   Mg2 +免费 PBS 来复制移植的 PBS 的条件。</li>
		<li>再用塔莫西芬治疗动物六天，再给镇痛药（如卡洛芬）服用两天。</li>
		<li>从移植后14天开始切除肌肉。按照《议定书》第 4 和第 5 部分的详细说明处理和分析样本。</li>
	</ol>
</li>
</ol>2. 肌肉萎缩小鼠模型移植
这种移植检测允许评估小鼠肌肉萎缩症模型中IDEM的移植程度。动物，如下列处理，也可以评估功能改善的疾病表型。功能测试可以从移植后两周开始进行。为了增强适应力，请考虑每三周进行一次移植前跑步机运动（如第3号议定书所述）和/或连续细胞注射3次（即总共3次注射/肌肉）。
<ol><li>肌肉内移植<ol>
<li>在移植和预处理细胞在移植日前在生长介质中加入1μM 4OH-tamoxifen（含血量配方）24小时，在腹膜内注射3.5微克/克的10毫克/毫升塔莫西芬（脂溶性制剂）进行预处理。</li>
		<li>通过尝试分化分离，在232 x g下对细胞进行计数和离心5分钟。</li>
		<li>在 Ca2+和 Mg2 +无 PBS、离心机中清洗细胞颗粒，然后在 Ca2+中重新插入颗粒 - 和 Mg2+无 PBS 中重新注入浓度为 106 细胞/30 μl。</li>
		<li>使用碘或氯西丁消毒剂对动物的皮肤进行镇痛和消毒（可选）。这也将有助于本地化 头骨前部 （TA）、 胃肠 炎（GC）和 四头肌股骨 （QC;特别是 大块的间歇性）肌肉。</li>
		<li>使用带 29 或 30 G 针的注射器将 30μl 的细胞悬浮注射到肌肉中。对于 TA，在插入近肌腱（颅道方向）下方插入5毫米的针头2毫米，相对于头骨有15°的倾向，在收回针头时慢慢注射细胞悬架（用2毫米的针头在肌肉内清空注射器）。对于 GC 和 QC，重复与 TA 详细相同的程序，主要区别是针头在 GC 跟腱肌腱的肌腱结上方 2 mm 上方的细胞插入，以及 QC 的脱毛肌腱上方 2 毫米（股骨倾斜度为 15° ）。在从肌肉中取出针头时注意，以避免将细胞悬架溢出到针的轨道上。 故障排除：在低受精的情况下，考虑注射幼鼠（1-2周大）小鼠7 与3 x 105 细胞/10 μl（注意，在这种情况下，塔莫西芬需要皮下管理）。</li>
	</ol></li>
	<li>动脉内移植 该协议的这一部分允许评估在动脉循环中交付的 MIDEM 和 HIDEM 的能力，穿过血管壁，并有助于在注射部位下游肌肉的骨骼肌肉再生。<ol>
<li>预处理动物和细胞，如上文所述，在第2.1.1步。</li>
		<li>通过尝试脱脂和过滤与40μm细胞过滤器分离（从细胞悬架中去除集群，万一一夜之间接触塔莫西芬可以驱动融合和形成肌管从相邻的细胞）计数和离心机在232 x g5分钟</li>
		<li>在 Ca2+和 Mg2 +无 PBS 中清洗细胞颗粒，然后在 Ca2+中重新插入颗粒 - 和 Mg2+无 PBS 与 0.2 国际 肝素钠（可选）单位至106 个细胞/50微克的最终细胞浓度。在溶液中加入 10% 专利蓝色染料（最终浓度：1.25 毫克/毫升普通盐水或 Ca2+- 和 Mg2+无 PBS），以便可视化细胞悬架的分布。</li>
		<li>根据特定机构动物设施外科手术指南，给小鼠进行麻醉和镇痛。</li>
		<li>剃须内分区域（也称为股骨或斯卡帕的三角形），用碘或氯西丁消毒剂对皮肤进行消毒。</li>
		<li>做一个5-7毫米的切口，并本地化股骨束：静脉，动脉和神经（神经横向躺在动脉和静脉中间）。</li>
		<li>轻轻取出用钳子覆盖捆绑包的连接筋膜。</li>
		<li>通过在股骨之间轻轻引入钳子尖（或30G针）和逐渐扩大孔，将股骨静脉和神经从动脉中分离出来。 故障排除：股骨静脉是脆弱的：注意不要捏它与钳子在其脱离股动脉。出血时，用无菌纱布排干血液，并使用微烧灼剂促进血液转移。</li>
		<li>用钳子的一尖抬起动脉，用另一个尖端夹住动脉。</li>
		<li>用装有 30 G 针的注射器刺穿动脉。在夹紧区域下游注入 50 μl 的细胞悬浮液，输液速度约为 5 μl/秒。 故障排除：在注射前小心地恢复注射器中的细胞：避免细胞沉淀和注射器内的气泡形成至关重要。股动脉的直径略小于30G针：在插入针头时小心不要截断动脉。专利蓝色染料允许识别注射的有效性：如果正确注射，整个肢体将迅速变成浅蓝色。</li>
		<li>慢慢取出针头和钳子从动脉恢复血液的肢体。</li>
		<li>使用无菌纱布施加压力，避免出血和/或根据需要烧灼。</li>
		<li>缝合伤口，监测动物，直到麻醉恢复。</li>
		<li>管理镇痛3天，每天检查伤口（在伤口感染的情况下，与动物设施人员讨论这个问题，并按要求施用抗生素）。</li>
	</ol></li>
</ol>3. 移植萎缩动物的效果测量：跑步机测试
从细胞移植后两周开始，通过跑步机测试评估治疗小鼠运动能力的功能改善是可能的。此测试允许评估经过治疗的小鼠的运动耐受性/耐力。当鼠标在休息区放置超过 5 秒时，在连续 3 次机械刺激（每 5 秒 1 次）后，不会尝试重新接触跑步机，因此被视为疲劳。基线测量大约在治疗前一个月开始，用于评估每个测试动物的改善情况。此测试之后可以进行额外的检测，以监测纤维脆弱性、力改善、移植细胞的移植、分化以及移植肌肉的形态改善（参见讨论）。
<ol><li>在第一次测量之前，将动物（通常为三组：治疗、未经治疗和野生类型/非营养不良控制）适应锻炼：将跑步机设定为6米/分钟的速度10分钟。每隔一天重复一次手术一周。 故障排除：在一天的同一时间记录所有测量结果，以避免由于昼夜周期而出现偏差。</li>
	<li>将动物放入跑步机中，跑步机以前设置的倾斜度为 10°。</li>
	<li>打开跑步机，起跑速度为6米/分钟（在动物不愿意开始运动的情况下提供温和的机械刺激）。</li>
	<li>启动计时器，每 2 分钟提高速度 2 米/分钟。</li>
	<li>一旦动物躺在休息区超过5秒，而无需尝试重新接触跑步机，用棍子轻轻触摸它，以刺激运动的重新启动（见上文）。</li>
	<li>记录每种动物的性能（时间和距离）。</li>
	<li>每周或每 10 天重复测量至少 3 次。</li>
	<li>移植后重复相同的程序。</li>
	<li>分析性能数据，比较每种动物的测量结果和基线性能。我们建议将图表中的值绘制为相对于基线的平均电机容量百分比，并通过单向或双向 ANOVA 测试进行分析，然后进行适当的测试后进行比较。</li>
</ol>故障排除：使用至少5个年龄、基因型和性别匹配的动物/组，并在移植后重复测量至少3次。
4. 移植肌肉细胞移植和分化评估
移植和控制肌肉是在适当时间点收获的（短期移植分析为<2天，中期为2-3周，长期分析为>1个月）。如果移植的细胞被贴上GFP的标签，新分离的肌肉的移植可以通过紫外线设备的立体显微镜下的直接荧光来评估。
<ol><li>沿着垂直轴铺设和定向，在特拉加昌斯牙龈中新分离的肌肉（6% w/v）</li>
	<li>将样品脱水在预制的同丙烷中一分钟，将样品冷冻在液氮中至少2分钟，并立即将其放置在-80°C以储存。</li>
	<li>用低温统计器处理样品，在极化滑梯上获取 7 μm 厚的部分。采样幻灯片上的大部分肌肉，收集约 8-10 张幻灯片，其中 30-40 节/滑梯。建议将一系列部分收集到 1.5 毫升管中，以执行分子生物学/生物化学检测。</li>
	<li>根据实验设置，评估具有不同免疫荧光染色成分的细胞移植。例如，在Sgca-null/scid/bg小鼠进行HIDEM移植的情况下，污渍部分有：a） 抗拉明A/C的抗体，以检测移植的人类细胞核：b） 抗拉米宁的抗体，可视化肌肉的整体结构：和 c） 一种抗体对 Sgca 检测捐赠者衍生的蛋白质恢复缺乏营养不良的动物。</li>
	<li>使用荧光显微镜量化每个肌肉部分的供体核数量和每节供体衍生的骨骼肌泡数量。</li>
</ol>5. 肌肉组织病理学
组织学分析允许对移植肌肉的形态结构进行评估。组织结构的结构改善有望作为细胞治疗方法的结果。
<ol><li>修复新分离的肌肉与4%的副甲醛1小时在4°C。</li>
	<li>用上升的蔗糖梯度（例如 7.5-15-30%w/v）使样品脱水。</li>
	<li>将肌肉过夜放在最高的蔗糖溶液中。</li>
	<li>将样品嵌入组织 Tek OCT 中，将其置于预切的同丙烷中，直到 OCT 变为固体（避免样品完全浸入），将样品冷冻在液氮中至少 2 分钟，并立即将其放置在 -80 °C 以进行储存。</li>
	<li>用低温统计器处理样品，获得上述 7 μm 厚的部分。</li>
	<li>根据标准制造商的协议，用血氧林和欧辛或马森的三色染色部分。</li>
</ol>血氧素和肌素染色可计算再生肌肉的标志，例如：a） 肌瘤的数量：b） 横截面区域：c） 含有中央核的肌泡数量。Masson 的三色用于计算纤维素指数，通过从图像的总面积中减去骨骼肌瘤所占用的总面积来计算：由此产生的区域主要反映肌肉的连接和脂肪渗透。图像上的所有分析都可以使用带有测量工具和单元计数器插件的 ImageJ 软件 （NIH） 执行。

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Invest. 101, 2119-2128 (1998).</li><li>Chaouch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+skin+fibroblasts+expressing+conditional+MyoD+as+a+renewable+and+reliable+source+of+converted+human+muscle+cells+to+assess+therapeutic+strategies+for+muscular+dystrophies:+validation+of+an+exon-skipping+approach+to+restore+dystrophin+in+Duchenne+muscular+dystrophy+cells.">Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: validation of an exon-skipping approach to restore dystrophin in Duchenne muscular dystrophy cells.</a> Hum. Gene Ther. 20, 784-790 (2009).</li><li>Kimura, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-lineage+regulated+myogenesis+for+dystrophin+replacement:+a+novel+therapeutic+approach+for+treatment+of+muscular+dystrophy.">Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy.</a> Hum. Mol. Genet. 17, 2507-2517 (2008).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell Stem Cell. 10, 678-684 (2012).</li><li>Wu, S. M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+potential+of+induced+pluripotent+stem+cells+for+regenerative+medicine.">Harnessing the potential of induced pluripotent stem cells for regenerative medicine.</a> Nat. Cell Biol. 13, 497-505 (2011).</li></ol>

F.S.T.已提交国际专利申请（否）PCT/GB2013/050112）包括本文中描述的战略的一部分。他得到了专业和DWC有限公司的资助，用于他的研究。

iPSC可以无限期地扩展，同时保持其自我更新的潜力，从而被引导到广泛的细胞血统12。由于这个和其他原因，iPSC衍生的干细胞/祖细胞被认为是自体基因和细胞治疗方法13的有希望的来源。作者先前发表的一篇著作报告了iPSC中小鼠和人类肌原祖先的生成，其表型与围网衍生的MAB（IDEMs）相似，并在小鼠肌肉营养不良7模型中对肌肉再生的有效贡献。
最大...

中生代细胞（MABs）是来自1-3个潜细胞子集的血管相关肌肉祖先。MAB比卫星细胞4 等规范性肌肉祖先的主要优势在于它们在动脉内传递时穿过血管壁的能力，从而有助于细胞治疗协议中的骨骼肌肉再生。这一特点已被评估和确认在肌肉萎缩症的穆林和犬模型1，5，6。这些临床前研究为基于杜琴肌肉营养不良儿童的捐赠者HLA相同MAB的动脉内移植（EudraCT 2011-000176-33;目前正在意大利米兰圣拉斐尔医院进行）的一人第一阶段I/II临床试验奠定了基础。细胞治疗方法的主要障碍之一是"药物产品"（细胞）的增殖潜力有限，需要数十亿个细胞来治疗整个身体的肌肉。此外，最近已经证明，不可能从受某些形式的肌肉营养不良症影响的患者那里获得MAB，因为它发生在肢体-腰肌营养不良2D（LGMD2D：奥米姆#608099）7.
为了克服这些限制，最近建立了一个协议，从人类和穆林iPSC中衍生出类似MAB的干细胞/祖细胞。这个过程产生容易膨胀的细胞群与血管基因特征和免疫嗜血型高度类似于成人肌肉衍生的MAB。在表达肌原调节因子MyoD时，穆林和人类IDEM（分别是MIDM和HIDEM）都会经历末期骨骼肌肉分化。为了实现这个目标，IDEM可以用能够驱动MyoD表达的载体进行转换，无论是构成还是以一种不可推断的方式8-10。使用含有肌激素受体（MyoD-ER）的扁桃体载体，从而允许其核转移后，塔莫西芬（雌激素模拟）管理11。这一策略导致形成富营养多核肌结核，具有高效率7。值得注意的是，IDEM是非肿瘤性的，移植后可以移植后在宿主肌肉内移植和分化，并且可以使用不同的载体（如 扁豆病毒或人类人工染色体）进行基因矫正，为未来的自体治疗策略铺平道路。上述出版物7中描述了IDEM的衍生、定性和移植。本协议文件详细说明了为评估IDEM的 体外 肌原分化而进行的检测以及随后的结果测量，以测试其移植在小鼠肌肉再生模型中的疗效。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>MegaCell DMEM</td>
			<td>Sigma</td>
			<td>M3942</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Sigma</td>
			<td>I3390</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Euroclone</td>
			<td>ECS0090L</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Lonza</td>
			<td>DE14801F</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Calcium/Magnesium free</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutammine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilline/Streptomicin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (Insulin-Transferrin-Selenium)</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acid solution</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Fer-In-Sol</td>
			<td>Mead Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ferlixit</td>
			<td>Aventis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid</td>
			<td>Sigma</td>
			<td>01257-10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>Sigma</td>
			<td>L5900-10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Human bFGF</td>
			<td>Gibco</td>
			<td>AA 10-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Grow factors-reduced Matrigel</td>
			<td>Becton Dickinson</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium heparin</td>
			<td>Mayne Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Patent blue dye</td>
			<td>Sigma</td>
			<td>19, 821-8</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>TAAB</td>
			<td>P001</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>4-OH Tamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td></td>
		</tr>
		<tr>
			<td>pLv-CMV-MyoD-ER(T)</td>
			<td>Addgene</td>
			<td>26809</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma</td>
			<td>C9759</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tragachant gum</td>
			<td>MP biomedicals</td>
			<td>104792</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopenthane</td>
			<td>VWR</td>
			<td>24,872,323</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>27,480,294</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarized glass slides</td>
			<td>Thermo</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4382</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Bio-Optica</td>
			<td>04-010802</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti Myosin Heavy Chain antibody</td>
			<td>DSHB</td>
			<td>MF20</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti Lamin A/C antibody</td>
			<td>Novocastra</td>
			<td>NLC-LAM-A/C</td>
			<td></td>
		</tr>
		<tr>
			<td>4/11/13</td>
			<td>Cappel</td>
			<td>559762</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma fluka</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti Laminin antibody</td>
			<td>Sigma</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>MATERIALS AND EQUIPMENT</td>
		</tr>
		<tr>
			<td>Adsorbable antibacteric suture 4-0</td>
			<td>Ethicon</td>
			<td>vcp310h</td>
			<td></td>
		</tr>
		<tr>
			<td>30G needle syringe</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>Columbus instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steromicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane unit</td>
			<td>Harvad Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optics</td>
			<td>Euromecs (Holland)</td>
			<td>EK1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Vet Tech</td>
			<td>C17A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Swann-Morton</td>
			<td>11REF050</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Fine Scientific Tools</td>
			<td></td>
			<td>5/45</td>
		</tr>
		<tr>
			<td>High temperature cauteriser</td>
			<td>Bovie Medical</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>MEDIA COMPOSITION</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Media composition is detailed below. 
			HIDEMs growth medium:
			<ul>
				<li>MegaCell Dulbecco&#39;s Modified Eagle Medium (MegaCell DMEM)</li>
				<li>5% fetal bovine serum (FBS)</li>
				<li>2 mM glutamine</li>
				<li>0.1 mM &#946;-mercaptoethanol</li>
				<li>1% non essential amino acids</li>
				<li>5 ng / ml human basic fibroblast growth factor (bFGF)</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
			</ul>
			Alternatively, if some of the above reagents are not available, HIDEMs can be grown in:
			<ul>
				<li>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</li>
				<li>10 % FBS</li>
				<li>2 mM glutamine</li>
				<li>0.1 mM &#946;-mercaptoethanol</li>
				<li>1% Non essential amino acids (NEAA)</li>
				<li>1% ITS (insulin / transferrin / selenium)</li>
				<li>5 ng / ml human bFGF</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
				<li>0.5 &#956;M oleic and linoleic acids</li>
				<li>1.5 &#956;M Fe2+ (Fer-In-Sol)</li>
				<li>0.12 &#956;M Fe3+ (Ferlixit)</li>
			</ul>
			MIDEMs growth medium:
			<ul>
				<li>DMEM</li>
				<li>20 % FBS</li>
				<li>2 mM glutamine</li>
				<li>5 ng / ml human bFGF</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
			</ul>
			Differentiation medium:
			<ul>
				<li>DMEM</li>
				<li>2 % Horse Serum (HS)</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
				<li>2 mM glutamine</li>
			</ul>
			 
			 
			</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. List of Reagents, Materials, Equipment, and Media.

transplantation of induced pluripotent stem cell-derived mesoangioblast-like myogenic progenitors in mouse models of muscle regeneration

患者衍生的iPSC可能是未来自体细胞治疗协议的宝贵细胞来源。iPSC 衍生的肌原干细胞/祖细胞类似于腹膜衍生的间质细胞（iPSC 衍生的中生代细胞（类似 IPSC 的干细胞/祖细胞：IDEMs）可以从受不同形式的肌肉营养不良患者产生的 iPSC 中建立。患者特异性IDEM可以通过不同的策略（如 扁桃体载体、人为染色体）进行基因校正，并在肌生成调节器MyoD过度表达后增强其肌原分化潜力。然后，通过特定的分化检测 在体外 评估这种致肌潜力，并通过免疫荧光进行分析。在显示急性和慢性肌肉再生的两个具有代表性的小鼠模型进行肌肉内和动脉内移植后，在 体内进一步评估IDEM的再生潜力。然后，在移植的小鼠中，不同的功能测试证实了IDEM对宿主骨骼肌肉的贡献。特别是，通过跑步机测试研究动物运动能力的改善。然后，通过对移植肌肉的一些组织学和免疫荧光检测来评估细胞的移植和分化。总的来说，本文描述了目前用于评估IDEM分化能力的检测方法和工具，重点是移植方法和后续结果措施，以分析细胞移植的功效。

报告的代表结果遵循图 1中工作流程中描述的主要体外/体外测定。48小时后4OH-塔莫西芬管理MyoD阳性核在肌电图-ER转导IDEM中可识别（图2A）。然后，细胞融合并分化成多核肌管（图2B）。当肌肉内移植到急性肌肉损伤的穆林模型中时，IDEM 有助于组织再生（图 3）。跑步机运动耐受性测试评估了IDEM在肌肉营养不良的murine模型基因和细胞治疗环...

Watch this Scientific Journal Video about Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration at JoVE.com

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

诱导多能干细胞（iPSC）衍生的肌原祖是细胞治疗策略治疗肌肉营养不良的有希望的候选人。该协议描述了评估急性和慢性肌肉再生小鼠模型中 iPSC 衍生中生肌肉细胞（一种肌肉祖先）的移植和分化所需的移植和功能测量。

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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JoVE Journal

Bioengineering

9.4K 次观看.  University College London. 诱导多能干细胞（iPSC）衍生的肌原祖是细胞治疗策略治疗肌肉营养不良的有希望的候选人。该协议描述了评估急性和慢性肌肉再生小鼠模型中 iPSC 衍生中生肌肉细胞（一种肌肉祖先）的移植和分化所需的移植和功能测量。

视频: Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

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

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

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

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

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

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

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

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

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

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

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

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

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

由人诱导多能干细胞改造的同基因肾小球芯片

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

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

从多能干细胞生成人体血管类器官

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

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...