本研究由上海市科技委员会 (CN) 赞助的上海创新基金 (1402H294300) 资助, Dr.。感谢 Dr. 广东周 (中国国家组织工程中心) 对本论文的技术支持和科学建议。我们还要感谢 Mr. 矿泉夏 (上海第九人民医院) 在动物福利方面的帮助。

涉及动物的程序由当地机构动物保育和伦理委员会批准, 以尽量减少动物的痛苦。本协议由上海市交通大学医学院附属机构动物护理与使用委员会 (IACUC) 批准, 并与本协议编号 [2017] 063. 
 1. 手术诱发大鼠膝骨关节炎模型的建立 <ol> <li> 为此外科手术, 使用 8-12 周老雄性大 (SD) 大鼠, 体重介于250和300克之间。
	<li> 麻醉40毫克/千克 tiletamine 和40毫克/千克唑通过肌肉注射的动物. </li>
	<li> 将动物置于处于加热阶段的左侧面位置。用剃刀把右膝彻底剃掉。用 10% providone 碘溶液和70% 乙醇进行消毒, 再重复两次, 再涂上 10% providone 碘溶液, 用手术垫覆盖非手术区. </li>
	<li> 使用外科手术刀, 沿髌腱侧向2厘米切口以暴露 jointcapsules. </li>
	<li> 使用外科手术刀将内侧副韧带横断面。反映股骨半月板. </li>
	<li> 在手术过程中, 在关节软骨表面滴入无菌0.9% 氯化钠溶液, 防止软骨干燥. </li>
	<li> 用镊子抓住内侧半月板, 用手术刀从胫骨附件的最窄点切开半月板 ( 图 1A )。避免损伤软骨. </li>
	<li> 用 4-0 的可吸收缝合线关闭关节囊. </li>
	<li> 监控手术鼠, 直到他们恢复知觉. </li>
	<li> 注射20毫克/千克庆大霉素, 以防止感染和0.05 毫克/千克丁丙诺啡通过肌肉注射的途径, 以减轻疼痛后手术. </li>
	<li> 在一个温度控制的动物设施中, 在12小时的光照/暗循环下维持 3-8 周的大鼠 KOA。动物继续接受止痛药, 包括但不限于丁丙诺啡 (0.05 毫克/千克, 贝聿铭) 和庆大霉素 (20 毫克/千克, 贝聿铭), 如果疼痛症状持续. </li>
</ol> 2。大鼠 KOA 模型的组织学评价 <ol> <li> 牺牲过量 CO 的大鼠 2 吸入 (在腔室中增加每分钟容积约30% 的填充率与二氧化碳到现有空气中, 使动物失去知觉在呼吸停止后2-3 分钟内维持 CO2 流至少1分钟。在指定的时间点 (代表性的结果显示在8周的 KOA 诱导范围从3到8周) 后, 手术. 和 #160; </li> <li> 解剖膝盖关节周围的皮肤和肌肉, 用手术刀切断膝关节. </li>
	<li> 将4% 甲醛 (粉煤灰) 的膝关节固定在室温下3天。然后, decalcify 20% 乙酸乙酸 (edta) 中的样品, 在4和 #176 ° C 下进行约4周, 每3天更换一次 edta. 
	警告: 甲醛是有毒的, 穿戴适当的个人防护设备 (PPE), 如手套和眼镜. </li>
	<li> 使用自动组织处理器脱水样品。设置菜单开始一个冲洗周期如下: 70% 乙醇为 1 h;80% 乙醇和95% 乙醇分别为 1 h;其次2循环100% 乙醇为 1 h;随后, 二甲苯每次两次1小时, 石蜡3次在58和 #176; C 每次1小时. 
	注意: 二甲苯是有毒的, 所以佩戴适当的 PPE, 如手套和眼镜. 
	注: (可选) 使用 Coplin 罐进行手动脱水, 其设置与自动组织处理器相同. </li>
	<li> 将完整的关节面的内侧部分向下放置, 并将其嵌入到中心的石蜡块中. 
	注: 可选择使用镊子手动将样品嵌入石蜡块中. </li>
	<li> 开始切割5和 #181; 从关节内侧缘的矢状段到30和 #181; m 间隔, 由旋转切片2个连续部分. </li>
	<li> 在玻璃幻灯片上安装剖面并 deparaffinize 二甲苯中的幻灯片。在2个连续部分之间, 染色一张幻灯片与苏木精和 #38; 曙红 (H 和 #38; E) 染色: 0.1% 苏木精为 15 min, 1% 乙酸溶液为十年代和0.5% 次曙红为 10 min。和染色的其他幻灯片与红 o/快速绿色染色: 0.1% 苏木精为15分钟, 0.02% 快速绿色为3分钟, 1% 醋酸溶液为十年代和0.1% 红 O 为 3 min. 
	注意: 二甲苯有毒, 佩戴适当的 PPE, 如手套、眼镜. </li>
	<li> 使用骨关节炎研究协会国际 (OARSI) 大鼠组织病理学评估系统对彩色幻灯片进行评分 6 . </li>
</ol> 3。人骨髓间充质干细胞与荧光染料的标签做了 <ol> <li> 准备在100% 乙醇浓度为1毫米的细胞标记溶液。保护细胞标签的解决方案从光和存储在室温下6月. </li>
	<li> 在 Dulbecco 和 #39 中使用标准的单元格培养程序扩展 haMSCs; s 修饰的鹰和 #39; s 培养基 (DMEM), 辅以1% 青霉素和链霉素和10% 胎小牛血清 (FCS) 在10厘米的 petri 皿中. </li>
	<li> 分离 haMSCs 与2毫升0.25% 胰蛋白酶与1毫米 EDTA 在大约80% 汇合之前. </li>
	<li> 中和胰蛋白酶与4毫升培养基和旋转的细胞在 800 x g 在室温下3分钟. </li>
	<li> 在5毫升无血清培养基中, 以 1 x 10 6 细胞/毫升的密度将细胞挂起. </li>
	<li> 通过添加50和 #181 对单元格进行标记; 我在37和 #176 的5毫升无血清培养基中做了细胞标记液; C 为 50 min. </li>
	<li> 将标记的单元格在 800 x g 处旋转3分钟. </li>
	<li> 用5毫升磷酸酯缓冲盐水 (PBS) 在 800 x g 上再清洗两次电池, 每次3分钟. </li>
	<li> 使用 Cy5 过滤器设置的荧光显微镜检查标签 haMSCs. </li>
	<li> 将台盼蓝染色的单元格计数并获得 2.5 x 10 6 100 和 #181 中的活细胞; L PBS 注射. </li>
</ol> 4。 在体内 荧光成像跟踪 haMSCs <ol> <li> 手术后八周 (步骤 1), 麻醉 KOA 鼠肌肉注射25毫克/千克 tiletamine 和25毫克/千克唑. </li>
	<li> 用剃刀彻底剃掉右膝, 露出关节区域。用70% 乙醇对关节区域进行消毒. </li>
	<li> 使用 26 G 注射器针, 注入 2.5 x 10 6 标记的单元格在100和 #181 中挂起; L PBS 位于由髌韧带内侧侧、股骨内侧髁和胫骨内侧髁形成的三角形区域中心 ( 图 1B ). </li>
	<li> 打开图像软件并初始化 在体内的 生物发光成像系统. </li>
	<li> 将大鼠置于成像系统中央阶段的仰卧位, 并关闭室的门. </li>
	<li> 检查 #34; 荧光 #34; 复选框, 然后选择 640 nm 的荧光滤光片进行励磁, 680 nm 发射 ( 图 2A ). </li>
	<li> 选择视野到 d. 设置像素分到中 (8), F/停止到2和曝光时间自动。保持最佳在整个研究中获得可比较结果的曝光时间 ( 图 2A ). </li>
	<li> 将动物从舞台上移除, 并从麻醉中恢复. 
	注意: 将动物放在覆盖着2-3 层毛巾的加热垫上. </li>
	<li> 选择荧光测量的辐射效率单位 ( 图 2B )。选择感兴趣的区域 (ROI) 分析和量化每个图像的荧光信号 ( 图 2C ). </li>
	<li> 重复此过程 在体内 依次对每只老鼠进行成像, 以研究 haMSCs 在关节中的纵向保留. 
	注: 和 #160; 我们建议成像动物和 #39; 荧光信号的活体成像系统每两天一次注射后第一周和每周一次, 直到信号无法检测. </li>
</ol>

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孟里、明浩和文王是目前的员工和细胞生物医学组的股票期权持有者 (纳斯达克: CBMG)。其他作者声明没有利益冲突。

我们迫切需要干细胞治疗的安全标准和分布研究, 才能将再生干细胞治疗 KOA 从长凳到床边。然而, 疾病的病理环境在移植 haMSCs 的持续和分布中起着重要作用10。最近, 我们的小组表明, 关节内注射 haMSCs 在病理 KOA 环境中的持续时间比正常情况下注射在8。炎症和退行性微环境可能会招募骨髓间充质干细胞进行修复, 随后有利于保留注入的 haMSCs1<s...

膝关节骨关节炎 (KOA) 是一种退行性疾病, 由关节软骨丧失和渐进炎症, 这已成为一个主要的慢性病在世界各地的老年人1。然而, 目前的治疗使用抗炎药物, 物理补充剂和手术程序可能只提供暂时缓解症状性疼痛2。
人脂肪源性间充质干细胞 (haMSCs) 已成为一种有希望的再生治疗膝关节骨性关节炎, 由于其多能分化潜能的软骨再生和免疫调节特性3, 4。与药理学途径, 以研究的作用机制的行为在体内, 跟踪活 haMSCs 在小 KOA 动物模型目前是有益的建立的理由和可行性 haMSC 治疗前的临床应用。在临床前测试中, 内侧切除 (MM) 破坏关节的机械负荷诱导大鼠 KOA, 提供了一个相对可行的模型, 具有一致的重现性5。KOA 诱发的早于前十字韧带横断, 或与部分内侧切除6相结合。因此, 在由 MM7、8引起的大鼠中, 通常会对注射 haMSCs 与 KOA 的病理微环境之间的长期相互作用进行评估。
虽然 haMSCs 的治疗效果已被广泛报道, 有关知识的体内持续的植入 haMSCs 通过关节内 (IA) 注射是稀缺的9,10。因此, 已经开发了各种细胞标记方法, 包括 immunohistology11、荧光12、绿色荧光蛋白13转染、氧化铁标记为磁共振成像 (MRI)14和众多荧光细胞染料8、15、16。与 labor-intensive 组织学分析相比,在体内非侵入性成像采用光学器件来检测标记有荧光信号的细胞的 real-time 分布和动态,1017。对于功能性活细胞成像, cytocompatible 荧光标记是一种先进的无放射性跟踪技术, 以揭示细胞活动后, 干细胞移植18。此外, 多色荧光亲油性染料具有相对于氨基活性亲水染料或荧光蛋白的优势, 包括其改进的细胞通透性和增强的荧光量子产率19。
因此, 这里所包含的协议使用红色激光激发带有亲油性 carbocyanines 的细胞 (DilC18(5)), 这是一个远红色荧光 Dil (dialkylcarbocyanines) 模拟20。红移激发和发射光谱的确有避免 autofluorescent 干扰, 并允许深层成像在很长一段时间的活动物8。这种跟踪细胞在体内的方法是有效的监测移植的干细胞, 如 haMSCs, 在动物模型, 这是必不可少的了解和改善现有的干细胞再生疗法。

<table>
	<tbody>
		<tr>
			<td>Matrx VMR animal anesthesia system</td>
			<td>Midmark</td>
			<td>VIP3000</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture</td>
			<td>Shanghai Jinhuan</td>
			<td>KC439</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Pritech</td>
			<td>LD-9987</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium chloride solution</td>
			<td>Hunan Kelun Pharmaceutical Co., Ltd.</td>
			<td>H43020455</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td>Dilute to final concentration of 10% in PBS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>Dilute to final concentration of 20% in PBS</td>
		</tr>
		<tr>
			<td>0.1% Hematoxylin Solution, Mayer&#8217;s</td>
			<td>Sigma-Aldrich</td>
			<td>MHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Eosin Y solution, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma-Aldrich</td>
			<td>S8884</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Excelsior ESTM Tissue Processor</td>
			<td>Thermo Fisher</td>
			<td>A78400006</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Histocentre&#8482; 3 Tissue Embedding Center</td>
			<td>Thermo Fisher</td>
			<td>B64100010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully Automated Rotary Microtome</td>
			<td>Leica</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>Molecular Probes, Life 
			Technologies</td>
			<td>V-22887</td>
			<td></td>
		</tr>
		<tr>
			<td>D-MEM High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO, Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Corning</td>
			<td>V118877</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>Optima MAX-TL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr>
			<td>Titetamme</td>
			<td>Virbac (Zoletil 50)</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>Zolazepam</td>
			<td>Virbac (Zoletil 50)</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile hyposermic syringe for single use 26G</td>
			<td>Shanghai Misawa Medical Industry</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging 4.0 software</td>
			<td>PerkinElmer</td>
			<td>None</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye

为了支持人脂肪源性间充质干细胞 (haMSC) 治疗膝骨关节炎 (KOA) 的临床应用, 我们研究了 haMSCs 在动物模型中的细胞持久性和分布的有效性。我们展示了一种用亲油性荧光染料标记 haMSCs 细胞膜的方法。随后, 由体内成像系统动态监测经手术诱导的 KOA 大鼠的关节内注射标记细胞。我们雇用的亲油性 carbocyanines 做了 (DilC18 (5)), 一个远红色荧光 Dil (dialkylcarbocyanines) 模拟, 它利用红色激光, 以避免激发自然绿色自发荧光从周围的组织。此外, 红移发射光谱的确有允许深层成像在活体动物和标记程序没有造成细胞毒作用或功能损害 haMSCs。这种方法已被证明是一种有效的跟踪方法的 haMSCs 在大鼠 KOA 模型。该方法的应用也可用于确定临床前研究中其他来源的骨髓间充质干细胞的最佳用药路线和剂量。

为了诱导 KOA 模型, MM 在 SD 大鼠的右膝关节中进行 (图 3)。术后八周, 对大鼠进行了牺牲, 并用 H 和 #38 对膝关节的连续段进行了评价; E 和红 O/快速绿色染色 (图 4)。对于 H 和 #38; E 染色, 关节软骨的表面在手术膝部比正常关节表现出更粗糙的边界而不进行手术。对于红/快速绿色染色, 我们观察到在手术关节中, 与正常对照组相比, ?...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

该协议描述了一种有效的方法来监测人脂肪来源的骨髓间充质干细胞 (haMSCs) 的细胞持久性和分布的远红色荧光标记的大鼠膝骨关节炎 (KOA) 模型通过关节内 (IA) 注射。

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

Research

JoVE Journal

Developmental Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

8.9K 次观看.  Cellular Biomedicine Group, China. 该协议描述了一种有效的方法来监测人脂肪来源的骨髓间充质干细胞 (haMSCs) 的细胞持久性和分布的远红色荧光标记的大鼠膝骨关节炎 (KOA) 模型通过关节内 (IA) 注射。

视频: In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

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 ...

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

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

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

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

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 ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...