这项工作是由美国国立卫生研究院DK095137（THC到）的支持。我们感谢现任和前任实验室成员是谁所描述的方法的发展和成熟作出了贡献。

伦理学声明:密歇根委员会对动物的使用和注意事项大学（UCUCA）已批准按照指南实验动物的护理和使用（学院实验动物研究，国家研究理事会）的所有方法和协议。小鼠均保持在密歇根大学的动物饲养，并给予食物和水自由进入并保持在12小时黑暗/光照周期。 1.准备<ol><li>制备原代培养介质用DMEM，10％FBS，1×P / S / G，和1x抗生素/抗真菌剂。这将被用来保持组织中之前消化。将股票和分装在37ºC水浴。 </li><li>制备的III型胶原酶溶液5毫升在5毫克/毫升溶于HBSS（+钙+镁）为每种类型的脂肪垫的被隔离，最多5只小鼠。例如，从一个单一的基因型分离SQ和可见光时，使10毫升，如果野生型和突变型的SQ和可见光，使20毫升调节pH值至7.4，STE激怒过滤器。分装成50ml试管。从光一边远位置。 </li><li>使用70％的乙醇消毒手术区域。擦拭，并用蓝色的垫覆盖。喷上蓝色垫些乙醇。 </li><li>使用22克针牵制的吸水垫的保丽龙板，并用剪刀修剪。喷雾用乙醇垫然后设置蓝色垫板并与另一个蓝色垫，直到开始清扫覆盖。 </li><li>填充两个50ml试管用乙醇和发生在邻近于外科领域的立场。放置在乙醇剪刀和镊子。 </li><li>填另一50ml管中，用PBS加1×防反冲洗乙醇关闭手术工具。 </li><li>在油烟机，标签60毫米菜肴每个组织类型，并填写与文化传媒加热到37ºC。放置邻近于手术区板。 </li></ol> 2.皮下（SQ）脂肪垫的分离<ol><li>安乐死鼠标异氟醚和气胸过量。 </li><li>平缓鼠标喷用70％乙醇打下平卧。针脚的爪子泡沫板与22克针。 </li><li>使下腹部皮肤的一小截。拿着切的顶部，镊子，拿剪刀和皮肤腹膜分开。 </li><li>一旦皮肤是从腹部到胸部的腹膜分离，通过在沿所述主体的一侧的皮肤侧向切口反映皮肤从朝向头部腹膜程。 </li><li>反映剩余的皮肤保持腹股沟的脂肪远离身体和牵制到板22克针。 </li><li>切换到精细剪刀和镊子。抓住用钳子腹股沟脂肪垫在腹膜附近的起源和剪断掉皮肤和脂肪腹股沟向腹股沟避免污染皮肤的SQ进展之间。 </li><li>放置曝晒腹股沟脂肪垫在标有60mm培养皿。 </li></ol> 3.内脏（VIS）脂肪垫的分离<ol><li>以类似的方式执行步骤2.5，切开腹膜打开以暴露内脏脂肪垫和肠道。 </li><li>移动肠道远朝胸部。 </li><li>抓住VIS脂肪垫在远端，轻轻往上拉。解剖出VIS脂肪垫，同时注意排除附睾（或子宫，如果使用雌性小鼠）组织。 </li><li>发生在标盘，并重复步骤3.3为剩余的VIS垫。 </li></ol> 4.脂肪垫的胶原酶消化<ol><li>移动60毫米菜的组织培养罩，吸了媒体。 </li><li>剁碎脂肪垫用弯剪，然后添加到胶原酶溶液。请务必用乙醇和PBS冲洗剪刀和镊子组织类型之间。 </li><li>在300rpm和37ºC10-20分钟摇动直至组织被消化。如果SQ的一些作品仍然可见这是可以接受的。这将在以后的步骤来解决。 </li><li> 25毫升培养基添加到胶原酶溶液以停止collagenase治疗和吸管上下10次，10毫升血清吸管驱散消化的组织。 </li><li>使用100微米的细胞滤网株细胞。离心机在300×g下10分钟。 </li><li>小心倒出了媒体不要打扰颗粒和5毫升无菌水添加到颗粒，轻轻吸暂停细胞。等待2分钟，以溶解红细胞。 </li><li>通过一个新的100微米的细胞滤网加入25毫升含10％FBS和应变细胞介质。 </li><li>离心机在300×g下10分钟。倒出培养基，并在1ml培养基重悬沉淀。 </li><li>计数使用1血球细胞:1台盼蓝。 </li><li>板1×10 6个细胞在一个阱上的6孔板中。 </li></ol> 5.磁性细胞分离<ol><li>约4至6小时细胞粘附后，冲洗细胞两次用HBSS（-Ca，-Mg）。分离使用0.05％胰蛋白酶粘附细胞。稀释胰蛋白酶细胞悬浮于1ml分离缓冲液和自旋为5分钟，在300×g的。 </li><li>吸上清。悬浮细胞于500μl缓冲液中。删除10微升等分试样并使用血球计数细胞。 </li><li>自旋细胞在300×g下5分钟。 </li><li>吸完全上清。悬浮细胞在90微升缓冲液中。添加10微升抗SCA1-FITC初级抗体。拌匀并在黑暗中在4ºC孵育10分钟。 </li><li>洗涤细胞用1毫升缓冲液中，离心细胞，在300×g下5分钟。完全取出上清液。 </li><li>悬浮细胞在80μl的缓冲液中。加入20微升抗FITC微珠。拌匀并在黑暗中在4ºC孵育15分钟。 </li><li>洗涤细胞用1毫升缓冲液中，离心细胞，在300×g下5分钟。 </li><li>在500微升缓冲液中取出上清，重新悬浮颗粒。 </li><li>放置列在磁性支架并用500μl缓冲液冲洗柱。 </li><li>细胞悬液添加到列。避免气泡而吹打。 </li><li>收集未标记SCA1 - &lt;/ SUP&gt;细胞并用500μl的缓冲液洗涤柱3次。只有增加新的缓冲区时，水库是空的。避免气泡而吹打。 </li><li>从磁性支架上取下柱并放入15毫升锥形管中。 </li><li>加入1ml通过柱水库推柱塞提供缓冲柱和洗脱SCA1 +。 </li><li>降速细胞组分在300×克5分钟。重悬细胞于1ml培养基中。 </li><li>除去来自标记的和未标记的级分10微升等分试样并使用血球计数细胞。 </li><li>板1以及一个6孔板的每一个单元级分。 </li></ol> 6.流式细胞仪SCA1 高 ACSs中的免疫磁珠分离的验证<ol><li>冲洗细胞两次用HBSS（-Ca，-Mg），并解离用0.05％胰蛋白酶的细胞。 </li><li>离心细胞，在300×g离心5分钟。重悬细胞于1ml培养基和计数使用血球。 </li><li>获得"10 6 </SUP&gt;细胞于1ml培养基，并在300×g离心5分钟离心。 </li><li>除去上清液，并重复步骤6.3两次。 </li><li>悬浮细胞用1毫升2％山羊血清的+ 2％BSA中并在室温30分钟块。 </li><li>离心细胞，在300×g离心5分钟。 </li><li>除去封闭液。 </li><li>添加大鼠IgG2a的Alexa Fluor 647（0.25微克，1:400）或抗SCA1的Alexa Fluor 647（0.25微克，1:400），在100μlPBS中，用2％山羊血清和2％BSA + PBS在4℃下在黑暗中30分钟。 </li><li> 1毫升冷PBS加入细胞。离心机300 xg离心在4℃下5分钟。 </li><li>除去上清液，并重复步骤6.9两次。 </li><li>重悬细胞于1ml PBS中。通过100微米的细胞过滤细胞悬浮液中进行流式细胞术分析制剂。 </li></ol>

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作者什么都没有透露。

在此，我们展示了隔离，并从不同的脂肪垫鼠携带者的免疫细胞分离和体外实验。所提出的方法是有效的的大量SCA1阳性的ASC的，这是过度的ASC 9,14的技术复杂和昂贵的FACS介导的隔离有利的快速隔离。不像FACS，免疫细胞分离不允许靶细胞群体的鉴定使用多个抗原。然而，如果表面抗原表征良好，利用免疫磁性分离的增加不依赖于使用的FACS设备15，其仍然不小院所许多生物学?...

干细胞抗原-1（SCA1，或Ly6A / E）最初被鉴定为通过造血细胞和间质干细胞5,6-表达的细胞表面标记。从小鼠脂肪库获得脂肪组织的基质血管级分（SVF）为含有成纤维细胞，巨噬细胞，血管内皮细胞，神经细胞，以及脂肪细胞祖细胞7的细胞的异质群体。脂肪细胞祖细胞，或脂肪干细胞（ASCs）是驻留在富含胶原蛋白的血管周围的细胞外基质（ECM）8非载脂细胞。大约SVF的50％由携带者，其特点为谱系阴性（林- ）和CD29 +:CD34 +:SCA1 + 9。大多数这些细胞是SCA1 +:CD24 -脂肪细胞祖细胞，其能够在体外脂肪细胞分化的;然而，只有细胞的级分（SVF的0.08％）构成SCA1 <sUP&gt; +:CD24 +细胞完全有能力和增殖分化为体内条件下9脂肪细胞。尽管使用SCA1 + SVF不脱离CD24识别CD24 +细胞的电位警告-的细胞，分离SCA1 +的ASC从用免疫细胞分离脂肪库是一种高效，实用的方法，以确定主脂肪细胞祖细胞的细胞自主表型。 在肥胖和糖尿病，组织纤维化和炎症的领域发挥在2型糖尿病3的开发和维护的关键作用。最近，德永等人 。表明，腹股沟（或皮下，SQ）和perigonadal（或内脏，VIS）隔离SCA1 高细胞C57BL6 / J脂肪库表现出体外 10个不同的基因签名和ECM重塑。 MMP14（MT1-MMP），膜 - t的典型构件YPE基质金属蛋白酶（MMP）家族通过其原活性1介导的白色脂肪组织（WAT）的开发。 可与分离和富集通过以下方案中的细胞进行的实验的例子包括三维培养，分化研究，胶原降解测定法，和RNA测序10,11。降解测定法应与酸提取胶原进行，以确保端肽11,12的保存。下面的协议将演示方法主要血管基质细胞从不同的脂肪库分离和使用免疫细胞分离脂肪细胞丰富的祖细胞。在细胞分选的有效性将用流式细胞仪，并通过使用SCA1-GFP小鼠表达SCA1 +细胞GFP，由SCA1启动子驱动13进行评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Type 3 Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>LS004182</td>
			<td>Tissue digestion</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>High-glucose culture medium</td>
		</tr>
		<tr>
			<td>Pen/Strep/Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td>Media antibiotic</td>
		</tr>
		<tr>
			<td>Anti-anti (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>Media antifungal</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x, pH 7.4)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.05%)</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Bioscience</td>
			<td>352360</td>
			<td>100-&#956;m cell strainer</td>
		</tr>
		<tr>
			<td>60mm plates</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14001-12</td>
			<td>Large</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14091-11</td>
			<td>Fine, curved tip</td>
		</tr>
		<tr>
			<td>Large Forceps</td>
			<td>FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G 5/8&#8221; needles</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>22G 1.5&#8221; needles</td>
			<td>BD</td>
			<td>305159</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Anti-Sca1 microbead kit (FITC)</td>
			<td rowspan="3">Miltenyi Biotec</td>
			<td rowspan="3">130-092-529</td>
			<td rowspan="3">FITC-anti-Sca1 1&#186;Ab and anti-FITC microbeads 2&#186;Ab</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>AutoMACS running buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue chux pads</td>
			<td>Fisher</td>
			<td>276-12424</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent pads</td>
			<td>Fisher</td>
			<td>19-165-621</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam board</td>
			<td></td>
			<td></td>
			<td>Use from 50ml tubes</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>R2a21</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>MSCA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a R-PE</td>
			<td>Invitrogen</td>
			<td>R2a04</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a anti-mouse F4/80 R-PE</td>
			<td>Invitrogen</td>
			<td>MF48004</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom tube</td>
			<td>BD Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (&#8211;Ca, &#8211;Mg)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca, +Mg)</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>For collagenase solution</td>
		</tr>
		<tr>
			<td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td>
			<td>Prepare in house12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Gibco</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>0.34N NaOH</td>
			<td>Prepare in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Corning</td>
			<td>2870-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>0.89M NaHCO3&#160;</td>
			<td>Gibco</td>
			<td>25080-094</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

从不同的脂肪垫SCA1 高的ASC的富集。 从SQ脂肪显示成纤维样分离出来的血管间质细胞，拉伸细胞形态，无论SCA1表达水平（ 图1A）。另一方面，VIS（eWAT衍生）SCA1 高和SCA1 低细胞证明其细胞形态明显的差异。像SQ（iWAT衍生）SCA1 高细胞，VIS（eWAT衍生）SCA1 高细胞显示拉伸，成?...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

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 ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Research

JoVE Journal

Developmental Biology

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

6.8K 次观看.  Biointerfaces Institute, University of Michigan. 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.

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

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

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. ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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 ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

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

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

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

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

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

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

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 ...