作者承认联合国教科文组织-fcav 的犬类绝育计划为提供卵巢而提供的。这项工作得到了 fapesp (第2012号进程编号----14293-0) 和 capes 的赠款。

本实验是在犬类绝育手术中 , 用 4 个母狗的卵巢进行的。这项试验得到了教科文组织-fcav 动物使用道德委员会的批准 (第026995号议定书)。
1. 实验准备
<ol><li>准备或购买500毫升无菌的杜尔贝克的磷酸盐缓冲盐水 (dpbs), 不含钙或镁。</li>
	<li>通过将40μg 的酶混合在 dpbs 的1毫升中, 制备胶原酶 i 库存溶液。使用0.2μm 注射器过滤器进行过滤, 并在-20°c 时存储2毫升。</li>
	<li>用10% 的胎儿血清和1% 的抗生素从 dulbecco 的改良鹰培养基 (dmem) 低血糖中制备培养基。</li>
	<li>收集无菌材料: 剪刀和钳子、组织培养瓶、盘子、管道、10毫升和5毫升移液器。</li>
	<li>使用标准的细胞培养实验室设备: 生物安全柜 (bsc)、5% co 2 和38°c 的细胞培养孵化器和离心机。</li>
	<li>用戴手套的手清洁 bsc: 用 70% etoh 用紫外线灯对其进行消毒。</li>
</ol>2. 从卵巢中分离间充质干细胞
<ol><li>手术后, 将 pbs 中的卵巢放在锥形管的冰上, 直到它们到达实验室。</li>
	<li>用10毫升的 pbs 填充两个100毫米 petri 菜。</li>
	<li>从试管中取出卵巢, 并将其转移到第一个与 pbs 的培养皿中。用混合运动清洗它们。</li>
	<li>轻轻地拿回卵巢, 把它放到另一道菜里。用无菌的推拿器和剪刀, 将组织切成非常小的碎片, 大约1毫米大小。</li>
	<li>将卵巢组织转移到35毫米的培养皿中, 加入2毫升的胶原酶。把纸巾再切一点。</li>
	<li>将培养皿放在38°c 的孵化器中 3小时, 每20分钟以圆周运动轻轻摇晃培养皿。</li>
	<li>3小时后, 从孵化器中取出培养皿。</li>
	<li>将菜的内容物转移到15毫升管中。添加5毫升的扩展介质。</li>
	<li>离心前轻轻倒置管。在 2100 x g 的时间内离心管 7分钟, 取出上清液, 用3毫升膨胀介质重新悬浮颗粒。</li>
	<li>将颗粒转移到 t25 瓶中。在38°c 的情况下, 将瓶放入 5% co2 的孵化器中3小时。 注: 该程序最重要的部分是在孵育3小时后更换介质。</li>
	<li>从孵化器中取出瓶子, 取出含有剩余组织的介质。在瓶中加入3毫升新鲜的膨胀介质。</li>
	<li>每48小时更换一次介质, 观察瓶子的细胞融合。 注: 该过程的主要步骤可以在图 1中观察到。</li>
</ol>3. 卵巢间充质干细胞的扩张
<ol><li>
细胞通道
	<ol>
<li>当细胞到达汇合处时, 从瓶子中取出介质。</li>
		<li>用3毫升的 pbs 清洗瓶子。删除 pbs。</li>
		<li>加入1.5 毫升的胰蛋白酶, 在38°c 的孵化器中放入孵化器 3分钟, 轻轻轻拍瓶子, 帮助细胞分离。 注: 细胞应在初始电镀后达到大约5-7天的融合。</li>
		<li>加入3毫升的膨胀介质, 并将内容物转移到锥形管。</li>
		<li>在 2100 x g的时间内离心管 7分钟, 取出上清液, 加入1毫升的膨胀介质。</li>
		<li>轻轻均匀的管的内容。</li>
		<li>取10μl 的样本进行细胞计数, 并将细胞的管放回孵化器。</li>
		<li>将混合物的10μl 放入血细胞计中。</li>
	</ol>
</li>
	<li>
对细胞进行计数
	<ol>
<li>使用显微镜的10倍目标, 并专注于血细胞仪的网格线。</li>
		<li>将单元格数到五个小方块中 (图 2)。</li>
		<li>将计数数乘以 50, 000 以估计每毫升的细胞数。</li>
	</ol>
</li>
</ol>4. 骨髓间充质干细胞从卵巢的分化
注: 差异化检测是根据 hill 等人制定的准则进行的.9个。
<ol><li>种子 1.0 x10 4 细胞每口井, 一式三份, 使用4井培养皿。</li>
	<li>添加1毫升的扩展介质。</li>
	<li>用圆周运动轻轻搅拌盘子。</li>
	<li>24小时后, 用理想的分化媒体取代培养基。</li>
	<li>对于成骨、脂质和软骨原分化, 用诱导培养基孵育细胞, 每 3 d 更换一次, 每 3 d 一次, 每项检测都使用特定的分化试剂盒。</li>
	<li>对于神经源性谱系分化, 在诱导培养基中孵育细胞 10 d, 每 3 d 更换一次培养基。这里使用的培养基含有 dmem 低葡萄糖、2 mm 丙戊酸、1μm 氢化可的松、10μm 福斯科林、5mm 氯化钾、5μgml 胰岛素和200μm 丁基羟基烟唑。</li>
	<li>对于内胚层前体, 按照试剂制造商的说明在内皮介质中孵育5d。</li>
	<li>对于原始生殖细胞谱系分化, 在诱导培养基中孵育细胞 14d, 每 3 d 更换一次培养基。这里使用的介质含有 dmem、10% fbs、1 mm 丙酮酸钠、10 ngml lif、1 mm 非必要氨基酸, 2 mm l-谷氨酰胺, 5μg/ml 胰岛素, 0.1 mm β-硫醇, 60μm 酯烷, 20μgml 转铁蛋白, 10ngml 小鼠表皮生长因子, 1 ng/ml 人碱性成纤维细胞生长因子, 40 ngml 人胶质细胞系源性神经营养因子, 青霉素 15 mgl。</li>
</ol>

<ol>
	<li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D., Atala, A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li><li>Ahmed, N., Thompson, E. W., Quinn, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li><li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17 (2005).</li><li>Gong, S. P., 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=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li><li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145 (2004).</li><li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li><li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391 (2017).</li><li>Dominici, M. L. B. K., 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li><li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li><li>Kuznetsov, S. A., 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=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li></ol>

作者没有什么可透露的。

在此, 我们提供的证据表明, 骨髓间充质干细胞可以从犬卵巢组织中分离出来, 这被认为是卵巢切除术后的生物废物。由于在卵巢中可以找到许多细胞类型, 我们提出了一种协议, 选择骨髓间充质干细胞的基础上, 他们的快速坚持塑料, 成功地选择细胞生长在单层生长与成纤维细胞样的形态。
第一次从骨髓中提取骨髓间充质干细胞的报告是根据骨髓间充质干细胞在培...

在过去的十年里, 以干细胞为重点的已发表的研究数量大幅增加, 这一研究工作是由发现强大的再生医学疗法的集体目标推动的。干细胞有两个主要的定义标记: 自我更新和分化。与胚胎干细胞1相比, 间充质干细胞负责正常的组织周转, 分化能力更加有限.最近, 许多研究表明骨髓间充质干细胞有广泛的分化, 目前正在讨论的一个话题是胚胎干细胞和成人干细胞之间的差异是否存在。
卵巢表面上皮是未承诺的细胞层, 分化相对较少, 表达上皮和间充质标记 3, 保留分化为不同类型细胞的能力.环境信号4。干细胞在卵巢中的确切位置尚不清楚;然而, 有人提出, 在中的生物祖细胞会产生生殖细胞5。免疫学研究假设这些细胞的基质起源为 6或位于或接近卵巢表面7。由于间充质干细胞表达了在细胞粘附中发挥重要作用的众多受体, 因此设计了一项实验来检验选择具有快速粘附的细胞群可以清楚地分离出大量细胞的假设在本质上可以被描述为间充质。最近, 我们的小组报告了 mscs 的来源, 基于他们的粘附能力的培养前3小时培养培养培养皿的塑料表面, 以获得纯化的细胞群表现出快速粘附 9.在这里, 我们描述了从卵巢组织中分离间充质干细胞的方法。

<table><tbody><tr><td>DPBS&amp;nbsp;</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Collagenase I</td><td>Thermo Fisher</td><td>17100017</td><td></td></tr><tr><td>Tissue flask</td><td>Corning</td><td>CLS3056</td><td></td></tr><tr><td>DMEM low glucose</td><td>Thermo Fisher</td><td>11054020</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher</td><td>12484-010</td><td></td></tr><tr><td>TrypLE express</td><td>Thermo Fisher</td><td>12604021</td><td></td></tr><tr><td>StemPro Adipogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007001</td><td></td></tr><tr><td>StemPro Chondrogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007101</td><td></td></tr><tr><td>StemPro Osteogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007201</td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Kit</td><td>StemCell</td><td>5110</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>CD45</td><td>AbD Serotec</td><td>MCA 2035S</td><td></td></tr><tr><td>CD34</td><td>AbD Serotec</td><td>MCA 2411GA</td><td></td></tr><tr><td>CD90</td><td>AbD Serotec</td><td>MCA 1036G</td><td></td></tr><tr><td>CD44</td><td>AbD Serotec</td><td>MCA 1041</td><td></td></tr><tr><td>Nestin</td><td>Milipore</td><td>MAB353</td><td></td></tr><tr><td>&amp;beta;-Tubulin&amp;nbsp;</td><td>Milipore</td><td>MAB1637</td><td></td></tr><tr><td>DDX4</td><td>Invitrogen</td><td>PA5 -23378</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR80F</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR120F</td><td></td></tr></tbody></table>

derivation and differentiation of canine ovarian mesenchymal stem cells

在过去的十年里, 人们对间充质干细胞 (mscs) 的兴趣增加了, 因为它们易于分离、扩张和培养。最近, 研究表明, 这些细胞具有广泛的分化能力。卵巢是细胞疗法的一个有希望的候选, 因为它富含骨髓间充质干细胞, 并经常在卵巢切除术后作为生物废物丢弃。本文介绍了从犬卵巢中提取的骨髓间充质干细胞的分离、扩展和分化过程, 而无需细胞分选技术。该方案是再生医学的重要工具, 因为这些高度可微化的细胞在临床试验和治疗用途中具有广泛的适用性。

犬卵巢间充质干细胞分离:
图 1总结了卵巢 msc 隔离过程。手术后, 组织切碎, 胶原酶消化, 和培养基改变3小时后的培养, 假定 msc 群体具有快速的塑料粘附性能, 成功地从犬卵巢组织中分离出来。收获的细胞迅速粘附在培养皿的塑料表面, 并成长为形态均匀的单层培养, 具有典型的 ms...

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

在此, 我们描述了一种从犬卵巢组织中分离、扩张和分化间充质干细胞的方法。

犬卵巢间充质干细胞的提取与分化

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

该程序的总体目标是分离，扩大和区分从犬卵巢组织获得的中质茎销售。这个实验是用犬类绝育计划选择性手术后捐赠的四只母狗的卵巢进行的。这项实验得到了圣保罗州立大学动物使用伦理委员会的批准。代表性结果。讨论. 结论.

derivation-differentiation-canine-ovarian-mesenchymal-stem

Research

JoVE Journal

Developmental Biology

5.6K 次观看.  São Paulo State University (UNESP). 该程序的总体目标是分离，扩大和区分从犬卵巢组织获得的中质茎销售。这个实验是用犬类绝育计划选择性手术后捐赠的四只母狗的卵巢进行的。这项实验得到了圣保罗州立大学动物使用伦理委员会的批准。代表性结果。讨论. 结论.

视频: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

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

科研

发育生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

生物学

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

癌症研究

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

医学

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...