Name: derivation and differentiation of canine ovarian mesenchymal stem cells
Uploaded: 2018-12-16T13:00:00
Description: Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

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?...

<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>

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。

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			<td height="21" style="height:21px;width:221px;">DPBS&#160;</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">14190144</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Collagenase I</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">17100017</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tissue flask</td>
			<td style="width:151px;">Corning</td>
			<td style="width:123px;">CLS3056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DMEM low glucose</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">11054020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FBS</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12484-010</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:221px;">TrypLE express</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12604021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Adipogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Chondrogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Osteogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">STEMdiff Definitive Endoderm Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:123px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Penicillin-Streptomycin</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD45</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2035S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD34</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2411GA</td>
			<td></td>
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			<td height="21" style="height:21px;width:221px;">CD90</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1036G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD44</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1041</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Nestin</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">&#946;-Tubulin&#160;</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB1637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DDX4</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:123px;">PA5 -23378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR80F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR120F</td>
			<td></td>
		</tr>
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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.

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