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>

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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养量 11的前48小时内的粘附能力编写的;然而, 据报道, 这种方法有可能分离异质种群, 表型和功能, 至少对于骨髓12.该培养方法还通过塑料粘附能力选择细胞, 并从具有多种细胞类型的组织中分离出间充质细胞, 而无需对理想的细胞进行分类。由于间充质干细胞表达了许多受体, 在细胞粘附中发挥着重要作用, 因此假设如果选择了具有快速粘附的细胞群, 就会导致细胞在细胞粘附中更加均匀。第一个段落, 具有可靠的 msc 配置文件。有代表性的结果提供了证据, 证明一个较短的持续时间的塑料粘附检测可以用来分离形态均匀的人群的骨髓间充质干细胞在第一段, 而无需等待更先进的通道或细胞分类。
此协议中的一个关键步骤是在区域性开始3小时后更改媒体。如代表性结果部分所示, 从犬卵巢中获得的干细胞符合为骨髓间充质干细胞的表征而制定的三个标准: 它们表现出类似于纤维细胞的形态, 对 msc 标记物的存在呈阳性。能够分化为成骨、脂肪生成和软骨生等间充质谱系。本实验中分离出的细胞也成功地分化为内皮前体、外胚层和原始生殖细胞谱系。细胞应在最初电镀后5-7天左右达到融合。来自老年动物的细胞生长速度可能比幼小动物慢, 其中, 粘附在塑料上的细胞较少。在这些情况下, fbs 在介质中的浓度可以增加到15%。
因此, 根据具有更快速粘附能力的细胞选择骨髓间充质干细胞是一种廉价、简化和有效的过程。从治疗的角度来看, 最重要的是要强调, 这些细胞是再生医学的一个有希望的工具, 特别是因为它们的分化范围很广。

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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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
	</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 msc 样形态 (图 3)。
卵巢 msc 的表征在体外
msc 表征的目标是确保分离的细胞符合标准 msc 标准。这些因素包括坚持塑料, 间充质表面标记物的阳性检测, 缺乏造血表面标记, 以及进行中胚层分化的能力。
如图 3所示, 卵巢衍生细胞附着在塑料上, 并表现出类似于成纤维细胞的形态, 满足了定义骨髓间充质干细胞的第一个标准。此外, 分离细胞显示犬 msc 标记 cd44、cd90 和 cd105 的转录表达。此外, 早期通过流式细胞仪分析和免疫细胞化学检测 msc 特异性细胞表面抗原 cd90 和 cd44。同样的技术也证实了 cd45 和 cd34 标记的缺失, 它们是造血特异性细胞表面抗原 (图 4)。这进一步证实, 卵巢包含表达 msc 特异性表型9 的细胞。本实验中用于犬 msc 特性鉴定的抗体列于材料表.
骨髓间充质干细胞表征的下一步是证明它们有能力分化为中胚层谱系。在针对特定血统的差异化协议中进行了30天的接触和培养后, 进行了染色, 以确定对不同谱系的承诺 (图 5a)。通过冯·科萨染色确认了对成骨系的承诺, 该染色确定了钙沉积。萨夫林 o 蛋白聚糖染色确认软骨原化承诺, 油红 o 脂质染色确认脂原承诺。继续研究这些细胞的可微性, 他们能够接受分化到外皮系, 如免疫染色两个神经外皮干细胞标记: β-肾素和奈斯汀, 接触后卵巢衍生的骨髓间充质干细胞到神经元谱系分化协议 10天 (图 5c)。在接触特定分化协议5天后, 观察到了 sox17 和 cd184 的转录, 以及特异性于内皮谱系的细胞形态 (图 5b)。最后, 在卵巢源性骨髓间充质干细胞暴露于生殖细胞诱导培养基14天后, 确定了 oct 4 和 ddx4 (图 5d)。这些结果共同支持了来自卵巢组织的骨髓间充质干细胞在特定分化协议中的强大和多样化的分化能力, 而暴露在特定的分化协议中。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58163/58163fig1.jpg"> 图 1: 隔离过程的主要步骤.(1) 手术后, 将卵巢转移到带 pbs 的无菌管, 在冰上。(2) 为了清洗卵巢, 应将卵巢转移到带有 pbs 的100毫米板上。(3) 将组织切成小块, 大小约1毫米。(4) 为了将细胞从组织中解脱出来, 将组织块转移到35毫米的板上, 并在孵化器中添加胶原酶进行3小时的消化。(5) 将板材内装物转移到锥形管进行离心。(6) 将颗粒放入 t25 瓶中, 孵育 3小时 (7) 培养后的前 3小时, 改变培养基。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58163/58163fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58163/58163fig2v2.jpg"> 图 2: 血液细胞仪.要统计的五个小方块用红色标着。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58163/58163fig3.jpg"> 图 3: 细胞形态.当培养基在培养开始3小时后改变时, 形成了同质的间质细胞群, 表现出纤维细胞样的形态。相反, 当第一次培养基改变是在48小时后完成的, 除了成纤维细胞一样的细胞之外, 还可以观察到其他类型的细胞。刻度杆 = 1, 000μm。这一数字已从 hill等人处修改。9.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58163/58163fig3large.jpg" target="_blank">请点击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58163/58163fig4.jpg"> 图 4: 卵巢 msc 的分子图谱.(a) 分离细胞表现出典型的 msc 标记特征, 通过实时 pcr (cd90、cd105 和 cd44) 对三个 msc 标记呈阳性。(b) 通过蛋白质分析, cd44 在这些细胞中呈阳性, 缺乏两种造血标志物 (cd34 和 cd45) 的表达。刻度杆 = 70μm。这一数字已从 hill等人处修改。9.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58163/58163fig4large.jpg" target="_blank">请点击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58163/58163fig5.jpg"> 图 5: 卵巢 msc 的鉴别.分离的细胞能够分化为 (a) 中胚层 (软骨原、成骨和脂肪生成; 鳞片棒 = 70μm)。此外, 细胞能够分化为 (b) 内皮前体 (刻度条 = 70 微米) 和 (c) 神经源谱系 (刻度条 = 50μm)。有趣的是, 这些细胞经历了 (d) 原始生殖细胞分化, 显示出卵巢间充质干细胞的广泛分化能力。这一数字已从 hill等人处修改。9.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58163/58163fig5large.jpg" target="_blank">请点击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.