作者没有什么可透露的。

为这项研究收集的所有人类子宫内膜活检都是从圣路易斯华盛顿大学, 妇产科学系通过一个机构审查委员会批准的书面同意表格获得的。
1. 准备
<ol><li>准备500毫升的1x 汉克平衡盐溶液 (HBSS), 加入100的 U/毫升青霉素和100µg/毫升链霉素到含有瓶子的介质 (进一步引用为 HBSS + 培养基)。</li>
	<li>通过添加1种抗生素-HEPES 溶液, 制备500毫升 Dulbecco 的改良鹰培养基: 营养混合物 F-12 (DMEM/F12) 与苯酚红、l-谷氨酰胺和15毫米 4-(2-羟乙基)-1%-piperazineethanesulfonic 酸 (防霉) (最终浓度是100的 U/毫升青霉素, 100 µg/毫升链霉素, 0.25 µg/毫升 Fungizone) 的媒体包含瓶 (进一步引用为人类胚胎干细胞隔离介质)。</li>
	<li>用苯酚红制备500毫升的 DMEM/F12, l-谷氨酰胺, 15 毫米 HEPES 通过添加10% 胎牛血清 (血清), 1x Na2HCO3, 1% 青霉素 (1万 U/毫升) 和链霉素 (1万µg/毫升) (笔链球菌) 的媒体包含瓶 (进一步引用为人类胚胎干细胞媒体)。</li>
	<li>用 l-谷氨酰胺、2.4 克/升碳酸氢钠和 HEPES 2% 炭剥胎牛血清 (csFBS) 和1% 支链球菌, 在含有瓶子的培养基上制备500毫升的降血清培养基 (进一步引用为蜕膜化培养基)。</li>
	<li>在含有瓶子的介质中加入2% 炭剥胎牛血清 (csFBS), 制备500毫升苯酚红 DMEM/F12 (进一步引用为变化介质)。</li>
	<li>在15毫升聚丙烯锥管中制备 10 nM 雌二醇 (E2)、1µM 甲羟孕酮 (MPA) 和50µM 循环腺苷磷酸 (营区), 并在体外添加2毫升的整除以前制备的蜕膜化培养基蜕膜化化验 (进一步被引用作为 EPC 媒介)。</li>
	<li>在50毫升聚丙烯锥管中制备50毫升的冷冻介质, 90% 的血清和10% 二甲基亚砜 (亚砜) (进一步引用为冷冻介质)。</li>
	<li>预重25毫克胶原酶和5毫克的 DNase I 在50毫升锥形聚丙烯管和贮存在20°c。</li>
</ol>2. 人子宫内膜活检采集
<ol><li>在 HBSS 的 IRB 批准的诊所, 从9天12天的月经周期中收集到的人子宫内膜活检标本。</li>
	<li>用1毫升的 prewarmed 37 °c HBSS + 中和轻轻摇动去除多余的血液和粘液的活检标本。</li>
</ol>3. 人子宫内膜间质细胞的分离 (人类胚胎干细胞)
注意: 这种隔离过程是在一个生物安全柜下的无菌环境中进行的。
<ol><li>将经灭菌的镊子移植到50毫升锥形聚丙烯离心机管中, 含10毫升新鲜 HBSS。</li>
	<li>九十年代在室温下离心活检 500 x g。<ol>
<li>如果细胞颗粒破裂, 重新旋转活检在 2000 x g 九十年代。</li>
	</ol>
</li>
	<li>使用1毫升吸管去除介质, 并用10毫升37°c prewarmed 人类胚胎干细胞隔离介质清洗, 然后在九十年代的 500 x g 处离心。</li>
	<li>使用1毫升吸管去除培养基, 并大力添加3毫升新鲜人类胚胎干细胞隔离介质, 以去除活检。</li>
	<li>将活检醒酒为含有5毫升人类胚胎干细胞隔离培养基的培养皿。</li>
	<li>用 #5 钳和细直缝剪刀将活检切成小块, 大约20分钟。</li>
	<li>通过添加10毫升的人类胚胎干细胞隔离培养基, 制备 DNase i 和胶原酶酵素, 预重 DNase I (0.5 毫克/毫升) 和胶原酶 (2.5 毫克/毫升)。和吸管混在一起。</li>
	<li>吸管切割组织样品使用1毫升吸管成一个新的50毫升锥形聚丙烯管。用7毫升的人类胚胎干细胞隔离介质冲洗, 加入聚丙烯管以获得完整的样品。</li>
	<li>将样品在室温下离心 800 x 克2分钟, 用吸管轻轻取出上清液。</li>
	<li>过滤 DNase I 和胶原酶溶液通过0.2 µm 过滤器附加到10毫升注射器直接到球团。</li>
	<li>漩涡为十年代到并用重悬颗粒。</li>
	<li>消化在37°c 水浴为1.5 小时, 涡流为十年代完全地每10分钟, 直到组织被消化。</li>
	<li>过滤样品通过40µm 细胞过滤器堆积在50毫升锥形聚丙烯管, 以消除上皮细胞和组织碎片。 注: 基质细胞在50毫升锥形聚丙烯管中。</li>
	<li>离心机50毫升圆锥聚丙烯管在 800 x g 在室温下2.5 分钟。用1毫升的吸管轻轻地取出上清液。</li>
	<li>并用重悬10毫升的人类胚胎干细胞隔离介质中的颗粒。</li>
	<li>离心机在室温下为 800 x 克, 2.5 分钟。吸出上清, 然后并用重悬细胞在6毫升的人类胚胎干细胞培养基和吸管向前和反转混合。</li>
	<li>慢慢地在3毫升的密度梯度介质上悬浮细胞, 将剩余的血细胞从基质细胞中分离出来, 小心不要混合层。</li>
	<li>离心15毫升聚丙烯管在 400 x g 在室温下30分钟。</li>
	<li>收集5毫升的上清在一个新的50毫升聚丙烯管, 并添加一个额外的5毫升人类胚胎干细胞媒体并用重悬细胞。</li>
	<li>离心机在室温下为 800 x 克, 在室温下为2.5 分钟, 其次是清除上清, 并添加6毫升人类胚胎干细胞培养基。涡流样品十年代和混合吹打向前和反转5次。</li>
	<li>整除10µL 重悬浮细胞到1.5 毫升离心管细胞计数。</li>
	<li>添加10µL 0.4% 台盼蓝染色整除细胞, 并与吸管混合。加载整除的10µL, 并使用 hemocytometer 计数单元格。</li>
	<li>在15毫升人类胚胎干细胞培养基中, 将细胞在适当的烧瓶中, 依赖于总细胞计数。<ol>
<li>如果细胞总数小于 100万, 则用6毫升的人类胚胎干细胞培养基将25厘米瓶中的所有细胞板。如果细胞总数超过 100万, 则将所有细胞在75厘米的烧瓶中, 用15毫升的人类胚胎干细胞培养基。</li>
	</ol>
</li>
	<li>培养细胞在 5% CO2孵化器在37°c 至少6小时, 以确保适当的细胞黏附力和改变媒体人类胚胎干细胞媒体。</li>
	<li>培养隔离人类胚胎干细胞在 5% CO2孵化器在37°c 在3-5 天期间, 直到细胞形成一层数达到 80-90% 融合在被镀的瓶。</li>
	<li>每两天更改人类胚胎干细胞媒体。<ol>
<li>温暖的人类胚胎干细胞介质在37°c 水浴为15-20 分钟。</li>
		<li>从烧瓶中吸取介质, 并将新鲜的人类胚胎干细胞介质添加到烧瓶中 (工作容积: 每25厘米或75厘米瓶的8毫升或16毫升)。</li>
		<li>地方烧瓶回到 5% CO2孵化器在37°c 直到80-90% 融合达到。</li>
	</ol>
</li>
	<li>一旦人类胚胎干细胞成为 80-90% 汇合, 转移细胞从 25 cm 到 75 cm 烧瓶或从一个 75 cm 烧瓶到三 75 cm 烧瓶。 注意: 人类胚胎干细胞可以被冻结和储存, 以便以后使用, 以便将来进行试验。</li>
</ol>4. 冻结和解冻干细胞
<ol><li>从75厘米的烧瓶中吸取培养基, 加入2毫升的胰蛋白酶-EDTA。</li>
	<li>在 5% CO2孵化器孵化2-3 分钟37摄氏度, 直到细胞从烧瓶中取出。</li>
	<li>添加7毫升的人类胚胎干细胞介质, 并通过吹打向前和反转混合。</li>
	<li>离心机在 800 x g 2.5 分钟, 并吸入上清。</li>
	<li>用细胞线、日期和通道号标记冻结管。</li>
	<li>在5毫升冷冻介质中重新悬浮细胞颗粒。</li>
	<li>整除1毫升的重新悬浮人类胚胎干细胞到每管和转移管到-80 °c 冰箱。</li>
	<li>在24小时内将管子输送到液氮中。 注: 如果计划在不久的将来解冻细胞, 将冰冻细胞储存在-80 摄氏度内一个月。</li>
	<li>为解冻干细胞预热人类胚胎干细胞介质20分钟。</li>
	<li>将8毫升预热的人类胚胎干细胞介质添加到25厘米的烧瓶中。</li>
	<li>从液氮罐或-80 °c 中取出冷冻管, 在37摄氏度水浴中将管浸入1-2 分钟, 解冻细胞。</li>
	<li>转移解冻人类胚胎干细胞到一个25厘米瓶与人类胚胎干细胞的媒体和孵化在一个 5% CO2孵化器隔夜在37摄氏度。</li>
	<li>第二天, 改变人类胚胎干细胞的媒体, 等待2-3 天, 直到人类胚胎干细胞成为汇合和转移细胞到75厘米的烧瓶, 如下文详细说明5号议定书。<ol>
<li>温暖的人类胚胎干细胞介质在37°c 水浴为15-20 分钟。</li>
		<li>从烧瓶中吸取介质, 并将新鲜的人类胚胎干细胞介质添加到烧瓶中 (工作容积: 每25厘米或75厘米瓶的8毫升或16毫升)。</li>
		<li>地方烧瓶回到 5% CO2孵化器在37°c 直到80-90% 融合达到。</li>
	</ol>
</li>
</ol>5. 人子宫内膜基质细胞 (人类胚胎干细胞) 的培养
注意: 这种培养过程是在一个生物安全柜下的无菌环境中进行的。
<ol><li>预热人类胚胎干细胞介质在37°c 的水浴中20-30 分钟。</li>
	<li>从烧瓶中吸取培养基, 加入0.25% 胰蛋白酶乙二胺 (EDTA) (1 毫升25厘米烧瓶或2毫升的75厘米烧瓶)。</li>
	<li>孵化在 5% CO2孵化器在37°c 2-3 分钟, 直到细胞去除。</li>
	<li>轻轻地敲击烧瓶壁, 将剩余的细胞赶走。添加7毫升的人类胚胎干细胞介质, 并通过吹打向前和反转混合。</li>
	<li>在室温下, 将细胞混合物吸管成50毫升锥形聚丙烯管, 离心机在 800 x g 处为2.5 分钟。</li>
	<li>在人类胚胎干细胞培养基中吸出上清并重新悬浮细胞颗粒。<ol>
<li>添加15毫升25厘米烧瓶或 45mL 75 厘米烧瓶 (每15毫升)。</li>
	</ol>
</li>
	<li>分别向每25厘米或75厘米的烧瓶中添加5毫升或15毫升细胞悬浮液。</li>
	<li>培养隔离人类胚胎干细胞在 5% CO2孵化器在37°c 在3-5 天期间, 直到细胞形成一层数达到 80-90% 融合在被镀的瓶。</li>
	<li>每两天更改人类胚胎干细胞媒体。<ol>
<li>温暖的人类胚胎干细胞介质在37°c 水浴为15-20 分钟。</li>
		<li>从烧瓶中吸取介质, 并将新鲜的人类胚胎干细胞介质添加到烧瓶中 (工作容积: 每25厘米或75厘米瓶的8毫升或16毫升)。</li>
		<li>地方烧瓶回到 5% CO2孵化器在37°c 直到80-90% 融合达到。</li>
	</ol>
</li>
</ol>6. 人子宫内膜基质细胞 (人类胚胎干细胞) 电镀和 siRNA 转染
注: 这种转染程序是在一个无菌环境下进行的生物安全柜。
<ol><li>从75厘米的烧瓶中吸取人类胚胎干细胞培养基, 加入2毫升0.25% 胰蛋白酶-EDTA 溶液。</li>
	<li>孵化在 5% CO2孵化器在37°c 2-3 分钟, 直到细胞去除。</li>
	<li>一旦细胞从烧瓶中脱落, 添加7毫升的人类胚胎干细胞培养基, 并轻轻地混合吹打向前和反转5次。</li>
	<li>在室温下, 将细胞混合物吸管成50毫升锥形聚丙烯管, 离心机在 800 x g 处为2.5 分钟。在10毫升的人类胚胎干细胞培养基中吸出上清并重新悬浮细胞颗粒。 注: 如果从一个以上的烧瓶电镀细胞, 只需增加人类胚胎干细胞介质体积。</li>
	<li>使用 Hemocytometer 计数单元格。<ol>
<li>整除10µL 再悬浮细胞成1.5 毫升离心管。</li>
		<li>添加10µL 0.4% 台盼蓝染色整除细胞, 混合吹打向前和反转。</li>
	</ol>
</li>
	<li>板 0.8 x 105细胞每井在6井板材。两天后, 细胞应60-70% 汇合, 以优化转染。</li>
	<li>对于每个 siRNA 转染样品, 用 siRNA (60 nanomoles) 制备复合物以转染试剂。</li>
	<li>稀释5µL 的转染试剂在150µL 的减少血清游离培养基和孵育5分钟。</li>
	<li>稀释 60 nanomoles siRNA 在100µL 减少血清游离培养基和孵育5分钟。</li>
	<li>5分钟后, 将稀释 siRNA 与稀释转染试剂溶液混合, 吹打向前和反转5次。 注: 如果转染一个或多个 siRNA 在多个井, 准备主混合10毫升聚苯乙烯管。</li>
	<li>室温下孵化 siRNA 转染试剂配合物5分钟。</li>
	<li>在这种孵化过程中, 每井添加1毫升的变化介质。</li>
	<li>经过5分钟的孵化, 离心管在 500 x g 2 分钟收集解决方案, 并添加250µL 的 siRNA 转染试剂复合物的每一个含有细胞和介质的井。</li>
	<li>混合通过轻轻摇摆和孵化细胞在37°c 在 5% CO2孵化器5小时。</li>
	<li>5小时后, 将媒体更改为人类胚胎干细胞媒体。</li>
	<li>在 5% CO2孵化器中孵育37摄氏度的细胞, 用于制备体外蜕膜化试验。</li>
</ol>7.体外蜕膜化测定
注: 本试验是在生物安全柜下的无菌环境下进行的。
<ol><li>在开始体外蜕膜化治疗前, 先准备 EPC 介质, 如卡姆登等所述。3 (见 1.6)。</li>
	<li>从步骤6.16 中从人类胚胎干细胞孵化的 post-48 小时的转染线中吸取介质。</li>
	<li>在每个井中加入2毫升的 EPC 介质。 注意: 作为一个控制, 留下一组细胞未处理的 EPC 媒体。相反, 在控制井中加入2毫升的人类胚胎干细胞介质。</li>
	<li>在 5% CO2孵化器孵化细胞在37°c 6 天。<ol>
<li>每48小时更换一次 EPC 媒体。<ol>
<li>在37摄氏度水浴中加热并准备 EPC 介质15-20 分钟。</li>
			<li>从井中吸取介质并添加新的 EPC 介质。</li>
			<li>把盘子放回 5% CO2孵化器在37°c。</li>
		</ol>
</li>
	</ol>
</li>
	<li>第六天后, 通过 ELISA 和 qRT PCR 方法分析基质细胞蜕膜化的程度 (如下所述)。</li>
</ol>8. 利用定量实时 pcr (qRT pcr) 对体外蜕膜化的验证
注: qRT PCR 完成前所述的 Kommagani et al10。
<ol><li>使用该协议从商业上可用的 rna 隔离套件中分离出人类胚胎干细胞的总 RNA。<ol>
<li>用加西亚-伊莱亚斯和al15中描述的 NanoDrop 或类似方法分析 RNA 的数量和纯度 (260/280)。</li>
	</ol>
</li>
	<li>通过商业上可用的反向转录试剂盒协议, 从总 RNA 的1µg 合成 cDNA。</li>
	<li>按照商业上可用的 qRT pcr 试剂盒协议中的描述, 运行 qRT pcr 反应。<ol>
<li>使用商业上可用的实时 PCR 主组合和基因特定探针以及内部系统控制 (18s、泌乳素和IGFBP1) 来执行反应。</li>
	</ol>
</li>
</ol>9. 利用 ELISA 法对体外蜕膜化的验证
<ol><li>利用市面上可用的催乳素 ELISA 试剂盒, 量化组织培养培养基分泌的催乳素水平。</li>
</ol>10. 利用罗丹明染色法对体外蜕膜化进行验证。
<ol><li>将无菌盖玻片插入12井板的每一个井中。</li>
	<li>重复步骤 6.1-6.5。</li>
	<li>板 0.8 x 105单元每井12井板。</li>
	<li>在 5% CO2孵化器孵化细胞在37°c 2 天。</li>
	<li>吸媒体。</li>
	<li>重复步骤 7.3-7.4。</li>
	<li>将4% 多聚甲醛 (粉煤灰) 中的细胞固定在室温下的磷酸盐缓冲盐水 (PBS) 中, 30 分钟。</li>
	<li>吸入固定液, 用1毫升 PBS 洗涤3次, 每5分钟。</li>
	<li>在 PBS 中加入0.1% 非离子表面活性剂聚乙二醇叔辛苯醚, 使固定细胞增加5分钟, 以提高渗透性。</li>
	<li>用1毫升 PBS 清洗3次, 每次5分钟。</li>
	<li>每盖玻片加100µL 罗丹明溶液, 室温孵育90分钟。<ol>
<li>准备1:100 稀释在 PBS 从使用罗丹明库存解决方案在1单位每5µL。</li>
	</ol>
</li>
	<li>用1毫升 PBS 清洗3次, 每次5分钟。</li>
	<li>装入包含 4 '、6-Diamidino 2-苯基吲哚 (DAPI) 的安装介质的幻灯片。</li>
	<li>使用共焦显微镜观察细胞。</li>
</ol>

<ol>
	<li>Maruyama, T., Yoshimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+for+differentiation+and+regeneration+of+the+uterine+endometrium.">Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium.</a> Endocrine Journal. 55, 795-810 (2008).</li><li>Yu, J., 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=Endometrial+Stromal+Decidualization+Responds+Reversibly+to+Hormone+Stimulation+and+Withdrawal.">Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.</a> Endocrinology. 157, 2432-2446 (2016).</li><li>Ahn, J. I., 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=cAMP-Response+Element-Binding+3-Like+Protein+1+(CREB3L1)+is+Required+for+Decidualization+and+its+Expression+is+Decreased+in+Women+with+Endometriosis.">cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.</a> Current Molecular Medicine. 16, 276-287 (2016).</li><li>Gellersen, B., Brosens, J. 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J., 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=Growth+regulation+by+estrogen+in+breast+cancer+1+(GREB1)+is+a+novel+progesterone-responsive+gene+required+for+human+endometrial+stromal+decidualization.">Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.</a> Molecular Human Reproduction. 23, 646-653 (2017).</li><li>Kommagani, R., 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=The+Promyelocytic+Leukemia+Zinc+Finger+Transcription+Factor+Is+Critical+for+Human+Endometrial+Stromal+Cell+Decidualization.">The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.</a> PLoS Genetics. 12, e1005937 (2016).</li><li>Large, M. J., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+embryo+implantation+and+endometrial+decidualization+by+progesterone+receptor+signaling.">The regulation of embryo implantation and endometrial decidualization by progesterone receptor signaling.</a> Molecular and Cellular Endocrinology. 358, 155-165 (2012).</li><li>Zhang, X. H., 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=The+mesenchymal-epithelial+transition+during+in+vitro+decidualization.">The mesenchymal-epithelial transition during in vitro decidualization.</a> Reproductive Sciences. 20, 354-360 (2013).</li><li>Kommagani, R., 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=Acceleration+of+the+glycolytic+flux+by+steroid+receptor+coactivator-2+is+essential+for+endometrial+decidualization.">Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization.</a> PLoS Genetics. 9, e1003900 (2013).</li><li>Ramathal, C. Y., Bagchi, I. C., Taylor, R. N., Bagchi, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+decidualization:+of+mice+and+men.">Endometrial decidualization: of mice and men.</a> Seminars in Reproductive Medicine. 28, 17-26 (2010).</li><li>Tamura, I., 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=Novel+Function+of+a+Transcription+Factor+WT1+in+Regulating+Decidualization+in+Human+Endometrial+Stromal+Cells+and+Its+Molecular+Mechanism.">Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism.</a> Endocrinology. 158, 3696-3707 (2017).</li><li>Vinketova, K., Mourdjeva, M., Oreshkova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Decidual+Stromal+Cells+as+a+Component+of+the+Implantation+Niche+and+a+Modulator+of+Maternal+Immunity.">Human Decidual Stromal Cells as a Component of the Implantation Niche and a Modulator of Maternal Immunity.</a> Journal of Pregnancy. , 8689436 (2016).</li><li>Wu, D., 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=Chronic+endometritis+modifies+decidualization+in+human+endometrial+stromal+cells.">Chronic endometritis modifies decidualization in human endometrial stromal cells.</a> Reproductive Biology and Endocrinology. 15, 16 (2017).</li><li>Garcia-Elias, 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=Defining+quantification+methods+and+optimizing+protocols+for+microarray+hybridization+of+circulating+microRNAs.">Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs.</a> Scientific Reports. 7, 7725 (2017).</li><li>Brosens, J. J., 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=Human+endometrial+fibroblasts+immortalized+by+simian+virus+40+large+T+antigen+differentiate+in+response+to+a+decidualization+stimulus.">Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus.</a> Endocrinology. 137, 2225-2231 (1996).</li><li>Koukouritaki, S. B., Margioris, A. N., Gravanis, A., Hartig, R., Stournaras, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+induces+rapid+actin+assembly+in+human+endometrial+cells+without+affecting+its+synthesis.">Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis.</a> Journal of Cellular Biochemistry. 65, 492-500 (1997).</li></ol>

这项工作得到了国家卫生研究院 (NIH)/国家儿童健康和人类发展研究所 (发育研究院) 赠款 (R00 HD080742) 和华盛顿大学医学院开办基金的资助, 以警服我们要感谢华盛顿大学生育诊所提供子宫内膜活检样本。

女性生殖月经周期的特点是孕激素水平在整个黄体期的上升, 从而诱导蜕膜化到圆形, 上皮样分泌细胞3,8。蜕膜化的启动是物种依赖性的。在人类中, 蜕膜化自发发生在孕激素浓度上升, 而小鼠需要囊胚存在10,11。这种不一致的蜕膜化例证了研究细胞分化的人细胞系的转化优势。通过原代细胞培养的体外检测, 经...

在月经初潮和更年期的阶段之间, 育龄妇女每月进行激素调节的子宫内膜增生、分化以及随后脱落的妊娠准备过程, 这一进程被称为1。 ,2。人类子宫内膜的这种物理修饰是必要的适当的胚胎植入到子宫壁1。子宫内膜的改变, 包括形态学和生物化学的适应, 通过卵巢类固醇激素雌激素和孕激素 (P4)3,4,5在整个月经周期中进行调解。在增生 (或卵泡) 阶段, 小鼠雌激素水平增加, 引发子宫内膜增厚。排卵后, 分泌 (或黄体) 阶段促进 P4 浓度的显著上升, 诱导子宫内膜基质细胞 (ESC) 从成纤维细胞样的外观到圆形, 上皮样蜕细胞的形态学改变。在一个被称为蜕膜化4,6的过程中。不正确的蜕膜化是导致植入失败和随后的早期胚胎流产4,7,8的根本原因。因此, 了解蜕膜化的分子机制有利于早期妊娠丢失的诊断和治疗。
目前, 利用几种方法探讨蜕膜化对子宫内膜基质细胞的潜在影响。在体内, 小鼠子宫可以通过机械刺激 (即, 划伤) 或注油在荷尔蒙引子宫9中人工诱导蜕膜化。与人类不同的是, 这种合成刺激通过提供囊胚存在的外观促进子宫腔的分化, 这是在啮齿目动物10,11中启动蜕膜化所需的步骤。因此, 由于与动物模型相关的翻译并发症和人类体内研究中的伦理困境, 蜕膜化模型是最成功的体外研究方法。
在这项研究中, 通过在当地的英语和西班牙报纸上放置广告来招聘科目。被确定为这项研究的合适人选的科目被引入与研究协调员会面, 其中讨论了潜在风险的充分披露。在确认对所涉潜在风险的完全理解后, 受试者的同意以书面和口头形式获得。主题同意包括准许 (1) 接受放血 (2) 长期保存他们的组织为将来研究目的和 (3) 同意创造原始的文化从被收集的组织标本。经同意后, 受试者获得一个表格, 以完成允许自我识别种族/族裔和/或保密权利。随后的访问将根据主题的月经周期来实现子宫内膜活检。被招募参加这项研究的志愿者反映了2012人口普查所记录的圣路易斯大都市地区的种族和种族人口, 不涉及任何弱势群体, 包括孕妇、胎儿、胚胎、18岁以下儿童或其他弱势群体。参加活检标本收集的资格要求包括 (1) 年龄在18-45 岁 (2) 之间, 有定期月经周期 (25-32 天) (3), 没有当前怀孕或使用荷尔蒙/宫内节育器避孕药具在注册前30天 (4) 没有当前的阴道感染或性传播疾病 (5) 没有目前的抗生素治疗, (6) 没有当前异常的 Pap 涂片。
在这项研究中, 人类子宫内膜基质细胞 (人类胚胎干细胞) 被培养和人工诱导通过补充激素 (雌二醇 (E2), 醋酸甲羟孕酮 (MPA) 和环腺苷来进行体外蜕膜化单磷酸 (营)) 到培养基中。在这种方法中, 蜕膜化的程度是根据激素治疗的总天数而改变的。与骨架重排, 激素补充诱导的生物化学适应, 其中蜕细胞经历分泌样素质2,4。标记基因的表达, 如催乳素和胰岛素样生长因子结合蛋白 1 (IGFBP1), 可以用来确认和量化人类胚胎干细胞蜕膜化的程度5,12,13,14. 重要的是, 这项议定书是否有能力进行基因特定的击倒, 也证明了这一点。

<table border="0" cellpadding="0" cellspacing="0" style="width:935px;" width="934">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Opti-MEM I (1X) Reduced Serum Medium</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">31985-070</td>
			<td style="width:271px;">For decidualization media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DMEM / F12 (1:1) (1X)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">15250-061</td>
			<td>For cell count</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PureLink RNA Mini Kit</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">12183018A</td>
			<td>For RNA Isolation/Purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan 2X Universal PCR Master Mix</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4304437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4374967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Eukaryotic 18S rRNA Endogenous Control</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:131px;">4319413E</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay Prolactin Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4331182</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay IGFBP1 Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4331182</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phalloidin-iFluor 488 Reagent - CytoPainter</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:131px;">ab176753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Human Prolactin ELISA Kit</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">EHIAPRL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">16% Paraformaldehyde</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:131px;">30525-89-4</td>
			<td>Fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lipofectamine RNAiMAX</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">13778150</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ProLong Gold antifade reagent with DAPI</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">P36935</td>
			<td>For mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Penicillin Streptomycin</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">F6765-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sodium Bicarbonate (7.5%)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">25080-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ficoll-Paque PLUS Reagent</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">45001749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">C0130-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">DN25-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Medroxyprogesterone 17-acetate</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">M1629-1G</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Estradiol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">E1024-1G</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">cAMP</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">A6885-100MG</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fine straight stitch scissors</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:131px;">15396-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay NCOA2 Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4351372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fetal Bovine Serum, heat inactivated</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">10-082-147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-antimycotic</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">21-021-cv</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical polypropylene Falcon tube</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dumont #5/45 Forceps</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:131px;">11251-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">100 x 15 mm Glass Petri dish</td>
			<td style="width:167px;">VWR</td>
			<td style="width:131px;">75845-546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">40 micron cell strainer</td>
			<td style="width:167px;">Midsci</td>
			<td style="width:131px;">229481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isotemp 215 Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">FS-215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LSE Centrifuge&#160;</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">6755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Human NCOA2 siRNA&#160;</td>
			<td style="width:167px;">Dharmacon</td>
			<td style="width:131px;">L-020159-00</td>
			<td>Gene specific qPCR probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Steri-cycle i160 CO2 Incubator&#160;</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">51030301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NanoDrop 2000</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">ND-2000</td>
			<td>For RNA quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffered Saline&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">50146771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">75 cm Canted Neck Cell Culture flask</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">430641U</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">6 well Non-Pyrogenic Cell Culture Plate</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pipet Controller Ultra</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">4099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Photoshop</td>
			<td style="width:167px;">Adobe</td>
			<td style="width:131px;">19.0.1.334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">25 cm Canted Neck Cell Culture flask</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">7200876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Triton X-100</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">X100-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">15 mL conical polypropylene Falcon tube</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">10 mL Syringe</td>
			<td style="width:167px;">BD</td>
			<td style="width:131px;">302995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1300 Series A2 Biological Safety Hood</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">1377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">accuspin Micro17 centrifuge</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">13100675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">7500 Fast real time PCR system</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">3052632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">EVOS FL Immunofluorescence Microscope</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:131px;">01414-155G-291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Leica Inverted Light Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:131px;">DMi1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Illrustrator</td>
			<td style="width:167px;">Adobe</td>
			<td style="width:131px;">22.0.1.253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Excel Spreadsheets</td>
			<td style="width:167px;">Microsoft</td>
			<td style="width:131px;">2016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Deckglaser 18 mm cover glasses</td>
			<td style="width:167px;">NeuVitro</td>
			<td style="width:131px;">GG-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Reichert Bright-Line Hemacytometer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2-mercaptoethanol</td>
			<td style="width:167px;">Fisher Bioreagents</td>
			<td style="width:131px;">BP176-100</td>
			<td>For RNA lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">1.2mL External Threaded Polypropylene Cryogenic Vial</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">430658</td>
			<td>For freezing HESC cells&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">D2650-100mL</td>
			<td>For freezing media</td>
		</tr>
	</tbody>
</table>

isolation of human endometrial stromal cells for in vitro decidualization

人子宫内膜间质细胞 (人类胚胎干细胞) 从成纤维细胞样的形态分化为分泌性的蜕片, 是胚胎植入子宫内衬的必要转变。不当蜕膜化是导致植入失败和随后的早期胚胎流产的根本原因。因此, 了解蜕膜化的分子机制有利于提高生育成功率。在基于活体的人工蜕膜化的研究中, 往往由于与人类研究相关的伦理难题以及动物模型中的平移并发症而受到限制。因此, 通过原代细胞培养的体外检测, 经常被用来探索通过激素调节蜕膜化的作用。本研究提供了一个详细的协议, 以分离人类胚胎干细胞和随后的人工蜕膜化通过补充激素的培养培养基。进一步, 本研究提供了一个精心设计的方法来击倒任何感兴趣的基因, 利用脂质基 siRNA transfections。该协议允许优化的文化纯度和产品的产量, 从而最大限度地利用这个模型作为一个可靠的方法, 以了解分子机制的基础蜕膜化, 并随后量化decidualized 子宫内膜基质细胞分泌的药物。

人类胚胎干细胞文化中的蜕膜化
分离后, 将人子宫内膜间质细胞培养成单层80-90% 融合, 并蜕膜化 10 nM E2、1µM MPA 和50µM 营 (EPC) 治疗体外诱导。与体外蜕膜化相关的形态学变化可视于图 1A。在蜕膜化, 干细胞接受骨架重排从拉长, 成纤维细胞细胞到圆上皮样细胞。因此, 罗丹明染色...

Watch this Scientific Journal Video about Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization at JoVE.com

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

本研究为人子宫内膜间质细胞的分离和培养进行体外蜕膜化试验提供了一个验证和优化的程序。此外, 本研究还提供了一种利用 siRNAs 在人子宫内膜基质细胞中有效地击倒特定基因的方法。

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

isolation-human-endometrial-stromal-cells-for-vitro

Research

JoVE Journal

Developmental Biology

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

10.1K 次观看.  Washington University School of Medicine. 本研究为人子宫内膜间质细胞的分离和培养进行体外蜕膜化试验提供了一个验证和优化的程序。此外, 本研究还提供了一种利用 siRNAs 在人子宫内膜基质细胞中有效地击倒特定基因的方法。

视频: Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

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

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

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

从人胎盘组织源分离和间充质干扩张/基质细胞

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

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 ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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 Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

角膜组织工程: 人角膜间质神经相互作用的体外模型

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.