Name: derivation, expansion, cryopreservation and characterization of brain microvascular endothelial cells from human induced pluripotent stem cells
Uploaded: 2020-11-19T14:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells at JoVE.com

这项工作得到了国家精神卫生研究所创新新科学家生物行为研究奖（BRAINS）奖R01MH113858（R.K.），国家卫生研究院奖KL2 TR002542（PL）的支持。国家精神卫生研究所临床科学家发展奖K08MH086846（R.K.），悉尼R Baer Jr基金会赠款（给P.L.）多丽丝杜克慈善基金会临床科学家发展奖（给R.K.），瑞安·利希特桑双极基金会（给R.K.），菲利斯和 杰罗姆·莱尔·拉帕波特基金会（R.K.）、哈佛干细胞研究所（R.K.）和史蒂夫·威利斯和伊莉莎·弗洛伊德（R.K.）。我们感谢安妮·卡图里亚博士对手稿的批判性阅读和反馈。

人类iPSC是根据马萨诸塞州总医院和麦克莱恩医院机构审查委员会批准的协议从健康捐赠者的成纤维细胞中重新编程的，其特征在先前的研究中被描述为44、45、46。
注:简言之，成纤维细胞通过基于mRNA的基因再编程47重新编程到iPSC。iPSC 保存在干细胞介质 （SCM） （见材料列表）中，并储存在 ±1.2 x 102 细胞/mL 的密度中，具有 1 mL 的 SCM， 10 μM 与 rho 相关的蛋白激酶抑制剂 （ROCKi） Y-27632 和 10% （v/v） 二甲基硫化物 （DMSO）， 在低温小瓶液氮在 -160 °C. 除非另有说明，否则以下所有程序均在生物安全柜中执行。
1. 地下室膜矩阵稀释和板涂层
<ol><li>稀释 （1:50） 生长因子减少地下室膜基质纯化从恩格尔布雷斯 - 霍尔姆 - 温暖肿瘤在杜尔贝科的改性鹰介质 （DMEM） 没有酚红色。</li>
	<li>涂层细胞培养板与适当数量的稀释地下室膜矩阵（即6井板=1mL，12井板=0.5 mL），并在37°C孵育这些板至少1小时。</li>
</ol>2. iPSC 维护
注:6井平底板中每口井的最大汇合量为~1.2 x 106 单元。
<ol><li>用 10 μM Y-27632 将冷冻保存的 iPSC 解冻到 SCM 中，并将板板涂在涂有稀释生长因子的 6 井板上，减少地下室膜矩阵。</li>
	<li>解冻后的前 24 小时内，在 SCM 中以 10 μM Y-27632 维护 iPSC。24小时后切换到新鲜介质。</li>
	<li>在 SCM 中保持 iPSC，直到细胞在通过之前达到 80-90% 的汇合度。<ol>
<li>通过将期望的分化密度乘以井的表面积（15，600 个单元/cm2），计算分化所需的 iPSC 数量。对于 6 井平底板，将 15，600 个单元/厘米2 乘以 9.6 厘米2， 共 149，760 个单元/井。</li>
	</ol>
</li>
	<li>要通过，用汉克斯的平衡盐溶液（HBBS）清洗细胞。然后，在37°C下用非酶乙烯二胺酯酸（EDTA）（见材料列表）孵育细胞5分钟。<ol>
<li>使用细胞刮刀轻轻地从细胞上抬起来。收集新鲜 SCM 中的细胞。</li>
		<li>将细胞板涂在涂有稀释的 SCM 的细胞培养板上，并保持步骤 2.3 中描述的细胞，或将其存储在 +1.2 x 106 细胞/mL 中，在 1 mL 的 SCM 中，10 μM Y-27632 和 10% DMSO （v/v） 在 -160 °C 的低温小瓶中储存在液氮中。</li>
	</ol>
</li>
</ol>3. iPSC 与 BMEC 的区分
注:非酶EDTA将细胞分离成团块。酶EDTA（见 材料表）将细胞分离成单细胞悬浮。视网膜酸（RA）应受到保护，免受光线照射。
<ol><li>用杜尔贝科的磷酸盐缓冲盐 （DPBS） 清洗 iPSC 一次。用酶EDTA孵化（6井板1 mL，12井板0.5 mL，24井板0.25mL）约5分钟，在37°C产生单细胞悬架。</li>
	<li>在室温下以300 x g（相对离心力）收集细胞和离心机5分钟。SCM 中含有 10μM Y-27632 的重新悬念细胞颗粒。</li>
	<li>使用 Trypan Blue 和自动细胞计数器或血细胞计设备确定细胞密度。SCM 中密度为 15，600 细胞/cm2 或 149，760 细胞/井的 6 井平底板（表面积为 9.6 厘米2/井），24 小时含有 10 μM Y-27632。</li>
	<li>通过将 SCM 更改为 E6 介质，在 24 小时后开始分化。在接下来的4天里每天更换E6介质。</li>
	<li>在分化的第 4 天，用稀释 （1:200） B27 补充剂、20 ng/mL bFGF 和 10 μM RA 补充的 hESFM 取代 E6 介质。在接下来的 48 小时内，不要更改此介质。</li>
	<li>准备 200 mL 的 hESFM 与稀释 （1:200） B27， 混合 1 mL 的 50 倍浓缩 B27 补充到 199 mL 的 hESFM.</li>
	<li>通过在 250μL 的特里斯缓冲区（5 mM 特里斯、pH 7.6、150 mM NaCl） 中重组 50 μg bFGF，准备 20 ng/mL 的 bFGF，以制作 200μg/mL 库存解决方案。通过将 200μg/mL bFGF 的 20 μL 与 200 mL 的 hESFM 混合，准备 200 mL 的 hESFM，包含 20 ng/mL bFGF。</li>
	<li>通过将 2.5 mL 的 DMSO 添加到 100 毫克 RA 粉末中，首先制作 40 毫克/mL 的 RA 库存解决方案，从而准备 10 μM RA。将此浓度稀释为 3 毫克/mL，以制作 10 mM 库存解决方案。通过在 200 mL hESFM 中混合 200μL 的 10μM RA，准备 200 mL 包含 10 μM RA 的 hESFM。</li>
</ol>4. 涂层胶原蛋白 IV （COL4） 和纤维素 （FN） 矩阵，用于净化 iPSC 衍生的 BMEC
<ol><li>将 2 毫升无菌水添加到 2 毫克 FN 中，使 1 毫克/mL FN 库存溶液。将 5 mL 的无菌水添加到 5 毫克 COL4 中，以制作 1 毫克/mL COL4 库存溶液。<ol>
<li>允许 FN 在 37 °C 下溶解至少 30 分钟，并在室温下溶解 COL4。</li>
	</ol>
</li>
	<li>将无菌水中的 FN 库存溶液稀释至最终浓度为 100μg/mL 和 COL4 库存溶液，最终浓度为 400μg/mL。</li>
	<li>涂上所需的板材（6 井板 = 1 mL 的 COL4/FN 溶液，12 井板 = 0.5 mL，24 井板 = 0.25 mL，以及 12 变井过滤板 = 0.25 mL），混合物为 400 gμ/mL COL4 和 100 μ/mL FN。</li>
	<li>在37°C下孵化板至少2小时或过夜;对于透水井过滤板，建议至少4小时。</li>
</ol>5. iPSC 衍生 BMEC 的亚培养和净化
注:根据分化第6天细胞的结合情况，用酶EDTA孵化可能需要超过15分钟的时间。
<ol><li>在分化的第6天，用DPBS清洗电池两次。在37°C下用1mL的酶EDTA孵育至少15分钟，直到获得单个细胞悬浮。</li>
	<li>在室温下，在300 x g下通过离心收集细胞5分钟。用稀释 （1:200） B27 补充剂、20 ng/mL bFGF 和 10 μM RA 重新悬生细胞颗粒。</li>
	<li>种子细胞涂在400μg/mL COL4和100μg/mL FN的混合物板上。种子细胞使用1口6井板与3口12井板、3口12井过滤板或6口24井板的油井的比例。</li>
	<li>种子无差别 iPSC 从同一细胞线到 COL4/FN 涂层 12 跨井过滤板作为 TEER 分析的负控制。</li>
	<li>经过24小时的分培养后，仅用B27补充剂将介质改为hESFM。在此步骤之后，无需进行介质更改。</li>
</ol>6. 发芽检测
<ol><li>收集第 8 天 iPSC 衍生的 BMEC，并在 100，000 个细胞/井上播种到 24 口平底板上，新涂有 200 μL/cm2 的地下室膜矩阵。</li>
	<li>用稀释 （1:200） B27 和 40 ng/mL 血管内皮生长因子 A （VEGFA165） 治疗这些细胞。</li>
	<li>每24小时观察一次细胞，每两天更换一次介质。</li>
</ol>7. 免疫细胞化学（ICC）
注:ICC在24口井平底板上进行。
<ol><li>经过48小时的分培养（第8天），用DPBS洗两次电池。用4%的甲醛（PFA）修复细胞20分钟。</li>
	<li>用 DPBS 清洗电池三次，每次洗涤 5 分钟。在 DPBS 的室温下用 5% 驴血清和 0.3% 三顿 X-100 （v/v） 预块细胞 1 小时。</li>
	<li>与主要抗体孵化:小鼠抗人类PECAM1（1:100，库存0.5毫克/mL），兔子抗人-TJP1（1:200，库存0.53毫克/mL），小鼠抗人-CLDN5（1:200， 库存 0.5 毫克/毫升），小鼠反人-OCLN （1:200， 库存 0.5 毫克/毫升）， 和兔子反人-SLC2A1 （1:100， 库存 0.2 毫克/毫升） 在 DPBS 含有 5% 驴血清过夜在 4°C.<ol>
<li>用 DPBS 冲洗一次细胞，然后每次用 DPBS 洗 5 次 5 分钟。</li>
	</ol>
</li>
	<li>培养具有二次抗体的细胞:驴抗兔亚历克萨氟555（1:200）和驴抗鼠488（1:200）在DPBS中含有5%的驴血清1小时。</li>
	<li>孵化后，在 DPBS 中加入 Hoechst 33342 三氢三氢稀释 （1:1000）， 10 分钟。<ol>
<li>取出 Hoechst 33342 溶液，用 DPBS 冲洗一次，每次用 DPBS 清洗 4 次，每次洗涤 5 分钟。</li>
	</ol>
</li>
	<li>在荧光显微镜上可视化细胞，以寻找细胞制造商的表达和定位。</li>
</ol>8. TEER 测量和分析
注:康宁12-Transwell过滤板配有由1.12厘米2 聚乙烯四甲酸酯膜和0.4微米孔隙组成的过滤器。TEER 测量是在技术（每口井 3 口）和生物复制（每细胞系 3 口井和/或条件）中获得的。
<ol><li>分培养后24小时（第7天），每24小时使用筷子电极和伏托毫米测量TEER。有关获取测量的具体说明，请参阅伏地仪用户手册。<ol>
<li>要测量 TEER，请在前一天晚上为伏托姆仪充电。将仪器和筷子电极用 70% 乙醇轻轻擦拭，然后放入安全罩中。</li>
		<li>按制造商的建议打开电源并校准欧米。</li>
		<li>插入筷子电极，用 70% 的乙醇冲洗电极，然后是 DPBS。</li>
		<li>将较短的末端电极放入跨井插入（顶点腔室），将较长的端放置到基底室中。</li>
		<li>首先测量仅涂有 COL4/FN 的空白井。然后测量其他油井。</li>
		<li>在测量不同条件（即测量不同的细胞系）时，用 70% 的乙醇快速冲洗筷子电极，然后是 DPBS。</li>
	</ol>
</li>
	<li>在记录了所有测量（Ω）后，用70%的乙醇冲洗筷子电极，然后用无菌水冲洗。轻轻擦拭电极，使其在安全罩中晾干。</li>
	<li>平均从空白井中将 TEER 值（Ω）三倍，并按条件从每个原始 TEER 值中减去此平均值。<ol>
<li>平均减去值并乘以 1.12 厘米2（12 转井插入的表面积）。</li>
		<li>使用从第 6 步转换的值来生成显示 TEER 值和 TEER 测量日标准误差的图形。</li>
	</ol>
</li>
</ol>9. Efflux 运输机活动和分析
注:Efflux 运输器活动检测在 24 井平底板上执行。感兴趣的埃夫勒克斯运输商包括 ABCB1 和 ABCC1。建议每个条件应与控制井（即没有相应抑制剂的空白井）一起进行三重处理。
<ol><li>经过48小时的亚培养（第8天），在37°C下用10μM Valspodar（ABCB1抑制剂）或10μM MK571（ABCC1抑制剂）孵育细胞1小时。<ol>
<li>通过在 DMSO 的 412 μL 中溶解 5 毫克粉末（1214.64 克/摩尔），并稀释到 10 μM 的工作浓度，准备 10 mM Valspodar 库存。例如，要使 10 mL 的 hESFM 与 10 μM 瓦尔斯波达尔，混合 10 μL 的 10 mm 瓦尔斯波达尔股票与 10 mL 的 hESFM。</li>
		<li>通过在 931 μL 中溶解 5 毫克粉末（537.07 克/摩尔）并稀释到 10 μM 的工作浓度，准备 10 mm MK571 库存。例如，要使 10 mL 的 hESFM 与 10 μM MK571 混合 10 μL 的 10 mm MK571 股票与 10 mL 的 hESFM。</li>
	</ol>
</li>
	<li>1小时后，用10微米罗达明123（ABCB1基板）或10 μM 2'，7'-二氯二氢氟二甲酸酯（H2DCFDA，ABCC1基板）孵育细胞，在37°C时，有或没有各自的抑制剂1小时。<ol>
<li>通过在 875 μL 的 DMSO 中溶解 10 毫克粉末（380.82 克/摩尔），并稀释到 10 μM 的工作浓度，准备 10 mM 罗达明 123。例如，要使 10 mL 的 hESFM 与 10 μM 罗达明 123，混合 10 μL 的 10 m M 罗达明 123 股票与 10 mL 的 hESFM。</li>
		<li>通过在 10.26 mL 的 DMSO 中溶解 50 毫克粉末（487.29 克/摩尔），并稀释到 10 μM 的工作浓度，准备 10 mM H2DCFDA。例如，要使 10 mL 的 hESFM 与 10 μM 罗达明 123，混合 10 μL 的 10 m M 罗达明 123 股票与 10 mL 的 hESFM。</li>
	</ol>
</li>
	<li>使用含有 5% 三元 X （v/v） 的 DPBS 用 0.5 mL 的 DPBS 和 lyse 清洗电池两次。</li>
	<li>使用多板或微板读取器测量解解细胞的荧光（见材料列表）。<ol>
<li>将荧光板读取器设置为 485 纳米激发和 530 纳米发射，并测量这些波长的荧光。</li>
		<li>对于运输机检测中未使用的油井，在用 4% PFA 固定电池以进行细胞核定量之前，先用 DPBS 清洗两次。</li>
	</ol>
</li>
	<li>在 DPBS 中用 Hoechst 33342 三氢三氢稀释 （1:1000） 孵化细胞 10 分钟。使用荧光显微镜在每个井中映像多个视觉场，以计算平均细胞核计数。</li>
	<li>使用斐济计算核，并在每个细胞的基础上使荧光值正常化。<ol>
<li>通过从各自的空白值中减去每个条件的原始荧光累积值来计算荧光的平均积累值。</li>
		<li>每个条件的平均减去值。</li>
		<li>将平均值与步骤 9.6.1 除以平均细胞计数。使用这些值使每个细胞的荧光值正常化。</li>
		<li>使用规范化值为每个抑制条件生成图形表示，并执行任何必要的统计分析。</li>
	</ol>
</li>
</ol>10. 通过、扩展和冷冻保存 BMEC
<ol><li>补充第8天BMEC培养物与新鲜的hESFM补充稀释（1:200）B27，并允许细胞在COL4/FN矩阵上再扩展两天。</li>
	<li>涂上新的 12-Transwell 过滤板和 24 井平底板，带 400μg/mL COL4 和 100 μg/mL FN，孵育 4 小时。</li>
	<li>第10天，用DPBS清洗细胞，在37°C下用1mL酶EDTA孵育至少15分钟，直到获得单个细胞悬浮液。</li>
	<li>在室温下，在300 x g下通过离心收集细胞5分钟。<ol>
<li>为了冷冻保存这些细胞，用新鲜的 hESFM 与 30% 胎儿牛血清 （FBS） 和 10% DMSO 重新悬浮细胞颗粒。</li>
		<li>将 iPSC 衍生的 BMEC 储存在低温保存小瓶中，在 -80 °C 的前 24 小时内存放在异丙醇容器中，然后放在液氮中，以 -160 °C 进行长期存储。</li>
		<li>要通过这些细胞，重新悬生细胞颗粒与新鲜的hESFM补充稀释（1:200）B27。</li>
	</ol>
</li>
	<li>种子细胞到第10.2步准备的涂层板上。种子细胞使用 6 井板的 1 口井与 12 口透水井过滤板的 3 口井和 24 口井板的 6 口井的比例。允许细胞生长和膨胀24小时。</li>
	<li>第 11 天，通过步骤 8 中列出的步骤测量 TEER。</li>
	<li>第 12 天，通过步骤 9 中列出的步骤执行 ICC。</li>
	<li>要解冻冷冻保存的 BMEC，可在 37摄氏度时将冷冻保存小瓶放在温水中或珠子浴中。然后将解冻的 BMEC 转移到 5 mL 的 hESFM，并辅以稀释 （1:200） B27。<ol>
<li>在室温下，在300 x g下通过离心收集细胞5分钟。以稀释 （1:200） B27、10 μM RA 和 10 μM Y-27632 补充的 hESFM 中的细胞重新悬索。</li>
		<li>24 小时后，将介质切换到 hESFM，并辅以稀释 （1:200） B27 和 10 μM Y-27632，无需 RA。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

修改和故障排除
在此协议中，我们在 iPSC 培养过程中使用常用的细胞外矩阵和细胞培养介质进行一些修改，以获取 BMEC（图1）。这些变化并没有影响从人类iPSC中提取BMECS的能力，如利普曼协议1所述。来自不同健康捐赠者的 iPSC 线用于证明此修改的协议显示的结果可与其他行1的先前研究相媲美。对于冷冻?...

血脑屏障功能 血脑屏障（BBB）形成了一个边界，限制物质从血液向大脑的移动。BBB由大脑微血管内皮细胞（BMECs）组成，形成血管内单层。BMEC与星形细胞、神经元、近层细胞、微胶质和细胞外基质一起形成神经血管单元。BMEC具有一个严格监管的半细胞结构，允许BBB保持高跨内皮电阻（TEER），这限制了被动扩散，并作为屏障完整性的指标1，2。BMEC还含有蛋白质，有助于细胞外运动，如内分泌、转细胞增多症和移位，以及白细胞在免疫反应3期间的外流。BMEC依靠流入和排泄运输机来滋养和清除废物，以维持大脑的平衡。例如， 溶质载体家庭 2 成员 1 （SLC2A1） 是负责葡萄糖在 BBB4中移动的涌入运输机，而 EFFLUX 运输器（如 ATP 绑定盒式子家庭 B 成员 1 （ABCB1） 和 ATP 绑定盒式子家庭 C 成员 1 （ABCC1） 负责将基板返回血流 3、5、6、7。ABCB1基质包括吗啡、紫杉醇4和抗精神病药，如奥兰沙平和利司酮8，而ABCC1运输机有各种基质，包括硫酸盐结合体、葡萄糖氨酸和葡萄糖苷结合物4。
BBB模型在精神疾病中的应用 BBB功能障碍已牵连到一些神经和精神疾病，包括精神分裂症和双相情感障碍9，10。最近，iPSC衍生的外体细胞模型被用来询问精神疾病的细胞和分子基础，但这些模型目前没有考虑到神经血管11，12，13所发挥的潜在作用。据推测，血液中循环的外周炎症细胞因子会对BBB14、15、16、17产生不利影响，但也有证据表明，准细胞18、19、20、21、22、转细胞23、24、25、26、27、28、29和细胞外矩阵20、29、30、31、32异常导致BBB功能障碍。BBB的破坏可能导致血液进入脑膜瘤和激活星形细胞和/或微胶质释放前炎细胞因子，这反过来启动炎症反应33，可以对大脑34产生有害影响。BMEC 是 BBB 的主要成分，检查这些细胞的结构和功能可以增进对神经和精神疾病中 BBB 功能障碍的理解。
替代 BMEC 型号 在制定从 iPSC1 、6 、35、36中推导 BMEC 的有效协议之前，研究人员曾使用不朽的 BMEC37来研究 BBB 功能。然而，许多这些模型未能达到理想的BBB表型，这种生理范围的TER值38，39。利用iPSC具有保留细胞从中提取个体的遗传背景的优点。科学家们正在积极研究建立iPSC衍生的外生微环境模型，这些模型可以重述人脑的结构和功能。研究人员已经开发出一些方法来推导结构和生理上与体内发现的BMEC相似的生物元。获得iPSC衍生BMEC纯化种群的方法需要采取一些不同的步骤，在过去几年中优化了协议1、6、35、36。一般来说，iPSC衍生的 BMECs 在基本 6 （E6） 介质中培养 4 天，然后在人类无皮血清介质 （hESFM） 中培养 2 天，辅以基本的成纤维细胞生长因子 （bFGF）、转氨酸 （RA） 和 B27 补充剂。然后，这些细胞在胶原蛋白 IV （COL4） 和纤维素 （FN） 基质上培养，以获得 + 90% 均匀的 BMECs1。
BMEC的身份通过免疫荧光得到确认，显示BMEC蛋白的共同表达，包括血小板内皮细胞粘附分子-1（PECAM1）、SLC2A1和紧密结蛋白，如紧密结蛋白1（TJP1）、奥克鲁丁（OCLN）和克劳丁-5（CLDN5）6。发芽检测已用于确认 iPSC 衍生 BMEC 的血管生成潜力。6 BMEC 的 BBB 完整性由体外TEER 值 （+2000Ω x cm2）37的存在和 ABCB1 和 ABCC11、6、36等流体运输机的可测量活性进行评估。利普曼小组最近的方法进展导致iPSC衍生的BMEC协议，减少了实验变异性和增强了可重复性1。然而，不知道它们是否可以扩展到和通过超越子培养阶段。我们修改后的协议旨在通过第 8 天之后通过 iPSC 衍生的 BMEC 并评估它们是否可以进一步扩大以保留低温保存后的 BBB 属性来解决这个问题。虽然没有研究描述iPSC衍生的BMEC的传递，但BMEC冷冻保存存在一个协议，该协议在经历冻结-结冰周期40后保留生理BBB特性。但是，尚不清楚低温后保存 BMEC 是否可以通过并保留 BBB 属性。
利用利普曼协议从iPSC中提取的BMEC已被用于模拟BBB在神经紊乱中的紊乱，如亨廷顿舞蹈症7。这些由 iPSC 衍生的 BMEC 也被用来研究细菌感染的影响，如奈塞里亚脑膜炎或B 组链球菌对血液-CSF 屏障和 BBB 的破坏分别41，42。此外，研究人员利用来自22q删除综合征精神分裂症患者的iPSC衍生的BMECs，观察到细胞间粘附分子-1（ICAM-1）的增加，这是BMEC中一种主要的粘附分子，有助于白细胞进入大脑43。综合起来，这些研究表明，iPSC衍生的BMECs对于研究复杂神经精神病的BBB干扰有用。

<table><tbody><tr><td>2&amp;prime;,7&amp;prime;-dichlorodihydrofluorescein diacetate</td><td>Sigma Aldrich</td><td>D6883-50MG</td><td></td></tr><tr><td>Accutase</td><td>Sigma Aldrich</td><td>A6964-100mL</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey anti-Mouse IgG</td><td>Life Technologies</td><td>A-21202</td><td></td></tr><tr><td>Alexa Fluor 555 Donkey anti-Rabbit IgG</td><td>Life Technologies</td><td>A-31572</td><td></td></tr><tr><td>B27 Supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>CD31 (PECAM-1) (89C2) Mouse mAb</td><td>Cell Signaling</td><td>3528S</td><td></td></tr><tr><td>CLDN5 (Claudin-5)</td><td>Thermo Fisher Scientific</td><td>35-2500</td><td></td></tr><tr><td>Collagen IV from human placenta</td><td>Sigma Aldrich</td><td>C5533-5mg</td><td></td></tr><tr><td>Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial&amp;nbsp;</td><td>Corning</td><td>&amp;nbsp;8670</td><td></td></tr><tr><td>Corning&amp;nbsp;Costar Flat Bottom Cell Culture Plates (6-wells)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Corning&amp;nbsp;Falcon Flat Bottom Cell Culture Plates (24-wells)</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts (12-wells)</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Countess slides</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>DMEM/F12 (without phenol red)</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;A1413202</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2438-50mL</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma Aldrich</td><td>D9663-10ML</td><td></td></tr><tr><td>DPBS (+/+)</td><td>Gibco/Thermo Fisher Scientific</td><td>14040-117</td><td></td></tr><tr><td>Epithelial Volt/Ohm (TEER) Meter (EVOM2) STX2</td><td>World Precision Instruments</td><td>N/A</td><td></td></tr><tr><td>Essential 6 Medium (Thermo Fisher)</td><td>Thermo Fisher Scientific</td><td>A1516401</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>F2442</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F2006-2mg</td><td></td></tr><tr><td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td><td>Thermo Fisher Scientific</td><td>A1413202</td><td></td></tr><tr><td>Hanks&#039; Balance Salt Solution with calcium and magnesium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>24020-117</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Human endothelial serum-free medium</td><td>Thermo Fisher Scientific</td><td>11111044</td><td></td></tr><tr><td>InCell Analyzer 6000</td><td>General Electric</td><td>N/A</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;Countess Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>MK-571</td><td>Sigma Aldrich</td><td>M7571-5MG</td><td></td></tr><tr><td>NutriStem</td><td>Stemgent</td><td>01-0005</td><td></td></tr><tr><td>Occludin</td><td>Thermo Fisher Scientific</td><td>33-1500</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Services</td><td>15710</td><td></td></tr><tr><td>Perkin Elmer Envision 2103 multi-plate Reader</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Recombinant Human VEGF 165</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Recombinant Human FGF-basic (154 a.a.)</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma Aldrich</td><td>R2625-100MG</td><td></td></tr><tr><td>Rhodamine 123</td><td>Sigma Aldrich</td><td>83702-10MG</td><td></td></tr><tr><td>SLC2A1 (GLUT-1)</td><td>ThermoFisher</td><td>PA1-21041</td><td></td></tr><tr><td>SOX17</td><td>Cell Signaling</td><td>81778S</td><td></td></tr><tr><td>TJP-1 (ZO-1)</td><td>ThermoFisher</td><td>PA5-28869</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787-50ML</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>T10282</td><td></td></tr><tr><td>Valspodar (Sigma) (cyclosporin A)</td><td>Sigma Aldrich</td><td>SML0572-5MG</td><td></td></tr><tr><td>Versene solution</td><td>Thermo Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Y-27632 dihydrochloride (ROCK inhibitor)</td><td>Tocris/Thermo Fisher Scientific</td><td>1254</td><td></td></tr></tbody></table>

derivation, expansion, cryopreservation and characterization of brain microvascular endothelial cells from human induced pluripotent stem cells

脑微血管内皮细胞（BMECs）可以与人类诱导的多能干细胞（iPSCs）分化，以开发用于研究血脑屏障（BBB）功能 的前体内 细胞模型。此修改的协议提供了详细步骤，使用与先前协议中报告的不同供体和试剂从人类 iPSC 中提取、扩展和低温保存 BMEC。iPSC用必需的6种介质治疗4天，然后用2天人类无内皮血清培养基补充基本成纤维细胞生长因子、视网膜酸和B27补充剂。在第6天，细胞被分培养到胶原蛋白/纤维素基质上2天。免疫细胞化学在第8天进行，使用CLDN5、OCLN、TJP1、PECAM1和SLC2A1进行BMEC标记分析。执行西方印迹以确认 BMEC 标记表达，以及缺少 SOX17，这是一种内皮标记。血管生成潜力通过发芽检测进行证明。从第 7 天开始使用筷子电极和伏托姆计测量跨内皮电阻 （TEER）。ATP 绑定盒式子家庭 B 成员 1 和 ATP 绑定盒式子家庭 C 成员 1 的 Efflux 运输器活动在第 8 天使用多板读取器进行测量。BMEC 的成功衍生通过相关细胞标记的存在、SOX17 的低水平、血管生成潜力、运输器活性和 TEER 值 ~2000 Ω x cm2来证实。BMEC 被扩展至第 10 天，然后传递到新涂层的胶原蛋白/纤维素板或低温保存。该协议表明，iPSC 衍生的 BMEC 至少可以扩展和通过一次。然而，在低温保存后，观察到低TEER值较低，BMEC标记的本地化较差。BMEC可用于与其他细胞类型（神经元、胶质、心室）的共培养实验， 用于三维大脑模型（器官芯片和水凝胶），用于脑器官的血管化，以及神经精神病中的BBB功能障碍研究。

BMEC 差异化 应精确遵循此协议中的几个关键步骤（图1）。第 1 天的 E6 介质使用很重要，因为它通常用于在相对较短的时间内从 iPSC 中提取神经直肠系，从而在多个细胞系36中产生可重复的结果。另一个重要步骤是在差异化的第 4 天，其中 E6 介质应切换到带有稀释 （1:200） B27、20 ng/mL bFGF 和 10 μM RA 以扩展 iPSC 衍生的 BMEC 的 hESFM。增加B27补充?...

Watch this Scientific Journal Video about Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells at JoVE.com

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

该协议详细介绍了一种经过调整的方法，用于提取、扩展和冷冻保存通过区分人类诱导的多能干细胞而获得的大脑微血管内皮细胞，并研究 前体内 的血脑屏障特性。

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

derivation-expansion-cryopreservation-characterization-brain

Research

JoVE Journal

Neuroscience

6.3K 次观看.  Massachusetts General Hospital. 该协议详细介绍了一种经过调整的方法，用于提取、扩展和冷冻保存通过区分人类诱导的多能干细胞而获得的大脑微血管内皮细胞，并研究 前体内 的血脑屏障特性。

视频: Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

科研

神经科学

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

发育生物学

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

<meta charset="utf8"></head><body>使用 iPSC 衍生的 HPC 生成具有驻留小胶质细胞的人脑类器官

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

<meta charset="utf8"></head><body>小胶质细胞发育的人脑类器官的衍生

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

小胶质细胞发育的人脑类器官的衍生

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

免疫与感染

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

摘要

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此视频中的章节

代的永生化小鼠脑微血管内皮细胞株作为

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

从胚胎前脑内皮细胞的分离和培养

生成基于人类 iPSC 的血脑屏障芯片

血源性内皮细胞与人多能干细胞的定向分化

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生

脑膜炎奈瑟菌诱导多能干细胞来源的脑内皮细胞的感染

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

摘要

探索更多视频

此视频中的章节

代的永生化小鼠脑微血管内皮细胞株作为

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

从胚胎前脑内皮细胞的分离和培养

生成基于人类 iPSC 的血脑屏障芯片

血源性内皮细胞与人多能干细胞的定向分化

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

脑膜炎奈瑟菌诱导多能干细胞来源的脑内皮细胞的感染