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&#8242;,7&#8242;-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&#160;</td>
			<td>Corning</td>
			<td>&#160;8670</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Costar Flat Bottom Cell Culture Plates (6-wells)</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;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>&#160;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&#39; Balance Salt Solution with calcium and magnesium&#160;</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&#160;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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.