Name: human microglia-like cells: differentiation from induced pluripotent stem cells and in vitro live-cell phagocytosis assay using human synaptosomes
Uploaded: 2022-08-18T14:30:00
Description: Watch this Scientific Journal Video about Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes at JoVE.com

作者感谢Michael Ward提供用于运动神经元分化的WTC11 hNIL iPSC系，感谢Jackson实验室提供用于小胶质细胞分化的KOLF2.1J WT克隆B03 iPSC系。我们还要感谢Dorothy Schafer在协议实施过程中的支持，Anthony Giampetruzzi和John Landers在活细胞成像系统方面的帮助，以及Hayden Gadd在修订期间的技术贡献和Jonathan Jung在这项研究中的合作。这项工作得到了UMASS Chan医学院的Dan和Diane Riccio神经科学基金和天使基金公司的支持。

注意：本协议中使用的所有试剂必须是无菌的，并且所有步骤必须在无菌条件下在生物安全柜中进行。所有iPSC细胞系以及维护和分化培养基均在材料表中进行了描述。下面图示的小胶质细胞分化方法基于先前发表的方案7，8，12，并在此描述新的修改。
1.小胶质细胞分化
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<ol>
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此处描述的分化方案提供了一种有效的方法，可在~6-8周内获得iPSC衍生的小胶质细胞样细胞，具有高纯度和足够的产量，以进行免疫荧光实验和其他需要更多细胞数的测定。该协议在 1 周内产生了多达 1 × 106 iMG，这允许蛋白质和 RNA 提取以及相应的下游分析（例如，RNASeq、qRT-PCR、蛋白质印迹、质谱）。也就是说，该协议的局限性是iMG的产量可能会限制某些应用。可以实施额外的修饰以积?...

小胶质细胞是中枢神经系统（CNS）中的常驻免疫细胞，在CNS的发展中起着至关重要的作用。小胶质细胞在成人大脑中对于维持体内平衡和积极应对创伤和疾病过程也很重要。累积证据表明，小胶质细胞是多种神经发育和神经退行性疾病发病机制的关键因素1，2。尽管目前关于小胶质细胞生物学的知识主要来自小鼠模型，但最近的研究阐明了小鼠和人类小胶质细胞之间的重要差异，强调了开发技术来研究人类小胶质细胞的遗传学和生物学功能的必要性3，4。从解剖的原代组织中分离小胶质细胞会严重改变小胶质细胞的特性5，这可能会混淆用这些细胞获得的结果。该方法的总体目标是将人iPSCs分化为iMG，从而提供细胞培养系统在基础条件下研究人小胶质细胞。此外，本文还包括使用全人模型系统的吞噬作用测定，作为研究iMGs功能的手段，既作为质量控制措施，又评估疾病背景下的iMG功能障碍。
最近在文献6，7，8，9，10中出现了从iPSC分化小胶质细胞的多种方案。某些方案的潜在缺点包括延长或长时间的分化，添加多种生长因子和/或复杂的实验程序6，9，10。在这里，展示了一种“用户友好”的分化方法，该方法通过将iPSC分化为称为原始巨噬细胞前体（PMP）的前体细胞来概括小胶质细胞个体发生的各个方面7，11。PMP如前所述生成，本文12中提供了一些优化。PMP模仿MYB非依赖性卵黄囊衍生的巨噬细胞，巨噬细胞在胚胎发育过程中通过在血脑屏障关闭之前侵入大脑而产生小胶质细胞13。为了最终将PMP分化为iMG，我们使用了基于Haenseler等人和Brownjohn等人的协议的快速简化的单一培养方法，并进行了一些修改以产生一种有效的小胶质细胞分化方法，其中iMG稳健地表达富含小胶质细胞的标记7，8。这种分化方法可以在具有iPSC培养专业知识的实验室中复制，其研究目标是使用人类模型系统研究小胶质细胞生物学。
iPSC来源的小胶质细胞是用于 体外 实验的人小胶质细胞的生物学相关来源，是研究小胶质细胞典型功能（包括吞噬作用）的重要工具。小胶质细胞是大脑和中枢神经系统的专业吞噬细胞，它们清除细胞碎片、聚集蛋白和降解的髓磷脂14。小胶质细胞还通过吞噬突触在突触重塑中发挥作用，并通过病原体的吞噬作用防御外部感染15，16。在该协议中，使用人突触体作为iMG吞噬的材料评估iMG的吞噬作用。为此，描述了分离源自人类i3LMN的突触体的描述。i3LMN衍生的人突触体用pH敏感染料标记，该染料允许在吞噬体加工和体外降解过程中定量位于酸性区室内的突触 体。显示了使用活细胞显微镜的吞噬作用测定，用于实时监测小胶质细胞吞噬的动态过程。该功能测定为使用完整的人体系统研究健康和疾病中小胶质细胞吞噬作用的可能缺陷奠定了基础。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies for immunofluorescence analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA1 rabbit antibody</td>
			<td>Wako Chemical USA</td>
			<td>NC9288364</td>
			<td>1:350 dilution</td>
		</tr>
		<tr>
			<td>anti-P2RY12 rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA014518</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>anti-TMEM119 rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA051870</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Antibodies for Western blot analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-Tubulin rabbit antibody</td>
			<td>Abcam</td>
			<td>ab6046</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>anti-Synaptophysin (SYP) rabbit antibody</td>
			<td>Abclonal</td>
			<td>A6344</td>
			<td>1:1,000 dilution</td>
		</tr>
		<tr>
			<td>anti-PSD95 mouse antibody</td>
			<td>Millipore</td>
			<td>MAB1596</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Borate buffer components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid (100 mM)</td>
			<td>Sigma</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3) BioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (75 mM)</td>
			<td>Sigma</td>
			<td>&#160;S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate (25 mM)</td>
			<td>Sigma</td>
			<td>221732</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m Cell Strainers</td>
			<td>&#160;Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 20 mm Tissue Culture Treated</td>
			<td>CELLTREAT</td>
			<td>229620</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter, Double End, Flat Blade &#38; Narrow Blade, Sterile</td>
			<td>CELLTREAT</td>
			<td>229305</td>
			<td></td>
		</tr>
		<tr>
			<td>low adherence round-bottom 96-well plate</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 24-well Flat Bottom Surface Modified Multiwell Cell Culture Plate</td>
			<td>Corning</td>
			<td>353847,</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 6-well Cell Clear Flat Bottom Surface-Modified Multiwell Culture Plate</td>
			<td>Corning</td>
			<td>353846</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 96-well Clear Flat Bottom Microplate</td>
			<td>Corning</td>
			<td>353872</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell dissociation reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>&#160;Corning</td>
			<td>25058CI</td>
			<td>dissociation reagents used for lower motor neuron differentiation</td>
		</tr>
		<tr>
			<td>TrypLE reagent</td>
			<td>Life Technologies</td>
			<td>12-605-010</td>
			<td>dissociation reagents used for microglia differentiation</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating reagents for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel GFR Membrane Matrix</td>
			<td>Corning&#8482;</td>
			<td>354230</td>
			<td>Referred as to extracellular matrix coating reagent</td>
		</tr>
		<tr>
			<td>CellAdhere Laminin-521</td>
			<td>STEMCELL Technology</td>
			<td>77004</td>
			<td>Referred as to laminin 521</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P7405</td>
			<td>Reconstitute to 0.1 mg/mL in borate buffer</td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>&#160;P3655</td>
			<td>Reconstitute to 1 mg/mL in borate buffer</td>
		</tr>
		<tr>
			<td>Components of iPSC media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mTeSR Plus Kit</td>
			<td>STEMCELL Technology</td>
			<td>100-0276</td>
			<td>To prepare iPSC media mixed the components to 1x</td>
		</tr>
		<tr>
			<td>Components of EB media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>Fisher Scientific</td>
			<td>PHC9534</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>iPSC media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Fisher Scientific</td>
			<td>BD 562822</td>
			<td>final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>PeproTech</td>
			<td>300-07</td>
			<td>final concentration 20 ng/mL</td>
		</tr>
		<tr>
			<td>VEGF</td>
			<td>PeproTech</td>
			<td>100-20A</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Components of PMP base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>final concentration 100 U/mL</td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>12001-988</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of PMP complete media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>final concentration 55 &#181;M</td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>PeproTech</td>
			<td>200-03</td>
			<td>final concentration 25 ng/mL</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td>final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>PMP base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of iMG base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>final concentration 100 U/mL</td>
		</tr>
		<tr>
			<td>Components of iMG complete media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>final concentration 55 &#181;M</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td>PeproTech or Biologend</td>
			<td>200-34 or 577904</td>
			<td>final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>iMG base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td>final concentration 5 ng/mL</td>
		</tr>
		<tr>
			<td>TGF-&#946;</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Components of Induction base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with HEPES</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA), 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of Complete induction media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound E</td>
			<td>Calbiochem</td>
			<td>565790</td>
			<td>final concentration 0.2 &#956;M and reconstitute stock reagent to 2 mM in 1:1 ethanol and DMSO</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D9891</td>
			<td>final concentration 2 &#956;g/mL and reconstitute stock reagent to 2 mg/mL in DPBS</td>
		</tr>
		<tr>
			<td>Induction base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Fisher Scientific</td>
			<td>BD 562822</td>
			<td>final concentration 10 &#956;M</td>
		</tr>
		<tr>
			<td>Components of Neuron media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Neuronal Culture System</td>
			<td>Gibco</td>
			<td>A3653401</td>
			<td>final concentration 1x for media and suplemment</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA), 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>iPSC lines used in this study</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KOLF2.1J: WT clone B03</td>
			<td>The Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WTC11 hNIL</td>
			<td>National Institute of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptosome isolation reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Syn-PER Synaptic Protein Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>87793</td>
			<td>Referred as to cell lysis reagent for isolation of synaptosomes</td>
		</tr>
		<tr>
			<td>Phagocytosis assay dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Live Ready reagent</td>
			<td>Invitrogen</td>
			<td>&#160;R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red, succinimidyl ester</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;P36600</td>
			<td>Referred as to pH-sensitive dye</td>
		</tr>
		<tr>
			<td>Other cell-culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% Solution</td>
			<td>AMRESCO INC</td>
			<td>K940-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>22144-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Sigma</td>
			<td>B9285</td>
			<td>Reconstitute to 40 mM in sterile water</td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Sigma</td>
			<td></td>
			<td>final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>DPBS with Calcium and magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>Referred as to DPBS</td>
		</tr>
		<tr>
			<td>KnockOut&#160; DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>Referred as to DMEM-F12 optimized for growth of human embryonic and induced pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td>Referred as to laminin</td>
		</tr>
		<tr>
			<td>Software and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 live cell imaging reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Microplate Reader and Imager Software</td>
			<td>Biotek</td>
			<td>version 3.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Therm Heat-Shake</td>
			<td>Benchmark</td>
			<td></td>
			<td>refer as tube shaker</td>
		</tr>
		<tr>
			<td>Water sonicator</td>
			<td>Elma</td>
			<td>Mode Transsonic 310</td>
			<td></td>
		</tr>
	</tbody>
</table>

human microglia-like cells: differentiation from induced pluripotent stem cells and in vitro live-cell phagocytosis assay using human synaptosomes

小胶质细胞是骨髓来源的常驻免疫细胞，在大脑微环境中维持稳态，已成为多种神经系统疾病的关键参与者。由于人类细胞供应极其有限，研究人类小胶质细胞在健康和疾病中是一项挑战。源自人类个体的诱导多能干细胞（iPSC）可用于规避这一屏障。在这里，展示了如何将人iPSCs分化为小胶质细胞样细胞（iMGs）以进行 体外 实验。这些iMG表现出小胶质细胞的预期和生理特性，包括小胶质细胞样形态，适当标志物的表达和活性吞噬作用。此外，还提供了用于分离和标记源自人 iPSC 衍生的下运动神经元（i3LMN）的突触体底物的文档。活细胞纵向成像测定用于监测用pH敏感染料标记的人突触体的吞噬，从而可以研究iMG的吞噬能力。本文描述的方案广泛适用于研究人类小胶质细胞生物学和小胶质细胞对疾病的贡献的不同领域。

为了使用该协议生成iMG，重要的是从未分化的iPSC开始，这些iPSCs显示出紧凑的集落形态和明确的边缘（图2A）。如EB形成部分所述维持的解离iPSC将形成球形聚集体，称为EB，其大小将增长到分化的第4天（图2B）。一旦EB被收集并在适当的条件下接种以产生PMP，它们就会附着在Matrigel涂层的板上，并且一层细胞将扩散并围绕球形聚集体（图2C...

Watch this Scientific Journal Video about Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes at JoVE.com

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

该协议描述了人诱导多能干细胞（iPSC）成小胶质细胞样细胞进行 体外 实验的分化过程。我们还包括从iPSC衍生的下运动神经元生成人突触体的详细程序，该神经元可用作使用活细胞成像系统进行 体外 吞噬测定的底物。

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

iPS衍生的小胶质细胞是用于体外实验的人类小胶质细胞的生物学相关来源，是研究健康和疾病中小胶质细胞生物学的重要工具。我们提出了一个简单的小胶质细胞分化方案，该方案需要最少的生长因子使用，并实现高纯度和产量。此外，我们还展示了一种完全人源化的吞噬测定。iPSC衍生的小胶质细胞样细胞对培养基蒸发非常敏感。应避免使用细胞培养板角落的孔，并用水填充所有空孔以提高存活率。一旦诱导多能干细胞（iPSCs）达到80%汇合度，通过用一毫升DPBS洗涤并在37摄氏度下加入一毫升解离试剂两分钟来解离菌落。使用细胞提升器通过多次刮擦以产生单细胞悬液来去除菌落。收集悬浮液，并将它们转移到含有9毫升DPBS的15毫升锥形管中。然后将细胞以500倍g离心一分钟，除去上清液，并重悬于一毫升Embryoid体或EB培养基中。取10微升细胞，用台盼蓝稀释一到两个。在血细胞计数器上计数细胞，并根据细胞计数，将细胞原液稀释至每100微升10， 000个细胞的最终稀释度。对于电镀细胞，每孔将 100 微升稀释的细胞加入低粘附性圆底 96 孔板中。将板以125倍g离心三分钟，并在37摄氏度和5%二氧化碳下孵育四天。第二天，使用多通道移液器，用新鲜培养基替换旧的半EB培养基。要生成原始巨噬细胞前体或PMP，请通过添加一毫升冰冷的基质胶包衣溶液来涂覆六孔板的孔。然后将板在 37 摄氏度和 5% 二氧化碳下孵育两小时，或过夜。在EB分化的第四天，使用一毫升移液器吸头将EB转移到Matrigel包被的孔中。上下移液以将EB从井中取出。然后保持板倾斜，使EB在井的边缘沉降。一旦所有EB都稳定下来，轻轻移液并更换旧培养基，同时用三毫升新鲜制备的PMP完全培养基将EB保持在孔的边缘。通过手动将板左右和从后到前洗牌，将细胞均匀分布在孔中。然后将板孵育七天，以使EB附着在孔底。七天后，在四倍放大镜的光场显微镜下检查EB，以确保它们附着在孔的底部。进行半培养基更换，并在第21天，用三毫升PMP完全培养基替换完全培养基。在第28天，在上清液中寻找称为PMP的圆形细胞。然后使用10毫升移液器和自动移液器收集含有PMP的培养基，而不会干扰EB。将培养基中的PMP转移到15毫升锥形管中。将收集的PMP以200倍g离心四分钟并吸出上清液，然后将其重新悬浮在一至两毫升小胶质细胞样细胞或IMG基础培养基中。然后如前所述计数血细胞计数器上的细胞。离心其余细胞，并将PMP稀释至所需浓度。然后使用新鲜制备的IMG完全培养基将细胞以每平方厘米10至5个细胞的密度接种在细胞培养处理的平板上。对于活细胞吞噬测定，将20至30乘以10至第四个PMP板放入96孔板和100微升IMG完全培养基中，并遵循分化过程10天。在测定当天，每孔除去40微升培养基，并加入10微升核染色溶液。将板孵育两个小时。在冰上解冻标记的突触体，并使用水超声仪轻轻超声处理一分钟。同样，立即将突触体放在冰上。在IMG完全培养基中以每50微升培养基一微升突触体稀释标记的突触体。对于阴性对照，在IMG完全培养基中制备60微摩尔的细胞松弛素D以抑制肌动蛋白聚合，从而抑制吞噬作用。然后向每个孔中加入10微升该溶液，终浓度为10微摩尔，并孵育30分钟。从培养箱中取出板，并在 10 摄氏度下孵育 10 分钟。将板保持在冰上，并加入50微升含有突触体的培养基。将板以270倍g在10摄氏度下离心三分钟，并将板保持在冰上直到成像采集。将板插入活细胞成像读数器，然后选择要分析的孔。然后选择20倍物镜。接下来，调整焦点、发光二极管或 LED、强度、积分时间以及明场和蓝色通道的增益。突触体荧光在初始时间点应可忽略不计。选择每个孔要在蒙太奇中获取的单个瓷砖的数量。在井中心采集16块瓷砖，成像约5%的总井面积。将温度设置为 37 摄氏度，并设置所需的成像时间间隔。接下来，打开分析软件。然后打开包含图像的实验，然后单击“数据缩减”图标。在菜单中，选择“成像处理”下的“成像拼接”，使用文本手稿中描述的参数从蒙太奇中的四乘四个单独的拼贴创建完整的图像。使用这些图像的 DAPI 和 RFP 通道定义强度阈值。打开图像，然后单击“分析”。在“分析”下，选择“蜂窝分析”，然后在“检测通道”下，在 DAPI 或 RFP 通道中选取拼接图像。接下来，转到“主掩码和计数”选项卡，并建立一个阈值和对象大小值，以正确选择 DAPI 通道中的细胞核或 RFP 通道中的突触体信号。要计算细胞核的数量，请转到“数据缩减”菜单，然后在“图像分析”下选择“细胞分析”。再次转到“主掩码和计数”选项卡，在“通道”下，选择 DAPI 拼接图像，然后使用文本手稿中描述的参数。接下来，转到“计算指标”选项卡，然后选择“单元格计数”。然后，要获取突触体信号的区域，请转到“数据缩减”菜单，然后在“分析”下选择“细胞分析”。再次，转到“主掩码和计数”选项卡，然后在“通道”下选择“RFP 拼接图像”，然后使用文本手稿中详述的参数。接下来，转到计算指标选项卡，然后选择对象总和面积。在“数据缩减”选项卡中，单击“确定”以允许软件分析所有采集的图像。分析图像后，导出每个时间点的对象总和面积和单元格计数值。然后将对象总和面积除以单元格计数以计算每个时间点的归一化面积。如果比较多种治疗或基因型，请使用给定的方程计算吞噬指数。未分化的iPSCs显示出紧凑的集落形态，边缘清晰。解离的iPSC形成称为EB的球形聚集体，并在分化的第四天之前逐渐增大。当EB被铺板以产生PMP时，它们附着在基质胶涂层的板上，并且一层细胞扩散并围绕球形聚集体。在第28天，悬浮液中出现具有大细胞质与细胞核比的圆形细胞。强烈建议在层粘连蛋白521包被的平板中培养iPSC而不是基质胶，因为层粘连蛋白521包被的板中的PMP产量更高。在PMP暴露于IMG培养基后，细胞在细长过程的存在下获得具有小细胞质的小胶质细胞样形态。此外，通过免疫荧光染色验证小胶质细胞的身份。通常，超过95%的细胞表达IB1，约90%的细胞表达P2RY12和TMEM119。蛋白质印迹分析证实，iPSC衍生了人突触体表达的突触标志物、突触素和突触后密度蛋白95的下运动神经元。与初始时间点相比，到10小时时，红色荧光信号很强烈，因为大多数突触体已被吞噬，并定位于酸性细胞内区室。在细胞松弛素D处理的IMG中，红色信号的强烈减少持续0小时和10小时表明检测到的信号是由吞噬事件引起的。吞噬突触体的面积随着时间的推移而增加，直到16小时。然而，来自细胞松弛素D处理细胞的红色信号面积不随时间增加。iPS衍生的小胶质细胞样细胞可用于各种应用，包括吞噬作用和神经发育和神经退行性疾病研究中的炎症反应。

Research

JoVE Journal

Neuroscience

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

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

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 ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

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 ...

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

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

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 ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

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

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 ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和体外活细胞吞噬测定中分化