Name: generation of induced pluripotent stem cells from turner syndrome (45xo) fetal cells for downstream modelling of neurological deficits associated with the syndrome
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Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndr

上述研究的财政支持由马尼帕尔高等教育学院提供。该生产线的表征部分在NCBS的M.M. Panicker实验室进行。我们感谢阿南德诊断实验室在核型分析方面的帮助。

FCV是在马尼帕尔医院伦理委员会的批准下从班加罗尔的马尼帕尔医院获得的。
注：所有缓冲液和溶液的组成见表1。
1. 从胎儿绒毛膜绒毛（FCV）中分离成纤维细胞
<ol><li>
胶原酶中的样本采集和组织分解
	<ol>
<li>在无菌条件下在磷酸盐缓冲盐水（PBS）中收集FCV，并将（室温下）转运到细胞培养设施。</li>
		<li>将绒毛转...

<ol>
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生成细胞遗传学异常胎儿组织的稳定细胞模型对于使有缺陷的表型永久化是必要的。iPSC途径是永久保存缺陷特性的最有效的细胞制备方法20。
多能干细胞（PSC）显示出自我更新和分化成特化细胞的特性，让人联想到早期裂解胚胎21。因此，PSC可以作为研究过早流产胎儿的早期分子，细胞和发育缺陷的优秀模型。
在本文?...

非整倍体导致人类出生缺陷/先天性畸形和妊娠丢失。~50%-70%的妊娠丢失标本显示细胞遗传学异常。在怀孕早期丢失的非整倍体胚胎不容易获得用于实验分析，因此需要开发其他密切代表人类胚胎发生的模型。来自被诊断患有遗传疾病的细胞的诱导多能干细胞（iPSCs）已被用于模拟代表性的遗传不规则性及其对胎儿发育的影响1，2，3，4。这些iPSC类似于发育中胚胎的外胚层细胞，可以概括胚胎形成的早期事件。它们允许理解和表征早期哺乳动物胚胎中细胞谱系和模式的发育程序。iPSCs以前来自皮肤成纤维细胞和羊膜细胞，来自非整倍性综合征的产前诊断测试，如单体X（特纳综合征），8三体性（Warkany综合征2），13三体性（Patau综合征）和部分三体11;22（伊曼纽尔综合征）提供了有关失败发展的宝贵见解4。
特纳综合征（TS）是一种罕见的疾病，其特征是女性不育，身材矮小，内分泌和代谢紊乱，自身免疫性疾病风险增加以及心血管疾病易感性5。虽然它是唯一可存活的单体综合征，但它对导致自然流产的发育中的胚胎也是致命的6。TS的存活个体表现出细胞中X染色体物质的改变程度。核型的范围从完全失去一条X染色体（45，XO）到像45，XO / 46，XX这样的马赛克;45，XO / 47，XXX，环形染色体的存在，Y染色体物质的存在等5。
该综合征的诊断通常通过对有症状个体的血液进行核型分析，并进行绒毛膜绒毛取样 （CVS） 以检测早期非整倍性综合征。由于非整倍性综合征约占自然流产的30%，因此在自然流产时对受孕产物（POC）进行核型分析是常规的。这些胎儿细胞包括具有细胞遗传学异常的绒毛膜绒毛和衍生自它们的iPSCs，为研究非整倍性综合征4，6提供了宝贵的生物材料来源。TS iPSCs先前已通过逆转录病毒重编程4从羊膜细胞，通过逆转录病毒重编程6从羊膜细胞，通过逆转录病毒重编程6从绒毛膜绒毛的成纤维细胞（通过产前诊断获得）建立，通过仙台病毒重编程从血液单核细胞7通过慢病毒重编程从TS个体的皮肤成纤维细胞4建立。.由于我们实验室的主要重点是了解发育障碍，因此我们从POC中产生了TS iPSCs，特别是自然流产的绒毛膜绒毛成分8。从该胎儿组织中分离出的所有细胞均具有45XO核型，并产生具有相同核型的iPSCs。这些iPSC是独一无二的，因为它们是第一个从流产胎儿产生的，并为研究非整倍性相关的妊娠失败提供了宝贵的资源。本文提供了通过外显体重编程从这种独特的细胞源产生iPSCs的详细方法。
iPSC生成的早期方法使用病毒转导和转座子来传递重编程因子。诱导细胞多能性的方法已经从使用整合逆转录病毒载体9、可切除慢病毒载体10、11和基于转座子的方法12发展到非整合性腺病毒载体13和仙台病毒载体14。基于逆转录病毒和慢病毒的重编程虽然有效，但涉及将重编程因子整合到宿主染色体中，导致插入突变，从而在iPSC中产生不可预见的影响。此外，基于病毒的重编程可防止 iPSC 的转化应用。已经探索了基于RNA的系统15和直接蛋白递送16，以完全消除与使用病毒和DNA转染相关的潜在风险。然而，这些方法已被证明是低效的。
2011年，Okita等人报道了通过shRNA增强TP53抑制的外生质粒来提高重编程效率。他们还用非转化性LMYC（与MYC相关的小细胞肺癌）代替了cMYC，以提高hiPSC的安全性。这些外生体质粒表达5种重编程因子：OCT4，LIN28，SOX2，KLF4，LMYC和shRNA用于TP5317，18。这些载体在染色体外保持，并在连续培养时从重编程的细胞中丢失，从而使品系在10-15次传代内无转基因。核细胞分离是一种特殊形式的电穿孔，可将核酸直接输送到宿主细胞的细胞核中。它是将重编程质粒递送到各种细胞类型的有效方法。外显体质粒具有成本效益，并补偿了细胞核摘除的高成本。该方法在优化条件下高效且可重复，可从各种体细胞中产生稳定的iPSC。在该协议中，我们描述了通过对外显体重编程质粒进行核切除术，从胎儿组织中分离的成纤维细胞产生iPSCs的方法。以下是从胎儿绒毛膜绒毛中分离成纤维细胞，质粒纯化，核切除，从重编程板中挑选集落以及建立稳定的iPSCs的详细方案。
必须确认新生成的iPSC中存在多能性状。这包括多能性相关因素的证明（例如，碱性磷酸酶表达，NANOG，SSEA4，Tra 1-80，Tra 1-81，E-钙粘蛋白;通常用免疫荧光或基因表达测定显示），通过体外分化测定鉴定三个胚层以验证其分化潜力，核型分析以确定染色体含量，STR分型以建立与亲本细胞的身份，验证外源性基因的丢失， 以及更严格的体内检测，如畸胎瘤形成和四倍体互补。在这里，我们描述了核型分析的表征方案，活细胞碱性磷酸酶染色，通过免疫荧光检测多能性相关生物标志物，体外分化测定和证明外源基因丢失的方法19。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.15% trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>27250018</td>
			<td>G Banding</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>4&#39;, 6 diamidino-2-phenylindole</td>
			<td>Sigma Aldrich</td>
			<td>D8417</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>Sigma Aldrich</td>
			<td>SRP3003</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Live Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14353</td>
			<td>AP staining</td>
		</tr>
		<tr>
			<td>AMAXA Nucleofector II</td>
			<td>Lonza</td>
			<td>-</td>
			<td>Nucleofection</td>
		</tr>
		<tr>
			<td>AmnioMAX II complete media</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>11269016</td>
			<td>Medium specific for foetal chorionic villi cell cultures</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>HiMedia</td>
			<td>TC021</td>
			<td>Plasmid purification</td>
		</tr>
		<tr>
			<td>Anti Mouse IgG (H+L) Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11059</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti Rabbit IgG (H+L) Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti Rabbit IgG (H+L) Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11035</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15240096</td>
			<td>Contamination control</td>
		</tr>
		<tr>
			<td>Anti-E-Cadherin</td>
			<td>BD Biosciences</td>
			<td>610181</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-Nanog</td>
			<td>BD Biosciences</td>
			<td>560109</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-OCT3/4</td>
			<td>BD Biosciences</td>
			<td>611202</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-SOX17</td>
			<td>BD Biosciences</td>
			<td>561590</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-SOX2</td>
			<td>BD Biosciences</td>
			<td>561469</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-SSEA4</td>
			<td>BD Biosciences</td>
			<td>560073</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Anti-TRA 1-81</td>
			<td>Millipore</td>
			<td>MAB4381</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>basic Fibroblast Growth Factor[FGF2]</td>
			<td>Sigma Aldrich</td>
			<td>F0291</td>
			<td>Pluripotency medium</td>
		</tr>
		<tr>
			<td>Bone Morphogenetic Factor 4</td>
			<td>Sigma Aldrich</td>
			<td>SRP3016</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3059</td>
			<td>Blocking</td>
		</tr>
		<tr>
			<td>Collagen Human Type IV</td>
			<td>BD Biosciences</td>
			<td>354245</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Collagenase blend</td>
			<td>Sigma Aldrich</td>
			<td>C8051</td>
			<td>Digestion of foetal chorionic villi</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>FastDigest EcoR1</td>
			<td>Thermo Scientific</td>
			<td>FD0274</td>
			<td>Restriction digestion</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2518</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>HiMedia</td>
			<td>S011</td>
			<td>G Banding</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>HiMedia</td>
			<td>AS001</td>
			<td>Fixative for karyotyping</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Insulin solution human</td>
			<td>Sigma Aldrich</td>
			<td>I9278</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>Insulin Transferrin Selenite</td>
			<td>Sigma Aldrich</td>
			<td>I1884</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>KAPA HiFi PCR kit</td>
			<td>Kapa Biosystems</td>
			<td>KR0368</td>
			<td>OriP, EBNA1 PCR</td>
		</tr>
		<tr>
			<td>KaryoMAX Colcemid</td>
			<td>Thermo Fisher Scientific</td>
			<td>15210040</td>
			<td>Mitotic arrest for karyotyping</td>
		</tr>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10829018</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>HiMedia</td>
			<td>M1151F</td>
			<td>Plasmid purification</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>Differentiation assays</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>HiMedia</td>
			<td>MB113</td>
			<td>Fixative for karyotyping</td>
		</tr>
		<tr>
			<td>Myosin ventricular heavy chain &#945;/&#946;</td>
			<td>Millipore</td>
			<td>MAB1552</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>NHDF Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VAPD-1001</td>
			<td>Nucleofection</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Fixing cells</td>
		</tr>
		<tr>
			<td>pCXLE-hOCT3/ 4-shp53-F</td>
			<td>Addgene</td>
			<td>27077</td>
			<td>Episomal reprogramming Plasmid</td>
		</tr>
		<tr>
			<td>pCXLE-hSK</td>
			<td>Addgene</td>
			<td>27078</td>
			<td>Episomal reprogramming Plasmid</td>
		</tr>
		<tr>
			<td>pCXLE-hUL</td>
			<td>Addgene</td>
			<td>27080</td>
			<td>Episomal reprogramming Plasmid</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>&#160; Thermo Fisher Scientific,</td>
			<td>&#160;15070063</td>
			<td>Pluripotency and Embryoid body medium</td>
		</tr>
		<tr>
			<td>Phalloidin- Tetramethylrhodamine B isothiocyanate</td>
			<td>Sigma Aldrich</td>
			<td>P1951</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>1 X PBS 1 tablet of PBS dissolved in 200mL of deionized water and sterilized by autoclaving 
			Storage: Room temperature. 
			PBST- 0.05% Tween 20 in 1X PBS. 
			Storage: Room temperature.</td>
		</tr>
		<tr>
			<td>Plasmid purification Kit- Midi prep</td>
			<td>QIAGEN</td>
			<td>12143</td>
			<td>Plasmid purification</td>
		</tr>
		<tr>
			<td>Potassium Chloride Solution</td>
			<td>HiMedia</td>
			<td>MB043</td>
			<td>Hypotonic solution for karyotyping</td>
		</tr>
		<tr>
			<td>QIAamp DNA Blood Kit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>Genomic DNA isolation</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td>Hepatocyte differentiation medium</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>HiMedia</td>
			<td>RM255</td>
			<td>Hypotonic solution for karyotyping</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>HiMedia</td>
			<td>MB031</td>
			<td>Permeabilisation</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>25300054</td>
			<td>Subculture of &#160;foetal chorionic villi fibroblasts</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>HiMedia</td>
			<td>MB067</td>
			<td>Preparation of PBST</td>
		</tr>
		<tr>
			<td>&#946; III tubulin</td>
			<td>Sigma Aldrich</td>
			<td>T8578</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td>Differentiation assays</td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from turner syndrome (45xo) fetal cells for downstream modelling of neurological deficits associated with the syndrome

染色体非整倍体可引起严重的先天性畸形，包括中枢神经系统畸形和胎儿死亡。产前遗传学筛查纯粹是诊断性的，并不能阐明疾病机制。虽然来自非整倍体胎儿的细胞是带有染色体非整倍性的宝贵生物材料，但这些细胞的寿命很短，限制了它们在下游研究实验中的应用。诱导多能干细胞（iPSC）模型的生成是细胞制备非整倍体性状永久保存的有效方法。它们是自我更新的，并分化成特化细胞，让人联想到胚胎发育。因此，iPSC是研究早期发育事件的极好工具。特纳综合征（TS）是一种与完全或部分缺失的X染色体相关的罕见疾病。该综合征的特征是不孕症，身材矮小，内分泌，代谢，自身免疫和心血管疾病以及神经认知缺陷。以下方案描述了从TS（45XO）胎儿组织中分离和培养成纤维细胞，通过细胞核切除递送表观体重编程质粒，然后进行表征，从而产生整合游离的TsiPSCs。重新编程的TSIPSCs最初通过活细胞碱性磷酸酶染色进行筛选，然后对多能性生物标志物进行广泛的探测。对选定的菌落进行机械解剖，传代几次，并使用稳定的自我更新细胞进行进一步的实验。细胞表达多能转录因子OCT4、NANOG、SOX2、细胞表面标志物SSEA 4和TRA1-81是典型的多能干细胞。最初的45XO核型在重新编程后保留。TSiPSCs能够形成胚状体并分化成内胚层，中胚层和表达外胚层的细胞谱系特异性生物标志物（（SRY BOX17），（肌球蛋白室重性CHAINα / β），（βIII管蛋白））。外源性外生体质粒自发丢失，在细胞中第15代后未检测到。这些TSiPSCs是一种宝贵的细胞资源，用于模拟导致与特纳综合征相关的神经认知缺陷的有缺陷的分子和细胞神经发育。

从具有45XO核型的自发流产胎儿中生成无整合的iPSC 我们从具有特纳综合征（TS）特异性45XO核型的FCV中分离出成纤维细胞，并用外显体重编程质粒对其进行核感染，以产生可用于综合征下游建模的TSIpSC，特别是相关的神经功能缺陷（图1a&b）。我们使用非整合的表观载体和核检点进行转染实验（图1 c&d）。我们跟踪?...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndr

Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

该协议描述了通过核切除递递递外体质粒，然后描述用于iPSC表征和神经元分化的方法，从胎儿组织成纤维细胞中产生整合的游离iPSCs。

从特纳综合征（45XO）胎儿细胞生成诱导多能干细胞，用于与该综合征相关的神经功能障碍的下游建模

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening ...

特纳综合征是一种与完全或部分缺失的X染色体相关的罕见疾病。该综合征的特征是不孕症、身材矮小、心血管疾病和神经认知缺陷。在某些情况下，它会导致胎儿死亡。虽然非整倍体胎儿的体细胞具有生物学价值，但它们的寿命很短，因此限制了它们在研究中的使用。因此，iPSC的产生是细胞制备非整倍体性状永久保存的有效方法。它们是自我更新的，可以分化成特殊的细胞类型，让人联想到早期胚胎发育。通过核假体递送非整合的外显体重编程质粒是产生完全重编程和稳定的iPSC的有效且可重复的方法。这种方法也可用于难以编程的细胞。在该协议中，我们描述了iPSCs从胎儿材料中再生的无症状。将收集的胎儿绒毛膜绒毛转移到无菌培养皿中。用含有1X抗生素抗真菌剂溶液的PBS洗涤几次。通过移液完全去除 PBS。将1ml胶原酶混合物加入绒毛中，并在37摄氏度下孵育10分钟或直到组织分解。孵育后，通过添加含有10%胎儿牛血清的培养基中和胶原酶。将培养皿的内容物转移到15ml管中。离心消化物以收集分解的绒毛和释放的细胞。小心地倾析上清液。在T25培养瓶和AmnioMAX培养基中将绒毛与释放的细胞一起分解，并生长直至获得汇合成纤维细胞培养物。扩增成纤维细胞和培养物，以制备用于后续转染和表征实验的储备。制备含有重编程质粒的碱培养物。根据制造商的说明，使用Midi质粒纯化试剂盒分离质粒。将每个托盘重新悬浮在适当体积的DNAse RNAse游离水中，以获得每毫升1微克的终浓度。按照制造商的说明，使用ECoR1限制性酶切试剂盒验证质粒。胰蛋白酶消化绒毛绒毛成纤维细胞。中和，然后离心以收集细胞。倾析上清液，并将细胞重新悬浮在5ml OPTI-MEM中。使用血细胞计数器计数细胞，并取出100万个细胞进行细胞核切除。离心机得到细胞托盘。使用Amaxa NHDF神经因子试剂盒进行细胞核切除。通过混合试剂盒中提供的0.5 mL补充剂和2.25 mL核因子溶液来制备神经因子试剂。除去100微升核因子溶液，并向其中加入1微克每个质粒。在此混合物中轻轻重新悬浮100万个细胞。将细胞DNA悬浮液转移到比色皿中，确保样品覆盖比色皿的底部，没有任何气泡。盖上比色皿，然后插入支架。选择核因子程序D-23以获得高效率，然后应用。从支架中取出比色皿，然后加入 1 mL AmnioMAX 培养基。使用试剂盒中提供的移液器将内容物轻轻转移到组织培养处理过的培养皿中。将细胞在37摄氏度的加湿二氧化碳培养箱中孵育。将细胞在AmnioMAX培养基中维持10天，然后转移到多能培养基20天。对于iPSC扩增，使用拉动的玻璃移液器手动解剖在重编程皿中形成的胚胎干细胞样菌落。适当传播 iPSC，每 5 到 7 天按摩一次。iPCSs多能标志物碱性磷酸酶阳性。通过将iPSC菌落切成小块并将其接种在胚状体培养基中的低附着培养皿中，产生胚胎样体。外胚层分化是通过在外胚层分化培养基中接种第四天的胚状体来诱导的。类似地，中胚层分化是通过在中胚层分化培养基中接种第八天胚体来诱导的。为了诱导内胚层分化，在内胚层分化培养基的单层中培养iPSCs。在重编程过程中监测成纤维细胞的形态变化。它们表达上皮细胞样形态并形成致密的菌落。这些细胞还具有高度细胞核与细胞质的比例，这是培养物中多能细胞的典型特征。iPSCs具有45 XO载流子类型，多能性标记OCT4，NANOG，SOX 2，SSEA 4，TRA-1-81和E-CADHERIN呈阳性。对细胞进行染色以形成具有代表性的生殖层标记物。因此，特纳综合征会出现神经认知缺陷。来自特纳综合症iPSC的神经元可以是一个很好的模型。这是在培养皿中建模的，以了解正常解离特纳综合征的新系列。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

从原生多能诱导干细胞中生成胚胎体表面的人原始生殖细胞样细胞

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...