Name: targeted and selective treatment of pluripotent stem cell-derived teratomas using external beam radiation in a small-animal model
Uploaded: 2019-02-17T13:00:00
Description: Watch this Scientific Journal Video about Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model at JoVE.com

作者要感谢国家卫生研究院 r01 hl134830 (pkn)、k08 hl135343 (ks) 和 5f32hl134221 (jwr);霍华德休斯医学院 (asl);和斯坦福心血管研究所 (asl) 的支持。

这项动物实验是在斯坦福大学的机构审查委员会和实验动物护理管理小组下批准和进行的。
1. ipsc 的细胞培养
<ol><li>在涂有基底膜基质 (例如, matrigel, 称为以下基质) 的6孔板上重新编程而获得的人的 ipsc。</li>
	<li>每天用在37°c 和 5% co 2 孵育的浓缩培养基 (见材料表) 改变 ipsc 的培养基.</li>
	<li>一旦细胞达到80–90% 的融合 (大约每 4天), 每口添加1毫升重组细...

<ol>
	<li>Sallam, K., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+biology:+insights+from+molecular+imaging.">Embryonic stem cell biology: insights from molecular imaging.</a> Methods in Molecular Biology. 660, 185-199 (2010).</li><li>Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenicity+as+a+clinical+hurdle+for+pluripotent+stem+cell+therapies.">Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.</a> Nature Medicine. 19 (8), 998-1004 (2013).</li><li>Amariglio, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-derived+brain+tumor+following+neural+stem+cell+transplantation+in+an+ataxia+telangiectasia+patient.">Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.</a> PLOS Medicine. 6 (2), e1000029 (2009).</li><li>Kuriyan, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+Loss+after+Intravitreal+Injection+of+Autologous+&amp;#34;Stem+Cells&amp;#34;+for+AMD.">Vision Loss after Intravitreal Injection of Autologous &amp;#34;Stem Cells&amp;#34; for AMD.</a> The New England Journal of Medicine. 376 (11), 1047-1053 (2017).</li><li>Berkowitz, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioproliferative+Lesion+of+the+Spinal+Cord+as+a+Complication+of+&amp;#34;Stem-Cell+Tourism&amp;#34.">Glioproliferative Lesion of the Spinal Cord as a Complication of &amp;#34;Stem-Cell Tourism&amp;#34.</a> The New England Journal of Medicine. 375, 196-198 (2016).</li><li>Zhang, W. Y., de Almeida, P. E., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation:+A+tool+for+monitoring+pluripotency+in+stem+cell+research.">Teratoma formation: A tool for monitoring pluripotency in stem cell research.</a> StemBook. , (2012).</li><li>Scott, C. T., Magnus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wrongful+termination:+lessons+from+the+Geron+clinical+trial.">Wrongful termination: lessons from the Geron clinical trial.</a> STEM CELLS Translational Medicine. 3 (12), 1398-1401 (2014).</li><li>Strauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geron+trial+resumes,+but+standards+for+stem+cell+trials+remain+elusive.">Geron trial resumes, but standards for stem cell trials remain elusive.</a> Nature Biotechnology. 28 (10), 989-990 (2010).</li><li>Coghlan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+mutations+put+stem+cell+trial+on+hold.">Unexpected mutations put stem cell trial on hold.</a> New Scientist. 227 (3033), 9 (2015).</li><li>Tang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antibody+against+SSEA-5+glycan+on+human+pluripotent+stem+cells+enables+removal+of+teratoma-forming+cells.">An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.</a> Nature Biotechnology. 29 (9), 829-834 (2011).</li><li>Lee, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cell+number+on+teratoma+formation+by+human+embryonic+stem+cells.">Effects of cell number on teratoma formation by human embryonic stem cells.</a> Cell Cycle. 8 (16), 2608-2612 (2009).</li><li>Tano, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+method+for+detecting+undifferentiated+human+pluripotent+stem+cells+as+impurities+in+cell+therapy+products+using+a+highly+efficient+culture+system.">A novel in vitro method for detecting undifferentiated human pluripotent stem cells as impurities in cell therapy products using a highly efficient culture system.</a> PLoS One. 9 (10), e110496 (2014).</li><li>Kuroda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+in+vitro+methods+for+detection+of+residual+undifferentiated+cells+in+retinal+pigment+epithelial+cells+derived+from+human+iPS+cells.">Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.</a> PLoS One. 7 (5), e37342 (2012).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113 (7), 1005-1014 (2006).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+embryonic+stem+cell+misbehavior+and+suicide+gene+ablation.">Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation.</a> Cloning Stem Cells. 9 (1), 107-117 (2007).</li><li>Kotini, A. G., de Stanchina, E., Themeli, M., Sadelain, M., Papapetrou, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+Mutations,+Ganciclovir+Resistance,+and+Teratoma+Formation+in+Human+iPSCs+Expressing+an+HSVtk+Suicide+Gene.">Escape Mutations, Ganciclovir Resistance, and Teratoma Formation in Human iPSCs Expressing an HSVtk Suicide Gene.</a> Molecular Therapy - Nucleic Acids. 5, e284 (2016).</li><li>Smith, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+susceptibility+to+DNA+damage+of+pluripotent+stem+cells+facilitates+pharmacologic+purging+of+teratoma+risk.">Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.</a> STEM CELLS Translational Medicine. 1 (10), 709-718 (2012).</li><li>Choo, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+against+undifferentiated+human+embryonic+stem+cells+by+a+cytotoxic+antibody+recognizing+podocalyxin-like+protein-1.">Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1.</a> Stem Cells. 26 (6), 1454-1463 (2008).</li><li>Yorke, E., Gelblum, D., Ford, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+safety+in+external+beam+radiation+therapy.">Patient safety in external beam radiation therapy.</a> American Journal of Roentgenology. 196 (4), 768-772 (2011).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+micro-computed+tomography-based+image-guided+conformal+radiotherapy+system+for+small+animals.">Development of a micro-computed tomography-based image-guided conformal radiotherapy system for small animals.</a> International Journal of Radiation Oncology &#8226; Biology &#8226; Physics. 78 (1), 297-305 (2010).</li><li>Slatkin, D. N., Spanne, P., Dilmanian, F. A., Gebbers, J. O., Laissue, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subacute+neuropathological+effects+of+microplanar+beams+of+x-rays+from+a+synchrotron+wiggler.">Subacute neuropathological effects of microplanar beams of x-rays from a synchrotron wiggler.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (19), 8783-8787 (1995).</li><li>Lee, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Report:+External+Beam+Radiation+Therapy+for+the+Treatment+of+Human+Pluripotent+Stem+Cell-Derived+Teratomas.">Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas.</a> Stem Cells. 35 (8), 1994-2000 (2017).</li><li>Berkowitz, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioproliferative+Lesion+of+the+Spinal+Cord+as+a+Complication+of+&amp;#34;Stem-Cell+Tourism&amp;#34.">Glioproliferative Lesion of the Spinal Cord as a Complication of &amp;#34;Stem-Cell Tourism&amp;#34.</a> The New England Journal of Medicine. 375 (2), 196-198 (2016).</li><li>Swijnenburg, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+stem+cells+reveals+patterns+of+survival+and+immune+rejection+following+transplantation.">In vivo imaging of embryonic stem cells reveals patterns of survival and immune rejection following transplantation.</a> Stem Cells and Development. 17 (6), 1023-1029 (2008).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+de+novo+imaging+of+human+embryonic+stem+cell-derived+teratoma+formation.">Noninvasive de novo imaging of human embryonic stem cell-derived teratoma formation.</a> Cancer Research. 69 (7), 2709-2713 (2009).</li><li>Priddle, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+human+embryonic+stem+cells+transplanted+in+vivo+in+murine+and+chick+models.">Bioluminescence imaging of human embryonic stem cells transplanted in vivo in murine and chick models.</a> Cloning and Stem Cells. 11 (2), 259-267 (2009).</li><li>Dale, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-rate+effects+in+targeted+radiotherapy.">Dose-rate effects in targeted radiotherapy.</a> Physics in Medicine &amp; Biology. 41 (10), 1871-1884 (1996).</li></ol>

"干细胞旅游" 受害者的临床前数据和传闻案例证实, 患上畸胎瘤的风险是与 psc 治疗23有关的一个严重缺陷。因此, 制定谨慎的方法来预防和治疗与干细胞疗法相关的肿瘤风险是促进再生干细胞疗法临床翻译的一个重要步骤。在本文中, 我们描述了一种方法的治疗靶向靶向 psc 相关畸胎瘤使用 ebrt 在小鼠模型中, 并显示了动态萎缩的辐照肿瘤使用 bi 成像。
我们?...

在过去几十年里, 组织再生干细胞疗法的发展遇到了一些障碍, 阻碍了有效临床部署的努力。这些障碍包括分娩部位细胞保留不良、干细胞免疫原性和肿瘤形成畸胎瘤1的可能性。致瘤是特别的临床关注, 因为它可能有潜在的危害干细胞移植受者2。由于不受管制的干细胞注射而形成的肿瘤的说法已经在多个临床环境中报告了 3,4,5。畸胎瘤形成的可能性是多能干细胞 (psc) 发展中最经常提到的临床问题, 并导致多个高调胚胎干细胞 (esc) 和诱导多能干细胞 (ipsc) 的延迟和取消试验6,7,8,9。因此, 有迫切需要一个翻译调查致力于提供适当的治疗, 如果这些医源性肿瘤出现。
到目前为止, 控制干细胞不当行为的大多数策略都集中在减少有致瘤潜力为 2,10的 psc 数量上。遗憾的是, 畸胎瘤的形成只需要少量的残留细胞 (例如, 1 x10 4至 1 x10 5细胞11), 远远低于目前可用的检测 12所引用的检测限值,13. 使用这些预分配方法的其他限制包括效率低和费用高、依赖可能不适合较新的组织工程方法的单细胞悬浮液以及细胞的潜在损伤生存和雕刻。
很少有研究涉及畸胎瘤形成后的治疗方案。也许最深入研究的策略是将 "自杀" 基因整合到干细胞中 14,15。这种方法包括基因操纵干细胞, 以纳入诱导凋亡激活基因, 可通过药物刺激后注射激活, 从而提供一种救援方法, 如果注射细胞产生畸胎瘤。然而, 这种方法存在重大缺陷, 包括私营保安公司基因改造的非目标效应和耐药的逐渐发展的可能性 16.类似的方法利用小分子通过抑制抗凋亡途径诱导psc 选择性细胞死亡17。其他群体使用多能性表面标记抗体, 如 podocalyxin-like (podxl)18, 将其靶向 psc 的细胞死亡。小分子或抗体传递的时间如果传递过早, 将对 psc 的治疗潜力产生重大影响, 如果传递得太晚, 可能缺乏治疗效果。此外, 还没有研究以这种方式使用的小分子和抗体的系统性影响。
治疗这些肿瘤的另一种方法依赖于使用外束放射治疗 (ebrt)。ebrt 是目前用于治疗实体肿瘤的主要方法之一.欧洲 brt 的创新, 包括质子束和立体定向放射外科的发展, 使病理结构的目标增强, 同时避免对正常组织的损害, 使保形欧洲爆炸器成为解决畸胎瘤的理想选择在解剖敏感的结构中形成20。此外, 这种方法避免了基因操纵或病毒转导的干细胞, 这两个充满了额外的临床安全性和有效性的问题15。最后, 微辐照器的发展使 ebt 在啮齿类动物21中的应用成为可能。
在本文中, 我们演示了如何通过在小鼠体内注射人的 ipsc 来创建小动物形成畸胎瘤的模型。然后, 我们展示了如何应用 ebrt 选择性地消除这些肿瘤在体内与最小的损害周围的组织。这种方法为 psc 衍生的畸胎瘤提供了有针对性的治疗, 同时避免了生物分子和肽的系统传递以及 psc 的基因操纵所产生的非目标效应。为了调查目的, 我们提供了一个可选的步骤, 通过生物发光成像 (bri) 来跟踪肿瘤对放射治疗的反应。

<table>
	<tbody>
		<tr>
			<td>Induced Pluripotent Stem Cell Control Line</td>
			<td>Stanford University</td>
			<td>Nguyen Lab</td>
			<td>Cell culture of iPSC</td>
		</tr>
		<tr>
			<td>Corning matrigel basement membrane matrix 354234</td>
			<td>Fisher Scientific</td>
			<td>CB-40234</td>
			<td>Cell culture of iPSC</td>
		</tr>
		<tr>
			<td>Essential 8 culture medium</td>
			<td>ATCC-The global bioresource center</td>
			<td>30-2203</td>
			<td>Cell culture of iPSC</td>
		</tr>
		<tr>
			<td>Tryple E</td>
			<td>Gibco</td>
			<td>12605-036</td>
			<td>Cell culture of iPSC</td>
		</tr>
		<tr>
			<td>Y27632 inhibitor 2 HCL (ROCK Inhibitor)</td>
			<td>Fisher Scientific</td>
			<td>S104950MG</td>
			<td>Cell culture of iPSC</td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Cyagen</td>
			<td>P170721-1001cjn</td>
			<td>Transduction of iPSC with double fusion reporter gene</td>
		</tr>
		<tr>
			<td>Polyrbrene Infection/Transfection Reagent</td>
			<td>Millipore Sigma</td>
			<td>TR-1003-G</td>
			<td>Transduction of iPSC with double fusion reporter gene</td>
		</tr>
		<tr>
			<td>Fluc-eGFP reporter gene driven by ubiquitin promoter</td>
			<td>Stanford University</td>
			<td>Sam Gambhir lab</td>
			<td>Transduction of iPSC with double fusion reporter gene</td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td>Transduction of iPSC with double fusion reporter gene and BLI</td>
		</tr>
		<tr>
			<td>Flow cytometer (BD FACSARIA III)</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td>Transduction of iPSC with double fusion reporter gene</td>
		</tr>
		<tr>
			<td>microplate spectrofluorometer (Glomax Navigator System)</td>
			<td>Promega Bio Systems, Sunnyvale, CA</td>
			<td>GM2000</td>
			<td>Transduction of iPSC with double fusion reporter gene</td>
		</tr>
		<tr>
			<td>Xenogen IVIS 200&#160;</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>BLI</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>CDS019936</td>
			<td>irradiation</td>
		</tr>
		<tr>
			<td>X-Rad SmART image-guided irradiator&#160;</td>
			<td>Precision X-ray Inc., North Branford, CT</td>
			<td>X-Rad SmART</td>
			<td>irradiation</td>
		</tr>
		<tr>
			<td>RT_Image software package</td>
			<td>Stanford University (http://rtimage.sourceforge.net/)</td>
			<td>RT_Image v0.2&#946;</td>
			<td>Irradiation</td>
		</tr>
	</tbody>
</table>

targeted and selective treatment of pluripotent stem cell-derived teratomas using external beam radiation in a small-animal model

在全球范围内, "干细胞旅游" 这一无管制的干细胞移植的受害者越来越多, 这引起了人们对干细胞移植安全性的担忧。虽然分化而非未分化细胞的移植是常见的做法, 畸胎瘤仍然可以产生于残留的未分化干细胞在移植时的存在或自突变在分化细胞。由于干细胞疗法通常被提供到解剖敏感的部位, 即使是小肿瘤也可能在临床上造成毁灭性的破坏, 导致失明、瘫痪、认知异常和心血管功能障碍。手术进入这些场所的机会也可能有限, 使患者几乎没有治疗选择。因此, 控制干细胞不当行为对干细胞治疗的临床翻译至关重要。
外部光束辐射提供了一种有效的手段, 提供有针对性的治疗, 以减少畸胎瘤的负担, 同时尽量减少对周围器官的伤害。此外, 这种方法避免遗传操作或干细胞的病毒转导-这与额外的临床安全性和疗效问题有关。在这里, 我们描述了一种在小鼠体内创建多能干细胞衍生畸胎瘤的方案, 并应用外束放射治疗在体内选择性地消融这些肿瘤。

注射小鼠通常会在4-8周后显示畸胎瘤的生长形成, bli 成像证实了这一点 (图 2)。当细胞分娩一个月后, 用18戈瑞的累积剂量照射, 肿瘤会急剧缩小, 导致荧光素酶信号显著下降 (图 2)。重要的是, 从辐照部位取5毫米的正常组织似乎没有任何重大损伤 (图 3)。
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Watch this Scientific Journal Video about Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model at JoVE.com

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

多能干细胞衍生畸胎瘤治疗策略的研究对干细胞治疗的临床翻译具有重要意义。在这里, 我们描述了一种协议, 首先, 在小鼠体内产生干细胞衍生的畸胎瘤, 然后, 选择性地靶向和治疗这些肿瘤在体内使用一个小动物辐照器。

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

该程序的总体目标是选择性地针对小鼠的干细胞衍生畸胎瘤，以评估小动物外束放射治疗的体内有效性。外部放射束疗法的主要优点是，它是干细胞相关特拉姆托马的靶向疗法，避免了系统提供疗法的离目标的不利影响。外束放射治疗的进步使得小畸胎瘤能够特定靶向，同时避免对正常组织的损伤，使这种疗法成为治疗辐射敏感畸胎瘤的的理想之选。该方法不仅能够深入了解细胞疗法的优化策略，而且具有在临床上适用于人类疗法的潜力。首先采取一个六井板包含 iPSC。为了诱导免疫缺陷动物形成四核糖核糖，在室温下用双融合报告基因转导的6孔板中每井加入1毫升重组细胞分离酶混合物，在室温下孵育5分钟。在孵育结束时，通过移液分散细胞，通过向每一井添加同等体积的细胞培养培养所来停止酶反应。将单细胞悬浮液拉在 15 毫升锥形管中进行计数。离心完成后，吸上经，将颗粒重新在30微升的Matrigel基质溶液中，将管子放在冰上。如果使用双聚变报告转染细胞，这些双阳性影响细胞也应该悬浮在30微升的基质中。接下来，将每个细胞群体装入装有28.5针的1毫升注射器中，并确认每个麻醉的8至10周大的麻醉性裸体接受者小鼠对头趾捏缺乏反应。然后，皮下将一个细胞基质混合物注入每个受接受动物的侧翼。在接种后的适当实验时间点，将每公斤二维他孕酮注射375毫克。10分钟后，根据标准生物发光成像方案，每个麻醉记者探针中的生物发光信号每隔5分钟使用1分钟采集窗口注射动物30分钟。对于畸胎激光照射，在适当的实验时间点，首先将麻醉接受动物放在图像引导的临床前照射器的床上，并使用 40 峰值千伏、2 毫安 X 射线束获取一组 400 个超过 360 度的投影微计算断层扫描图像，然后将图像重建为 0.2 毫米等向像素大小的体积图像。重建畸胎的体积图像至关重要。这些图像将有助于规划放射治疗，为特定治疗只向畸胎瘤，同时尽量减少周围组织的任何非目标辐射。然后使用微CT图像和RT图像软件包来规划放射治疗方案。连续三天进行治疗，共为目标肿瘤提供18个灰色。注射小鼠通常表现出畸胎瘤生长形成四至四周后肿瘤细胞注射，通过生物发光成像证实。当在细胞分娩一个月后用累积的18个灰色照射时，肿瘤将急剧缩小，导致荧光酶信号显著重合减少。重要的是，正如这些图像所说明的，从辐照地点采集的5毫升正常组织活检似乎没有受到任何重大损害。看完这段视频后，您应该清楚地了解如何在体内产生干细胞衍生的畸胎瘤，并有效安全地用外部光束辐射来治疗它们。这种简单的方法需要获得一个主体的高分辨率CT图像，之后可以规定一系列辐射束疗法来照射目标肿瘤，同时避免相邻组织。不要忘记，使用辐射可能极其危险，执行此过程时应始终采取预防措施，例如使用适当的防护罩。

Research

JoVE Journal

Medicine

Protocol for Long Duration Whole Body Hyperthermia in Mice

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

科研

医学

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

使用小动物看更衣室反转恢复（SALLI）的MRI大鼠心脏功能和心肌形态学评估

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System

放射规划助理-一个流线型的全自动放疗治疗计划系统

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

建立干细胞治疗心肌梗塞心力衰竭的猪模型

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...