Name: characterization of thymic settling progenitors in the mouse embryo using in vivo and in vitro assays
Uploaded: 2015-06-09T15:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays at JoVE.com

巴斯德研究所，INSERM，法新社国立德RECHERCHE ANR（格兰特的淋巴细胞&#39;），兴中未来投资计划和"香格里拉法甲勒驳癌症"的支持。

伦理声明:所有实验均根据巴斯德研究所伦理宪章，批准由法国农业部执行，欧盟的指导方针。一个机械手与小啮齿动物外科手术培训，由农业部法国教育部认证，完成所有手术干预。 注:见附件表1显示了5步计划程序。 1.选用胚胎<ol><li>使用36 C57BL / 6 CD45.2女性，女12例CD45.1，12 C57BL / 6 CD45.1男性和12 C57BL / 6 CD45.2男性。穿越女雌雄基因型会产生胚胎CD45.1 / 2和CD45.1作为TSP或CD45.2用作收件人胸腺叶源的来源。众议院单独所有男性。 </li><li>为了获得胚胎E18 TSP隔离，放置2女在笼子里有一只公CD45.1下午6时许，嫁接实验前21天。检查阴道塞的存在，第二天，中午之前。国家环保总局打分插入女性。 </li><li>以获得E14胚胎由TSP定植，则重复上述（1.2）的方法，用雄性CD45.2，接枝实验前17天。 </li><li>以获得的胚胎以分离E13的TSP，则重复上述（1.2）的方法，用雄性C57BL / 6 CD45.1，接枝实验前16天。 </li></ol> 2.解剖胚胎在一个水平层流罩注:在嫁接实验前两天。 <ol><li>至少5分钟期间通过颈脱位或由CO 2气体的渐进施用牺牲怀孕女性，在一个饱和密闭室。通过心肺运动最终逮捕确保死刑。湿用70％乙醇的腹部。 </li><li>做一个纵行切口在皮肤在中线腹部和提拉肌肤除了打开它。不接触消化道打开与镊子和剪刀腹膜。拉出双歧uter我们并从与剪刀阴道分离它。 </li><li>将子宫在含有40-50毫升DPBS一个90×15毫米的培养皿和切割横向每个胚的蜕膜之间。 </li><li>随着对精尖剪刀和细镊子取出子宫的肌肉膜，取出胚胎的胎盘和卵黄囊之间切割，取出amnios，这是保持胚胎。 </li><li>将胚胎在培养皿中90×15毫米含有的HBSS + 1％胎牛血清（FCS）。比E15更旧的胚胎，除去头上的一把剪刀之前，任何进一步的解剖。 </li></ol> 3.隔离胸腺<ol><li>根据双目放大镜，将胚胎，趴在仰卧位（腹面），在湿纱布床上用品。 </li><li>插入一个镊子纵向地沿胸骨的软骨，捏和打开胸电网。用弯钳，拉胸壁一边想象的Thymic裂片上气管的每一侧。 </li><li>小心地用细镊子捏下面各持有叶，将它们放置在含有1ml HBSS + 1％FCS培养皿。隔离裂片和带有两​​个1毫升注射器+＆G⅜"针除去周围结缔组织，而不破坏胶囊。 </li><li>在3毫升HBSS + 1％FCS的洗叶，以消除血细胞。 注:E15之前，胸腺裂片位于中间的气管和后来熔丝的每一侧，以构成双叶的器官。 </li></ol> 4.悬浮细胞<ol><li>梳理出的叶双目放大镜下，用安装在1毫升注射器2台26摹针，在HBSS + 1％FCS。通过尼龙网过滤细胞悬浮液。 </li></ol> 5.用染色荧光抗体和细胞分选注意:所有的抗体被预先滴定以获得种群最佳定义（titrat离子可以与抗体批次而变化）。 <ol><li>孵育胸腺细胞（E13或E18）为15-30分钟，在4℃下用100微升生物素化的抗体进行染色谱系阳性细胞的鸡尾酒（抗CD19，抗Gr1的，抗TER119，抗NK1.1 ，抗CD11c的，抗CD3，抗CD4，抗CD8，抗CD25）。 </li><li>洗去多余的抗体降速管用4ml的HBSS + 1％FCS在280 XG 7分钟，消除上清，重新悬浮在新鲜HBSS + 1％FCS沉淀。重复离心。 </li><li>在15分钟，在4℃下孵育E18生物素标记的细胞与链霉微珠和洗涤细胞的HBSS 1％FCS的两倍。重悬在2ml的HBSS + 1％FCS中细胞。最E13胸腺细胞是谱系阴性，因此不需要之前细胞分选的MACS富集。 </li><li>通过对LS柱的细胞，根据生产商的说明。当所有的细胞悬液进入列，加入2mL HBSS + 1％FCS。恢复将4ml柱的流过和离心将细胞在280×g下7分钟。 </li><li>重悬要么耗尽E18或总E13胸腺细胞在50μl的TSP抗体混合物（抗CD117的APC，抗CD135 PE，抗CD127 PECy7，抗CD24 FITC，抗CD44 APCCy7和链霉太平洋蓝的粒料），并孵育20分钟，在4℃下在黑暗中。 </li><li>用4ml的HBSS + 1％FCS和洗涤细胞重悬浮细胞于1ml HBSS + 1％FCS中用碘化丙锭。 </li><li>排序的TSP林- CD44 + CD117 + CD24 低 CD127 + CD135 +细胞在流式细胞仪（4激光器）到1.5 mL管含有200微升完全培养基+ 20％FCS。门首先根据其大小和粒度的FSC和SSC通道的细胞。消除死细胞（碘化丙啶染色），门出双峰和得分荧光指数尺度。大约100或600的TSP可以从一个E13或者一个E18胸腺，respectiv获得伊利。 </li></ol> E14殖民6.胸腺肺叶祖:悬滴法<ol><li>照射（30格雷= 30戈瑞）E14从胚胎CD45.2胸腺叶，培养前3小时。 注:E14或E15胸腺裂片已成功地被用作T细胞祖细胞的收件人。使用后妊娠天导致过早坏死因器官的大尺寸收件人胸腺裂片而胸腺裂片在较早妊娠天很差发展，可能是由于上皮细胞的发育不完全。 </li><li>离心机排序的TSP并重新悬浮细胞在培养基（OPTI-MEM，10％FCS的完全培养基，青霉素（50单位/ ml），链霉素（50微克/毫升）和2β巯基乙醇（50μM）），使得35微升包含500 TSP。 </li><li>放置细胞悬浮液的35微升滴60孔酶标板的每一个其它公。 注:35微升液滴可以熔化，如果他们被放置在相邻的孔中。 </li><li>将一个辐射波瓣每一滴的表面上。关闭寺崎板和转动板倒置以让细胞达到下降通过重力根尖极。 </li><li>击中轻轻倒置板的上侧，迫使瓣滑动到液滴的前端缘。孵育倒置酶标板在湿润的培养箱中48小时，在37℃+ 5％CO 2。定植后，无论是文化E14 thymi在FTOC 7，或移植在收件人成年小鼠8的肾囊。 </li></ol> 7.胚胎胸腺器官培养<ol><li>放置3个ml完全培养基在35毫米的培养皿。发布Isopore表面滤膜过滤漂浮在介质上。将重构裂片上用钳子（不超过8裂片上一个膜）膜的边缘。 </li><li>将含有胸腺裂片90毫米的培养皿内，连同35毫米的菜培养皿，无盖，含2水:毫升R，以确保最佳的湿度。孵育12天在37℃+ 5％CO 2。 </li></ol> 8.在移植肾包膜在无菌条件下注:肾实质被结缔组织形成胶囊包围。子囊区域是特别丰富的血液和淋巴管，从而提供一个合适的环境移植物的发展（ 例如胸腺裂片，胰岛或新生儿心脏）。移植物通常是在左肾，因为它比右肾更容易获得。 CD3 - / -雄性小鼠作为收件人从而避免移植物抗宿主反应，由于连接到Y染色体次要组织相容性抗原，因为E15之前性别决定是不容易做到。 <ol><li>消毒仪器和佩戴沿手术无菌手套。通过腹膜内注射氯胺酮10毫克/毫升+赛拉嗪1毫克的溶液麻醉小鼠/毫升在PBS中稀释（每10g体重50至100μl），用1ml注射器。通过检查缺乏叉指反射的验证麻醉。放置在每只眼睛一滴水力Optimune凝胶，以防止在麻醉过程中干燥。 注:溶液应即席制备和注射前温热至室温。麻醉持续至少20分钟。麻醉的程度应该都沿手术来控制。麻醉可以通过腹腔内注射一半剂量为每20分钟被延长。 </li><li>将鼠标在其右侧，下一个水平层流罩。消毒手术区域用70％乙醇和10％的碘化物dermic溶液。用钳子部的老鼠毛沿2厘米长度正好后腿的关节的上方。剃须手术区。 </li><li>用手术刀，使第一，皮肤组织的切口1.5厘米长度，然后用剪刀剪断肌肉组织。施加轻微压力，切口两侧，这将迫使肾脏出腹腔。 </li><li>应用生理盐水用棉芽，以保持湿润肾脏。如果您在暴露器官困难，用平头镊子拔出周围的脂肪组织到肾脏附后。用棉签放在下面的器官维持肾脏出了腹膜后腔。 </li><li>有两个细镊子，使胶囊2-3毫米的孔（避免触及实质，以防止出血）。在整个手术过程中保持暴露肾囊连续湿润。 </li><li>插入仔细裂片肾囊下，通过保持打开的胶囊的壁并朝向边缘极包膜下滑动接枝。将高达每肾脏6瓣。 </li><li>替换复古腹腔肾脏。缝合肌肉腱膜，然后用医生个人的结皮。湿用10％的碘化聚维酮溶液的伤口。把鼠标放在加热PAð在37℃。 </li><li>调查移植鼠标，直到完全恢复从麻醉中通过心脏，呼吸和运动晶须，粘膜颜色，直到总回收率看到自给自足的运动控制。 注意:我们建议注射的镇痛溶液（Buprenorphin，0.1毫克/公斤）内肌肉刚刚手术后和2天手术后，以防止手术后的疼痛。 </li><li>保持经营的动物隔离，直到完全恢复。检查隔日伤口的线圈，以验证和预防感染。从未观察到伤口感染下此过程。 注:外周血细胞从CD3每周分析- / -接枝小鼠允许我们检测来自群体源自使用CD45同种异型标记和T细胞谱系特异性抗体（ 图3）或者E13或E18祖胸腺。 </li></ol> TSP的后代通过流式细胞仪分析9 <ol><l我&gt;单独逗FTOCs获得单细胞悬液（见协议第4节）。 </li><li>染色细胞悬浮液用含有抗体识别CD45.1 PECy7，CD45.2 AmCyan，CD4 APCCy7，CD8 APC，CD3太平洋蓝，Vγ5FITC，PEVδ1的抗体混合物和孵化在第5节个人裂片。 </li><li>重悬的细胞在HBSS + 1％FCS +碘化丙啶和流式细胞仪中的流动分析（见结果在图2中）。 </li></ol>

<ol>
	<li>Rodewald, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+organogenesis.">Thymus organogenesis.</a> Annu Rev Immunol. 26, 355-388 (2008).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat Rev Immunol. 6, 127-135 (2006).</li><li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus.">MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.</a> Nature. 362, 70-73 (1993).</li><li>Douagi, I., Andre, I., Ferraz, J. C., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+T+cell+precursor+activity+in+the+murine+fetal+thymus:+evidence+for+an+input+of+T+cell+precursors+between+days+12+and+14+of+gestation.">Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation.</a> Eur J Immunol. 30, 2201-2210 (2000).</li><li>Calderon, L., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic,+context-dependent,+and+hierarchical+functions+of+epithelial+components+in+thymic+microenvironments.">Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments.</a> Cell. 149, 159-172 (2012).</li><li>Adolfsson, 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=Identification+of+Flt3++lympho-myeloid+stem+cells+lacking+erythro-megakaryocytic+potential+a+revised+road+map+for+adult+blood+lineage+commitment.">Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.</a> Cell. 121, 295-306 (2005).</li><li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li><li>Bhandoola, A., von Boehmer, H., Petrie, H. T., Zuniga-Pflucker, 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=Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors:+plenty+to+choose+from.">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26, 678-689 (2007).</li><li>Ramond, 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=Two+waves+of+distinct+hematopoietic+progenitor+cells+colonize+the+fetal+thymus.">Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.</a> Nat Immunol. 15, 27-35 (2014).</li><li>Allison, J. P., Havran, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+T+cells+with+invariant+gamma+delta+antigen+receptors.">The immunobiology of T cells with invariant gamma delta antigen receptors.</a> Annu Rev Immunol. 9, 679-705 (1991).</li><li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+thymus+organ+culture.">Fetal thymus organ culture.</a> CSH Protoc. , (2007).</li><li>Schmitt, T. M., Zuniga-Pflucker, 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=Induction+of+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro.">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li><li>Barbee, S. D., 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=Skint-1+is+a+highly+specific,+unique+selecting+component+for+epidermal+T+cells.">Skint-1 is a highly specific, unique selecting component for epidermal T cells.</a> Proc Natl Acad Sci U S A. 108, 3330-3335 (2011).</li><li>Douagi, I., Colucci, F., Di Santo, J. P., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+earliest+prethymic+bipotent+T/NK+progenitor+in+murine+fetal+liver.">Identification of the earliest prethymic bipotent T/NK progenitor in murine fetal liver.</a> Blood. 99, 463-471 (2002).</li><li>Malissen, M., 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=Altered+T+cell+development+in+mice+with+a+targeted+mutation+of+the+CD3-epsilon+gene.">Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene.</a> EMBO J. 14, 4641-4653 (1995).</li></ol>

作者宣称，他们有没有竞争的财务权益。

两个主要的测定法可用于分析T细胞分化的体外 。最近报道是造血祖细胞与骨髓基质细胞，OP9的共培养，表达Noth1的配位体，δ-样1或4 12，这2-D测定是容易执行的，高效的和灵敏的，允许分析在单个细胞水平。然而，它既不支持T细胞发育超出DP的阶段也没有γδDETC 13的产生，这两者需要与胸腺上皮的直接相互作用。 FTOCs长期以来一直用于分析T细胞发育<sup...

T淋巴细胞，轴承αβ或γδT细胞受体（TCR），区分在一个专门的机构，胸腺。充分发展胸腺分为两个不同的区域:皮质，其中胸腺祖发展和地点的胸腺细胞重新排列高效TCR的β和α链基因是从程序性死亡（被称为阳性选择的过程）获救;和髓质，在这里选择的胸腺细胞与太强的反应，以自配体被删除（负选择）1,2。胸腺来源于第三咽囊以为稍后由间充质细胞3包围的内胚层层。它是由起始于天胚胎E12，此后，连续招募所需的正常T细胞发育4造血祖细胞定植。胸腺移民通过连续的发育阶段发展，通过严格监管的程序，发起并策划迈ntained由胸腺Notch信号传导途径的相互作用时的活化与其配体，δ-样4，表达于胸腺上皮细胞（TECs的）5。 胸腺细胞发育开始于所谓的CD4 - CD8 -双阴性（DN）的阶段。 ，DN2（CD25 + CD44 +），DN3（CD25 + CD44 - ）和DN4（CD25 - CD44 - ） - DN胸腺细胞可以根据CD25和CD44的表达到DN1（CD44 + CD25）进一步细分。 CD24（HSA）和CD117（的c-Kit）进一步细分DN1隔成5个亚群，其中DN1a和b对应于早期胸腺祖细胞（ETP）。胸腺细胞重新排列T细胞受体δ，β和α链在DN阶段，并进行预-TCR选择（DN3，DN4阶段）。他们进一步迁移到的CD4 + CD8 +双阳性（DP）室，其中的TcRα链重新排列之前正和负塞莱ction。在这个阶段，大多数胸腺细胞被消除，只有一小部分（3-5％）达到的CD4 +或CD8 +成熟T细胞室。 淋巴分化途径过程通过造血干细胞的生成多潜能祖细胞（MPP）和淋巴引发的多潜能祖细胞（LMPP）失去的红细胞和巨核细胞潜力6阶段。 LMPP由缺乏分化血细胞标记物表型定义（谱系阴性，林- ），的c-Kit（CD117）的表达，的Sca-1和Flt3的/ FLK2（CD135）和没有白细胞介素的可检测水平的（ IL）-7受体α链（IL-7rα或CD127）。 LMPPs进一步分化为普通淋巴祖（CLP）7，到这个阶段已经失去了产生骨髓细胞的能力。电保留淋巴细胞（B和T细胞），NK细胞，DC和先天淋巴样细胞（ILC）的潜力，并从LMPP相差CD1的表达27，以及缺乏高水平的Sca-1的。 虽然胸腺解决祖细胞（TSP）的性质已经被广泛讨论8，它成为近日明确表示，TSP变化表型，分化潜能和功能，整个开发9。我们的体外和体内测定进行到TSP，通过FACS分离细胞从任E13（第一波）或E18（第二波）排序表征。胎儿胸腺器官培养（FTOC）与E13的E18和祖人数相等，不同的轴承异型标志殖民，使跟随他们类似的发展环境和子代细胞揭示内在特性，这两种类型的祖细胞之间的不同照射胸腺叶。无论是通过E13 E18或定植TSP胸腺叶允许开发无选择，由于双方的祖细胞之间的竞争。 在体内移植的殖民胸腺裂片进一步表明还牛逼他E13的成熟后代和E18 TSP 在体内生物不同的特性。从第一波的TSP快速生成T细胞，但引起低的数字αβγδ和T细胞。在后者我们检测Vγ5Vδ1树突上皮T细胞（DETC）中，具有一个不变的TcR，迁移到它们施加在伤口愈合的函数和胚胎发育10中仅产生的表皮。与此相反，TSP从第二波需要更长的时间，以产生高数量的TcR + T细胞和不能生成DETC。

<table><tbody><tr><td>90 x 15 mm and 35 x 15 mm plastic tissue culture Petri dishes.</td><td>TPP</td><td>T93100/T9340</td><td>Sampling</td></tr><tr><td>26 G &amp;frac38;&quot; 0.45 x 10 mm syringes with needles</td><td>BD Plastipak</td><td>300015</td><td>Cell suspension</td></tr><tr><td>Nylon mesh bolting cloth sterilized 50/50 mm pieces.</td><td>SEFAR NITEX</td><td>03-100/32</td><td>Filtration of cells</td></tr><tr><td>Ethanol 70%.</td><td>VWR</td><td>83801.36</td><td>Sterility actions</td></tr><tr><td>Iodide Povidone 10% (Betadine)</td><td>MEDA Pharma</td><td>314997.8</td><td>Sterility actions</td></tr><tr><td>Ketamine 100 mg/ml (stock solution)</td><td>MERIAL</td><td></td><td>Anesthesic</td></tr><tr><td>Xylazine 100 mg/ml in PBS (stock solution)</td><td>Sigma</td><td>X1251-1G</td><td>Anesthesic and muscle relaxant</td></tr><tr><td>Buprenorphin 0.3 mg/ml stock solution</td><td>AXIENCE</td><td></td><td>Morphinic analgesic</td></tr><tr><td>Ophtalmic gel (0.2% cyclosporin)</td><td>Schering-Plough Animal Health</td><td></td><td>Eye protection</td></tr><tr><td>DPBS (+ CaCl&lt;sub&gt;2&lt;/sub&gt;, MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>GIBCO Life Technology</td><td>14040-174</td><td>to isolate embryos</td></tr><tr><td>HBSS Hanks&#039; Balanced Salt Solution (+ CaCl&lt;sub&gt;2&lt;/sub&gt;, MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>GIBCO Life Technology</td><td>24020-091</td><td>to wash out the blood and dissect the embryos</td></tr><tr><td>OPTI-MEM I GlutaMAX I</td><td>GIBCO Life Technology</td><td>51985-026</td><td>Medium for cultures</td></tr><tr><td>Fetal calf serum</td><td>EUROBIO</td><td>CVFSVF00-0U</td><td>additive for cultures</td></tr><tr><td>Penicilin and streptomycin</td><td>GIBCO Life Technology</td><td>15640-055</td><td>Antibiotics for cultures</td></tr><tr><td>2-Mercaptoethanol</td><td>GIBCO Life Technology</td><td>31350010</td><td>additive for cultures</td></tr><tr><td>LEICA MZ6 Dissection microscope</td><td>LEICA</td><td>MZ6 10445111</td><td>Occular W-Pl10x/23</td></tr><tr><td>Cold lamp source</td><td>SCHOTT VWR</td><td>KL1500 compact</td><td>Two goose neck fibers adapted</td></tr><tr><td>Silicone elastomer</td><td>World Precision Instruments</td><td>SYLG184</td><td>Dissecting Pad</td></tr><tr><td>Spoon, round and perforated</td><td>Fine Science Tools</td><td>10370-18</td><td>Dissection tools</td></tr><tr><td>Fine iris scissors</td><td>Fine Science Tools</td><td>14090-09</td><td>Dissection tools</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15018-10</td><td>Dissection tools</td></tr><tr><td>Forceps: Dumont #5/45 Inox 11 cm</td><td>F.S.T.</td><td>11253-25</td><td>Dissection tools</td></tr><tr><td>2 pairs of fine straight watchmakers&amp;rsquo; forceps Dumont #5 11 cm fine tips.</td><td>Fine Science Tools</td><td>11295-20</td><td>Dissection tools</td></tr><tr><td>Polyamid thread with needle 6-0 C-3 3/8c</td><td>ETHICON</td><td>F2403</td><td>for sutures</td></tr><tr><td>Needle-holder</td><td>MORIA/F.S.T.</td><td>12060-02</td><td>for sutures</td></tr><tr><td>Heating pad</td><td>VWR</td><td>100229-100</td><td>To maintain mouse temperature during anesthesy</td></tr><tr><td>Membrane Isopore RTTPO2500</td><td>DUTSCHER</td><td>44210</td><td>For FTOC</td></tr><tr><td>Terasaki 60 wells plates</td><td>FISHER</td><td>1x270 10318801</td><td>For hanging drop technique</td></tr><tr><td>Gauze swabs steriles 7.5 x 7.5 cm</td><td>Hydrex</td><td>11522</td><td>To apply disinfecting solution</td></tr><tr><td>Fluorescence or biotin labelled antibodies</td><td>BD Biosciences, Biolegend or e-Biocsiences&amp;nbsp;</td><td>Clone Number see table below</td><td>Staining cells</td></tr><tr><td>MACS Columns/Streptavidin Microbeads</td><td>Miltenyi Biotec</td><td>130-042-401/130-048-101</td><td>Cell depletion</td></tr><tr><td>Mice</td><td>Charles Rivers Laboratories (CD45.1) and Janvier Labs&amp;nbsp; (CD45.2)</td><td>C57BL/6 CD45.1 OR CD45.2</td><td>Source of cells and thymic lobes</td></tr><tr><td>Mice</td><td>CDTA Orl&amp;eacute;ans, France</td><td>CD 3 epsilon Ko CD45.2</td><td>Grafting experiment</td></tr></tbody></table>

characterization of thymic settling progenitors in the mouse embryo using in vivo and in vitro assays

表征胸腺解决祖重要的是要了解T细胞发育的前期阶段，胸腺，必须设计一种T细胞置换淋巴细胞减少患者的策略。我们研究了胸腺从13鼠胚胎日和18 thymi沉降祖细胞由两个体外互补和体内技术，无论是基于"悬滴"的方法。这种方法允许辐照的殖民胸腺胎儿与叶和E13 / E18还是通过CD45异型标记，从而跟随他们的后代区分胸腺祖。定植混合种群允许分析的祖细胞生物学特性细胞自主的分歧，而与殖民人口要么尽可能去除选择性的竞争压力。殖民胸腺瓣也可以在免疫缺陷雄性受体小鼠允许成熟T细胞后代的体内分析，如叔种群动态接枝他外周免疫系统和不同的组织和器官的殖民化。胎儿胸腺器官培养透露，E13祖细胞迅速发展成为成熟的全部CD3 +细胞，引起了规范γδT细胞亚群，被称为树突状上皮性T细胞。相比之下，E18祖细胞有延迟分化和无法生成树突上皮T细胞。 / - -胸腺-接枝的CD3外周血监测小鼠进一步表明，E18胸腺沉降祖生成，随着时间的推移，更大的成熟T细胞比它们的E13对应的号码，无法在短期内胎儿胸腺可以理解的特征器官培养。

为了选择一个方法来消耗内源性的胸腺细胞，允许殖民祖最好的发展胸腺叶，我们比较了在照射后殖民胸腺叶T细胞重建的水平或5天的脱氧鸟苷（D-GUA）治疗。结果表明，尽管有在培养的第9天无差异，照射裂片含有更多的T细胞比具有的d卦处理，在这样12.天，照射是小于d卦治疗更为合适后获得T细胞发育胸腺定植（ 图1）。研究E13和E18的TSP的发育潜能，我们拓殖E14照射胸腺裂片与两种类...

Watch this Scientific Journal Video about Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays at JoVE.com

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

characterization-thymic-settling-progenitors-mouse-embryo-using-vivo

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

8.2K 次观看.  Institut Cochin. This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny. 

视频: Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

科研

发育生物学

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

在胸腺的鼠标采用双光子显微镜的原位成像

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

生物学

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

免疫与感染

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

癌症研究

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

<meta charset="utf8"></head><body>使用 iPSC 来源的类器官重建胸腺微环境

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>作者聚焦：利用人胸腺类器官推进胸腺上皮细胞和 T 细胞研究

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

在三维纤维蛋白水凝胶中形成人胸腺类器官

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

胸腺表征祖定居在小鼠胚胎使用

摘要

探索更多视频

此视频中的章节

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

在胸腺的鼠标采用双光子显微镜的原位成像

活体成像的鼠标使用2 - 光子显微镜的胸腺

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

对T细胞推导

隔离和不同的老年小鼠胸腺移植的移植。

在三维纤维蛋白水凝胶中形成人胸腺类器官

在三维纤维蛋白水凝胶中形成人胸腺类器官

在三维纤维蛋白水凝胶中形成人胸腺类器官

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应