我们感谢普华永在图准备方面的帮助。这项工作得到了中国国家自然科学基金 (81530004、31425016) 和中国科学技术部 (2016YFA0100500) 的资助。. 进行了实验并草拟了手稿;--编辑了手稿。两位作者阅读并批准了最后的手稿。

包括动物学科在内的所有程序都已获得中国科学院动物研究所伦理审查委员会的批准. 
 1. 材料准备 <ol> <li> 对0.65 和 #181 进行消毒; m 过滤器在紫外线灯下产生紫外线, 在清洁的长凳上为4小时, 并且在 2 h 标记以后转动滤光器。
	注意: 当使用 UV 光时, 请穿戴适当的保护. </li>
	<li> 121 和 #176 用高压灭菌器消毒不锈钢网格, 30 分钟. 
	注: 需要一定高度的不锈钢网格, 以支持气接口上的过滤器, 这可以用钢丝网 ( 图 1 A ) 实现. </li>
	<li> 将氟西汀稀释到10和 #181 的最终浓度; M 进入 M5300 长期培养基, 均匀混合. 
	注: 对于一个六井板, 2 毫升 M5300 长期培养基是适当的每一个井。氢化可的松在 10 -6 M 可以被选择性地稀释到培养基中. </li>
	<li> 将培养基与氟西汀加在10和 #181; M 成六孔板或碟, 并将灭菌不锈钢网放入介质中. </li>
	<li> 洗涤0.65 和 #181; m 过滤器与煮沸的水二次为3分钟每次在玻璃细胞培养盘为绝育。每次洗涤后, 将过滤器放入磷酸盐缓冲盐水 (PBS) 2 分钟, 并将湿过滤器放在气接口的不锈钢网格上, 如 图 1 A 所示 [示意图在中心]. 
	注: 用蒸压灭菌器产生的开水可实现无菌, 设备应及时取出灭菌器, 避免温度降低. </li>
</ol> 2。大会外植体培养 <ol> <li> 在 E10.5 或 E11 的胚胎中小心地将股东大会区域与镊子分开。
	<ol> <li> 用解剖剪刀将子宫从孕鼠中剥离出来, 并将胚胎从子宫中分离出来. </li>
		<li> 清除胚体中的卵黄囊、脐带和内脏. </li>
		<li> 小心移除背神经组织和周围肌肉组织. </li>
		<li> 用钳切断前、后肢部位的组织, 将其与胚体分离。 图 1 B-C 显示了解剖的周年大会的示意图表示和形态学. </li>
	</ol> </li> <li> 将被解剖的公司分别放在气接口 ( 图 1 ) 上的滤镜上, 并在 5% CO 2 中的区域性中, 37 和 #176; C 为 2-3 天. 
	注意: 不要将被解剖的周年大会放在钢网的钢丝上, 并确保过滤器在文化气界面。除了周年大会, 蛋黄囊和胎儿肝脏也可以培养与此系统 7 . </li>
</ol> 3。表达式分析 <ol> <li> mRNA 级别 <ol> <li> 将培养的公司收集到1毫升 PBS 和离心机在4和 #176; C, 310 x g 为 6 min. </li>
		<li> 除去上清液后, 根据制造商和 #39 的指示, 从收集的公司中提取总 RNA, 并采用单相苯酚溶液. </li>
		<li> 总 RNA (2 和 #181; g) 然后用 m-MLV 反转录酶反向转录, 以获得 cDNA 为模板。定量 real-time PCR 方法是用 SYBR 绿和 Gapdh 作为内部控制进行的. </li>
	</ol> </li> <li> 蛋白质级别 <ol> <li> 收集在步骤3.1.1 中如上所述的培养的年度股东大会。
		<li> 用细胞裂解缓冲液 (10 毫米的三盐酸盐, 10 毫米氯化钠, 0.5% NP-40) 制备含有蛋白酶抑制剂的蛋白质, 并用于西方印迹检测先前描述的蛋白质水平 13 . </li>
	</ol> </li> </ol> 4。菌落形成单位在文化 (CFU-C) 化验中 <ol> <li> 在培养 2-3 天后, 收集1毫升磷酸盐缓冲盐水 (pbs) 和离心机在4和 #176; C, 310 x g 为6分钟和离解与200和 #181; L 胶原酶 (0.1% 在 PBS) 在37#176; C. 
	注: 胶原酶产生单细胞悬浮液所需时间约20分钟。每5分钟摇晃一次含周年大会的管子可以加速消化过程。200和 #181; 0.1% 胶原酶用于每一个股东大会和200和 #181; l 10% 血清用于停止反应. </li>
	<li> 在超低附着的 M3434 介质中培养单细胞悬浮 24-井板. 
	注意: 为避免污染, 青霉素-链霉素溶液可选择性添加到培养基中。由于 M3434 介质是粘性的, 所以使用1.0 毫升无针头的注射器来混合细胞和培养基. </li>
	<li> 7-10 天后在37和 #176 培养; C 在 5% CO 2 中, 每种类型的菌落的数量, 包括爆裂形成单位-红 (BFU), CFU-granulo-单核细胞 (CFU-GM) 和 CFU-粒, 红细胞, 巨噬细胞, 巨 (CFU-GEMM)根据形态学进行区分, 并以倒置显微镜 ( 图 2 C ) 进行评分. </li>
</ol> 5。 在体内 移植试验 <ol> <li> 菌落形成单位-脾脏 (CFU S) 化验 <ol> <li> 2-3 天的外植体培养, 收集公司和准备单细胞悬浮通过孵化200和 #181; L 胶原酶 (0.1% 在 PBS) 为20最小 </li>
		<li> 在细胞注射前, 对8至10周的老雄性 C57BL/6 小鼠进行致死性照射 (9 X 射线), 4-5 小时. </li>
		<li> 将辐照的鼠标放入抑制剂以限制其活动. </li>
		<li> 用75% 酒精浸泡的棉签擦拭尾部, 以促进静脉舒张. </li>
		<li> 注射0.5 胚当量 (ee) 或 1 ee 单细胞悬浮剂, 静脉注射到经照射的小鼠尾静脉, 用1.0 毫升注射器. 
		注: 细胞悬浮与 pbs 和0.5 毫升 pbs 每接收者是一个适当的容量。25口径或26口径的针用于注射 1.0 cc 注射器。没有空气可以被引入到注射器。用消毒棉签按压注射部位止血. </li>
		<li> 十一天后移植, 收集并修复 Bouin 和 #39 中的受体小鼠的脾脏 (15 毫升1.22% 苦味酸酸饱和水溶液, 2 毫升40% 甲醇和1毫升乙酸) 1-2 天, 用80% 酒精清洗 1-2 天。从宏观上计算脾脏的可见菌落数, 并确定每个 ee 的菌落数量. 
		#8203; 注意: Bouin 和 #39 的固色剂, 穿戴适当的保护. </li>
	</ol> </li> <li> 长期移植化验 <ol> 2-3 #160;d 总是外植体培养, 收集公司, 并以200和 #181 孵化制备单细胞悬浮液; L 胶原酶 (0.1% 在 PBS) 为 20 min. <li>
		<li> 对8至10周的老雄性 C57BL/6 小鼠进行致死性照射 (9 x 射线), 在细胞注射前 4-5 小时. </li>
		<li> 将辐照的鼠标放入抑制剂以限制其活动. </li>
		<li> 用75% 酒精浸泡的棉签擦拭尾部, 以促进静脉舒张. </li>
		我注射0.5 胚当量 (ee) 或 1 ee 单细胞悬浮剂, 静脉注射到经辐照后的小鼠尾静脉, 用1.0 毫升注射器。每位获奖者注射 1 ee 2 和 #215; 10 5 骨髓细胞 (CD45.1 背景). 
		注: 注射器中不能引入空气。用消毒棉签按压注射部位止血. </li>
		<li> 四月后, 收集接受者的骨髓并将其用于重构试验。只有与 #8805 的收件人; 10% 捐助者派生的嵌被认为是成功重组的表现. </li>
	</ol>
	</li>
</ol>

<ol>
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众所周知, 成熟的干细胞在骨髓中可以重新填充受照射的接受者的血液系统。与这些功能干细胞不同的是, 在小鼠胚胎的周年大会上出现的干细胞是不成熟的。直接移植结果表明, I 型 (ve cad+ CD45- CD41+) 和类型 II (ve cad+ CD45+) 前干细胞具有没有重构能力20。利用股东大会的外植体培养体系, 在干细胞的新生中, 可以在移植试验中重建受体<sup clas...

造血干细胞 (干细胞) 是组织特异的成体干细胞, 具有多向的潜能, 包括红、髓细胞和淋巴细胞, 以及自我的能力。最近的研究表明, 最早的干细胞产生于一个专门的内皮细胞, 称为 hemogenic 内皮细胞 (他), 通过内皮到造血转移 (EHT) 在腹壁的背主动脉1,2 ,3,4。一旦从胚胎 (E) 10.5 到 E12.5 在小鼠胚胎中形成了主动脉-性腺-肾 (年会) 区域, 干细胞就会迁移到胎儿肝脏进行扩张, 最终在个体的生命中殖民骨髓以维持成人造血。5,6. 尽管这一研究已经进行了许多年, 但 HSC 产生和发展的基本机制仍未得到完全理解。
与体外不同的是, 斑马鱼胚胎的受精和发育, 小鼠胚胎的宫内发育使得在胚胎发生过程中的最终造血研究更不方便。虽然使用击倒 (ko) 小鼠的基因实验是常用的, 但某些高鼠的缺乏也限制了它们在造血研究领域的应用。此外,在体内抢救实验在 KO 小鼠中不易进行。自1996年以来, 在该领域的先驱在7中为造血研究开发了 "年度" 外植体培养。在这一文化体系的帮助下, 已经确定了股东大会区域的腹侧组织以促进 HSC 活动, 而背部组织则发挥相反的作用8,9。在 HSC 的发展中, 应用了 Mpl、蛋白、蛋白、BMP 和刺猬的信号, 确定了血清素、外植体培养系统的作用10、11、12、13、14. 重要的是, 它也是一种常用的方法来挽救突变体胚胎的造血缺损13,15。

<table><tbody><tr><td>durapore 0.65 &amp;micro;m filters</td><td>Millipore</td><td>R5BA63787</td><td>AGM explant culture</td></tr><tr><td>M5300 long-term culture medium</td><td>Stem Cell Technologies</td><td>5300</td><td>AGM explant culture</td></tr><tr><td>Trizol&amp;nbsp;</td><td>Tiangen</td><td>5829</td><td>RNA extration</td></tr><tr><td>M-MLV reverse transcriptase</td><td>Promega</td><td>90694</td><td>reverse transcription</td></tr><tr><td>SYBR Green</td><td>Qiagen</td><td>A6002</td><td>quantatitive real-time PCR</td></tr><tr><td>protease inhibitor</td><td>Roche</td><td>55622</td><td>Protein extration</td></tr><tr><td>collagenase</td><td>Sigma</td><td>C2674</td><td>digestion of AGM tissues</td></tr><tr><td>MethoCult GF M3434 medium</td><td>Stem Cell Technologies</td><td>3434</td><td>CFU-C assay</td></tr><tr><td>ultra-low attachment 24-well plates</td><td>Costar</td><td>3473</td><td>CFU-C assay</td></tr><tr><td>C57BL/6 CD45.2 mice</td><td>Beijing HFK Bioscience Co. Ltd</td><td></td><td>CFU-S assay</td></tr><tr><td>C57BL/6 CD45.1 mice</td><td>Beijing HFK Bioscience Co. Ltd</td><td></td><td>Long-term transplantation</td></tr><tr><td>phosphate buffered saline</td><td>Life Technologines</td><td>8115284</td><td>AGM Collection</td></tr><tr><td>Penicillin-Streptomycin solution</td><td>HyClone</td><td>SV30010</td><td>AGM explant culture</td></tr><tr><td>anti-CD45.2-PE-CY7</td><td>eBioscience</td><td>25-0454-80</td><td>Long-term transplantation</td></tr><tr><td>anti-CD45-FITC</td><td>eBioscience</td><td>11-0451-81</td><td>Long-term transplantation</td></tr><tr><td>UV lamp</td><td>Beijing jiangmorning yuan bio-technology co., LTD</td><td>039-14402</td><td>power: 30W</td></tr><tr><td>stainless steel mesh</td><td>AS ONE SHANGHAI Corporation</td><td>2-9817-10&amp;nbsp;</td><td>aperture diameter: 2.5 mm</td></tr><tr><td>hydrocortisone</td><td>Sigma</td><td>H0396</td><td>selectively diluted into M5300 medium</td></tr></tbody></table>

application of aorta-gonad-mesonephros explant culture system in developmental hematopoiesis

使用小鼠胚胎进行造血研究的局限性是在手术中增加了不便, 这主要是由于胚胎的宫内发育。尽管来自击倒 (ko) 小鼠的基因数据令人信服, 但在需要时为所有基因生成高鼠并不现实。此外, 执行在体内抢救实验, 以巩固从 KO 小鼠获得的数据是不方便的。为了克服这些局限性, 主动脉-性腺-肾 (年会) 外植体培养作为研究造血干细胞 (HSC) 发育的适宜系统而发展起来。特别是在抢救实验中, 可用于恢复高鼠的造血损伤。通过在培养基中加入适当的化学物质, 可以重新激活受损的信号, 或抑制上调的通路。利用这种方法, 可以进行大量的实验, 以确定 hsc 发展的关键调控因子, 包括 hsc 相关基因在 mRNA 和蛋白质水平、菌落形成能力和重构能力上的表达。这一系列的实验将有助于确定哺乳动物 HSC 发育所必需的基本机制。

最近发表的一篇报道说, 内皮细胞衍生的血清素通过抑制干细胞的亡途径, 促进了其生存.13。为了证实血清素对 HSC 发育的促进作用, 氟西汀被列入。作为一种选择性血清素再抑制剂 (SSRI), 氟西汀已被证明抑制血清素吸收在周围组织16,17,18。采用上述方法对氟西汀对 HSC 培养的影响进行了...

Watch this Scientific Journal Video about Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis at JoVE.com

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

本议定书描述使用培养的主动脉-性腺-肾的表达分析, 菌落形成单位在文化和脾脏, 并长期重建, 以确定影响的调节因子和信号通路的造血干细胞发育。这已被证明是一个有效的系统研究造血干细胞生物学和功能。

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

application-aorta-gonad-mesonephros-explant-culture-system

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.4K 次观看.  Chinese Academy of Sciences. 本议定书描述使用培养的主动脉-性腺-肾的表达分析, 菌落形成单位在文化和脾脏, 并长期重建, 以确定影响的调节因子和信号通路的造血干细胞发育。这已被证明是一个有效的系统研究造血干细胞生物学和功能。

视频: Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

科研

发育生物学

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

体外拟态正常与异常的人造血

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

生物工程

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

修改ES / OP9共培养协议，提供增强造血后代的表征

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

生物学

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

摘要

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运用

体外拟态正常与异常的人造血

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

小鼠胚胎肾的解剖与培养

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

修改ES / OP9共培养协议，提供增强造血后代的表征

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法