M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

该协议是按照欧洲的指导原则（EU二千〇十分之六十三）和保护用于科学研究的动物意大利（2014分之26）的法律进行。颈椎脱位安乐死用于卵巢隔离，它是由专家进行和训练有素的已批准的方案涉及本次研究中列出的个人。 1.动物<ol><li>保持成年3至6周大的雌性小鼠中的房间温度和湿度受控（具有12L的阶段:12D，并自由采食）。 </li><li>腹腔注入小鼠2.5 IU孕马血清促性腺激素（PMSG），通过模仿的卵泡刺激素（FSH）的影响，刺激卵泡生长。隔离卵巢的窦收集的卵母细胞48小时后。安乐死的雌性小鼠以前用PMSG注射，根据你的机构的指导方针。 </li><li>从体腔迅速取出卵巢和它们放入培养皿中续癌宁M2培养后续的卵母细胞隔离（见第3节）。 <ol><li>使在皮肤上的覆盖腹壁接着在腹膜切口通过使用无菌解剖剪刀切开。打开切口以暴露腹腔，用无菌镊子将胆一方和本地化子宫管，形成一个V形从膀胱开始。 </li><li>观察位于下方的肾脏卵巢。保持每个管，用无菌镊子和作一小切口在卵巢释放他们的底部。在含预平衡介质的培养皿的底部转移双侧卵巢。 </li></ol></li></ol> 2.激素制剂<ol><li>稀PMSG（在不同的库存浓度可提供）用无菌等渗盐水溶液以获得每0.1 ml的2.5 IU激素一个工作浓度为适当的注射体积。 注:激素溶液必须等分的0.5ml管中，并储存在-20℃下直至使用当天。不要将稀释分装比在-20°C三个月的时间。一旦解冻，如果连续注射执行，存储激素的解决方案的分装置于+4℃，4天之内使用。 </li></ol> 3.胃窦卵母细胞隔离 对于正确的卵母细胞的分离: <ol><li>在开始的卵母细胞的分离步骤之前平衡M2培养基13（M2），在5％的CO 2和37℃下至少1小时。准备两个培养皿（35毫米×10毫米），一个用1ml M2的用于卵巢穿刺和M2的从隔离后覆盖有矿物油（〜1.5毫升）中的卵母细胞转移，第二个具有小滴（20微升）子房（见3.4-3.6）。保持在孵化两培养皿在5％的CO 2和37℃下，直到准备使用。 </li><li>在卵母细胞分离方法使用立体显微镜。 </li><li>准备薄薄的嘴唇玻璃吸管收集的卵母细胞伸缩兴玻璃巴斯德吸液管保持水平（持用双手吸管的两端），在一个垂直的火焰本生灯到来（ 图1）。 <ol><li>一旦玻璃开始熔化，取吸管出来的火焰和执行快速牵引运动，以获得所需吸管。牢牢剪断多余的玻璃靠近窄点用指甲以调节长度和内部毛细管的直径的吸移管的。确保薄毛细管具有〜100微米的内径，比卵母细胞的直径稍大（〜80微米）。 </li><li>口移液管，通过使用适当的尖端连接吸气管至所述拉吸管的一端，并将另一端的嘴，用牙固定它。 </li></ol></li><li>使用无菌镊子收集卵巢和将它们存放在细胞培养的培养皿（35毫米×10毫米）的底部。覆盖它们用1ml M2的先前在37℃下平衡过和5％的CO 2（ 图2）。 </li><li>轻轻穿刺卵巢的窦性卵泡，用无菌胰岛素注射器针头释放胃窦的卵母细胞中的M2（图3）。 </li><li>轻轻口通过一个狭窄的巴斯德吸管除去毛囊细胞和任何其他可能的组织碎片（图4A），然后解决它们在小滴的M2（20微升）覆盖有适于胚胎培养矿物油的底部（先前吸管的卵母细胞制备; 图4B）。 注:通过几个不同滴M2来消除附着在卵母细胞连续移液的透明带的卵丘细胞是非常重要的。执行此步骤尽可能快，以避免在介质的pH的改变。 </li></ol> 4.胃窦卵母细胞染色与Hoechst33342染色注:窦卵母细胞可以在SN（环绕核仁），诺西（不环绕核仁），或者加以区分在任何其他中间形式的基础上，他们的染色质组织，Hoechst33342染色的体外活浓度（或特定的任何其他标记物为基础的AT）14染色后。事实上，SN卵母细胞表现出周围的核仁一个赫斯特阳性的环，而诺西不这样做，因此它的名字。 对于正确的卵母细胞染色: <ol><li>传送用嘴吸管卵母细胞成Hoechst33342染色（20微升）稀释在M2的液滴在50毫微克/微升体外活浓度在黑暗10分钟。 </li><li>用Hoechst33342染色的培养后，在小滴的M2（5微升）覆盖有矿物油，基于染色质组织（SN或NSN）分选的底部转移的每个单个卵母细胞用吸管嘴。使用配备Hoechst33342染色过滤器（发射波长486纳米）的任何荧光显微镜可视化的卵母细胞的染色质结构。 注意:根据所用荧光显微镜的型，it可以成为当务之急使用玻璃底培养皿进行图像采集。在激光共聚焦显微镜奥林巴斯Fluoview Fv10i在配备35毫米盘试样固定器只需要玻璃底菜进行图像采集，用0.17毫米标准厚度和范围内可以接受0.13 0.21毫米。 </li><li>激励的卵母细胞的时间不超过5-10秒，可能有足够的时间来可视化的存在或不存在围绕核仁一个环的（图5）。 </li><li>后整理，培养胃窦SN和诺基亚西门子的卵母细胞中的M2在5％的CO 2和37℃以诱导体外成熟或保持它们在适当的缓冲器，用于分子或细胞的分析。 </li></ol>

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The authors have nothing to disclose

本协议描述用来分离和分类鼠标胃窦GV卵母细胞的方法。有强调，除少数例外，没有特别的关键步骤。第一:此过程应当尽可能快地进行，以:1）保持卵母细胞的活力和b）避免在使用的培养基的pH变化。第二:卵母细胞应该是简要地兴奋（5-10秒），以Hoechst33342染色，以避免染色质损害和卵母细胞的随后死亡，特别是如果IVM和体外受精是以下步骤。迄今为止，这是SN和NSN小鼠卵母细胞胃窦排序的最快方?...

胃窦从卵巢的窦室分离的完全的卵母细胞生长（GV，生发泡）通常用于不同的目的，诸如，例如，在体外成熟直至中期II阶段背后的分子和细胞机制的研究。 尽管他们漂亮的外观最有腔卵母细胞，但不是所有的，都能够完成减数分裂并达到囊胚阶段;事实上，在许多哺乳动物物种，包括人类1,2，其中的大约30％阻止其发展在2-细胞阶段，如果发生受精3-5。迄今为止，只有少数参数用于卵母细胞能够维持从那些无法做到的胚胎发育之间进行区分。 例如，甲酚蓝染色（BCB）是一个有效的方法，以基于葡萄糖-6-磷酸脱氢酶活性的卵母细胞进行排序尽管这种分选方法的效用与BCB +或BCB-染色仍然依赖于实验室6-8。 另一成熟方法驻留在他们的染色质组织:如果沾上任何DNA嵌入染料（如Hoechst33342染色），胃窦的卵母细胞的70％表现出围绕核仁染料正环和被称为环绕核仁（SN）而剩余的30％的显示出更多的斑点积极的信号，被称为不环绕核仁（NSN）3-5。最近我们发现，如果没有细胞质晶格9（的CPL，重要的存储位点在两个哺乳动物卵母细胞和胚胎母体衍生mRNA和核糖体）的10和下调脑膜（必不可少的CPL形成11），加上存在在其胞质9,12脂滴，可以是有关的卵母细胞诺西无法继续进行的2细胞期其他原因。 不幸的是，很少有技术可应用于排序胃窦的SN和NSN和卵母细胞，因为这个原因，较少创伤的方法的开发（没有核染色，固定程序或操纵与嘴移液器）可能成为非常重要的是增加人和动物胚胎发育的速率。 在本文中，我们详细介绍了简单快捷的协议，用于隔离的广告排序SN和雌性小鼠NSN窦卵母细胞，它是给所有有兴趣的女性配子，卵母细胞成熟和胚胎发育的研究中，科学家。

<table>
	<tbody>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4527</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbrioMax M2</td>
			<td>Millipore</td>
			<td>MR-015-PD</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35mmx10mm</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Dish 35mm, high glass bottom</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Pasteur Borosilicate</td>
			<td>Corning</td>
			<td>7095D-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated micropillary pipettes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

下面的图片展示用于隔离窦卵母细胞来自小鼠卵巢，用Hoechst33342染色的方式从移液器准备到卵母细胞"排序启动方法。这种方法相当简单，并且可以在装有CO 2培养箱，立体显微镜，配有正确的过滤器，用于观察Hoechst33342染色的荧光显微镜任何实验室中进行图1示出了移液管制备的初始步骤:作为玻璃开始熔化并变软，吸移管的两端必须被拉开，并在同一?...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

在这里，我们提出了一个协议的基础上核仁组织小鼠卵母细胞胃窦表征。

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

13.2K 次观看.  Fondazione IRCCS Policlinico San Matteo. 在这里，我们提出了一个协议的基础上核仁组织小鼠卵母细胞胃窦表征。 

视频: Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...