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.

このプロトコルは、欧州（EU 2010分の63）、科学研究のために使用される動物の保護イタリア（2014分の26）法律の指針に従って実施されます。頸椎脱臼を安楽死は、卵巣の単離のために使用され、それは、この研究をカバーする承認されたプロトコルに記載されている個体を専門家によって行われ、訓練されます。 1.動物<ol><li>制御された温度および湿度の部屋で6週齢の雌マウスに大人3を維持（12Lの位相と：12Dおよび摂食自由摂取 ）。 </li><li>卵胞刺激ホルモン（FSH）の効果を模倣することにより卵胞の成長を刺激するために妊馬血清性性腺刺激ホルモン（PMSG）の2.5 IUを腹腔内にマウスを注入。後で洞卵母細胞の収集48時間のための卵巣を分離します。あなたの機関のガイドラインによると、以前にPMSGを注射したマウスを安楽死させる女性。 </li><li>すぐに体腔から卵巣を除去し、ペトリ皿の続きにそれらを置きますその後の卵母細胞の単離のためのM2培地をaining（セクション3を参照）。 <ol><li>無菌解剖ハサミを使用して、腹膜の切開に続いて腹壁をカバーする皮膚の切開を行います。膀胱から始まるV字形状を形成する、腹部を露出するように切開を開き、滅菌ピンセットを用いて、一方の側に勇気を移動させ、子宮管をローカライズ。 </li><li>腎臓の下方に位置する卵巣を観察します。滅菌ピンセットで各チューブを持ち、それらを解放するために卵巣の下部に小さな切開を行います。予め平衡化培地を含むペトリ皿の底に両方の卵巣を転送します。 </li></ol></li></ol> 2.ホルモンの準備<ol><li>適切な注入量0.1 1mlあたり2.5 IUホルモンの作用濃度を得るために、滅菌等張食塩水PMSG（異なるストック濃度で利用可能）を希釈します。 注：ホルモンソリューションを0.5 mlチューブに分注し、で保存する必要があります-2使用日まで0℃。 -20℃で3ヶ月よりも長い希釈アリコートを保管しないでください。解凍したら、シリアル注射が行われた場合、4℃のホルモンソリューションのアリコートを保存し、4日以内にそれらを使用しています。 </li></ol> 3.洞卵母細胞の分離 適切な卵母細胞の単離のために： <ol><li>卵母細胞の単離手順を開始する前に少なくとも1時間、5％CO 2、37℃でM2培地13（M2）を平衡化。 2ペトリ皿（35ミリメートル×10 mm）で、卵巣穿刺からの単離後の卵母細胞の転送のためにミネラルオイル（〜1.5ミリリットル）で覆われたM2の小滴を有する第二1（20μL）のためのM2の1ミリリットルのものを準備します卵巣（3.4から3.6を参照）。準備が使用されるようになるまで、5％CO 2、37℃のインキュベーター内で両方のペトリ皿にしてください。 </li><li>卵母細胞の単離手順中に実体顕微鏡を使用してください。 </li><li>ストレッチにより卵母細胞を収集するために、薄い口のガラスピペットを準備興ガラスパスツールピペットは、ブンゼンバーナーからの垂直燃焼（ 図1）を介して、（両手でピペットの先端を保持する）水平に保ちました。 <ol><li>ガラスが溶融し始めると、炎の外にピペットを取り、目的のピペットを得るために迅速に引き運動を行います。しっかりと長さと内部の毛細血管の直径を調整するための爪とピペットの狭い点に近い過剰ガラスをカット。細い毛細血管が卵母細胞の直径よりも僅かに大きい〜100μmの内径を有することを確認します（〜80ミクロン）。 </li><li>口のピペットには、適切なチップを使用して引っ張っピペットに吸引管の​​一端を接続し、歯とそれを確保、口の中にもう一方の端を置きます。 </li></ol></li><li>滅菌ピンセットを用いて卵巣を採取し、細胞培養用シャーレ（35ミリメートル×10ミリメートル）の底部にそれらを堆積させます。以前は、37℃で平衡化したM2の1mlでそれらをカバー、5％CO 2（ 図2）。 </li><li>ゆっくりM2（ 図3）で胞状卵母細胞を解放するために、滅菌インスリン注射針と卵巣の胞状卵胞を穿刺。 </li><li>そっと口は包細胞および他の可能性のある組織片（ 図4A）を削除し、以前に（胚培養に適したミネラルオイルで覆われたM2の小滴（20μL）の下でそれらを解決するための狭いパスツールピペットを通して卵母細胞をピペット準備; 図4B）。 注：これは、M2のいくつかの異なる滴を通して連続ピペットで卵母細胞の透明帯に接続されているすべての卵丘細胞を除去することが重要です。培地のpHの変化を避けるために、可能な限り迅速にこの手順を実行します。 </li></ol> Hoechst33342 4.胞状卵母細胞の染色注：洞卵母細胞は、SN（囲ま小体）、NSN（小体を囲まれていない）、またはに区別することができます他の中間形式で、Hoechst33342の超生体濃度（又は塩基の具体的な任意の他のマーカーAT）14で染色した後、それらのクロマチン組織に基づきます。 NSNがいない間、実際には、SN卵母細胞は、したがって、その名前、核小体の周りヘキスト陽性リングを示しています。 適切な卵母細胞染色のために： <ol><li>暗所で10分間、50 ngの/μLの濃度で超生体M2に希釈したHoechst33342（20μL）の液滴に口のピペットを用いて卵母細胞を転送します。 </li><li> Hoechst33342とのインキュベーションの後、クロマチン組織（SNまたはNSN）に基づいてソートするため、鉱物油で覆われたM2の小滴（5μL）の底に口のピペットを用いて各単一卵母細胞を転送します。卵母細胞のクロマチン構造を可視化するためにHoechst33342フィルタ（発光波長486ナノメートル）を装備した任意の蛍光顕微鏡を使用してください。 注：使用する蛍光顕微鏡の種類に応じて、私はtは、画像取得用のガラス底ペトリ皿を使用することが不可欠となることができます。 35mm皿のための試料ホルダーを装備した共焦点顕微鏡オリンパスFLUOVIEW Fv10iは0.17ミリメートルの標準的な厚さ0.13から0.21ミリメートル許容可能な範囲で、画像取得のための唯一のガラスボトムディッシュが必要です。 </li><li>そう、もはやよりも5〜10秒のために存在または核小体の周りにリングが存在しない場合（ 図5）を視覚化するのに十分な時間を卵母細胞エキサイト。 </li><li> 体外成熟を ​​誘導するか、または分子または細胞の解析のための適切な緩衝液中でそれらを保つために、5％CO 2、37℃でM2で、文化胞状SNとNSNの卵母細胞をソートした後。 </li></ol>

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The authors have nothing to disclose

このプロトコルは、マウス洞のGV卵母細胞を分離し、分類するために使用される方法について説明します。いくつかの例外を除いて、下線する特段の重要なステップはありません。 A）卵母細胞の活力を維持し、b）に用いられる培地中のpH変化を避ける：まず：にこの手順は、可能な限り迅速に実行する必要があります。第二：卵母細胞は、IVMおよびIVFは、次の手順がある場合は特に、クロマ...

卵巣の胞状コンパートメントから分離された胞状十分に成長した卵母細胞（GV、卵核胞）は日常、などなど、さまざまな目的に使用されている例えば、中期II段階まで体外成熟の背後に分子・細胞メカニズムの研究。 その美しい外観で最も胞状卵母細胞にもかかわらず、すべてではないが、減数分裂を完了し、胚盤胞段階に到達することができます。受精が3-5が発生した場合 、実際には、人間1,2を含む多くの哺乳動物種において、それらのおよそ30％が、2細胞期で、その開発を逮捕します。今日までに、いくつかのパラメータは、そうすることができないものから胚発生を維持することができる卵母細胞を区別するために使用されます。 例えば、クレシルブルー染色（BCB）はBCB +またはBCB-染色に関連し、このソート方法の有用性はあるが、グルコース-6-リン酸デヒドロゲナーゼの活性に基づいて、卵母細胞を分類するための有効な方法であり、まだ研究室に依存6-8。 もう一つのよく確立された方法は、彼らのクロマチン組織に存在する：（Hoechst33342のような）任意のDNAインターカレー色素で染色した場合、胞状卵母細胞の70％が核小体の周りの色素陽性リングを示し、小体（SN）囲ま呼ばれ、残りの30％ながら、より多くの斑点陽性シグナルを示し、仁（NSN）3-5囲まれていないと呼ばれています。最近では、ことを示した細胞質格子9（のCPL、哺乳動物の卵母細胞および胚の両方で母体由来のmRNAとリボソームのための重要な貯蔵部位）の不在10との存在と一緒（のCPL形成の11のために必須）MATERのダウンレギュレーション、その細胞質9,12における脂肪滴は、2細胞期を超えて進行するNSNの卵母細胞の無力に関連する他の原因であることができます。 残念ながら、いくつかのテクニックが胞状SNとNSN卵母細胞とをソートするために適用することができ、このため、（核染色、固定手順や口のピペットとの操作なし）少ない侵襲的方法の開発は、ヒトおよび動物の両方の胚発生の速度を向上させることが非常に重要になる可能性があります。 本稿では、詳細簡単かつ迅速プロトコルは、広告ソートSNと雌マウスからNSN胞状卵母細胞を単離するために使用し、それは女性の配偶子形成、卵成熟と胚発生の研究に興味を持つすべての科学者にアドレス指定されています。

<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.

以下の写真は、Hoechst33342を用いて卵母細胞「ソートにピペットの準備から始めて、マウスの卵巣から胞状卵母細胞を単離するために使用する方法を示しています。この方法は非常に簡単で、CO 2インキュベーター、実体顕微鏡およびHoechst33342の観察のために正しいフィルターを備えた蛍光顕微鏡を備えた任意の実験室で行うことができます。図1は、</stron...

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

ここでは、核小体の組織に基づいて、マウス胞状卵母細胞の特徴付けのためのプロトコルを提示します。

核小体クロマチン組織に基づいてマウス洞卵母細胞の単離とキャラクタリゼーション

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

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13.2K ビュー.  Fondazione IRCCS Policlinico San Matteo. ここでは、核小体の組織に基づいて、マウス胞状卵母細胞の特徴付けのためのプロトコルを提示します。 

ビデオ: 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.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

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

発生生物学

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.

ラットヘッドBranchiomeric筋肉から衛星細胞の単離とキャラクタリゼーション

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.

ゼブラフィッシュ胚からの単一細胞の単離とキャラクタリゼーション

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.

ヒト臍帯及び胎児胎盤由来間葉系間質細胞の単離および特徴付け

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.

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

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.

ニューラル ネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

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.

ヒト臍帯由来間葉系幹細胞から早産児と正期産児の分離と性状

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.

マウス胚性幹細胞からの神経前駆細胞とニューロンの分化と特徴付け

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.

未熟ネコの最小体積ガラス化

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;

牛の初期のアンタル卵胞からの卵母細胞のための体外培養戦略

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