1。胚の除去と胎盤切開<ol><li>開始するには、胚が最初に妊娠した母獣から分離する必要があります。 </li><li>動物は、承認された手順に従って安楽死させる - 私たちのケースで我々は、イソフルラン麻酔薬の致死量を使用するつもりです</li><li>最初に、エタノールで腹をスプレーし、ハサミで小さな切開を行います。 </li><li>腹部を明らかにし、腹膜を切り開くために両手で皮膚を離れて引き裂く。 </li><li>鉗子で、子宮角をピックアップし、それぞれの先端にはさみを使用してそれらを収集する。 PBSと氷の上で場所で満たされたペトリ皿の子宮角に置きます。また、胎盤を分離する前にPBSで数回洗浄してください。我々は現在、胎盤を分離することができます。 </li><li> two鉗子で、慎重に胚のconceptusesを周囲の子宮内膜組織を離れて剥離を開始。これはその後の解剖の手順を複雑にするので一つは、胚への構造的な損傷を与えていない穿刺またはそうでなければ注意する必要があります。 </li><li>氷上でPBSで新しいペトリ皿の中で孤立conceptusesを置き、すべてのconceptusesが子宮内膜から解放されるまで続けます。 </li><li>顕微鏡下と離れて胎盤から脱落膜ピールtwoピンセットで配置されたPBSで新しいペトリ皿一つ受胎を転送する。脱落膜の細胞は単一細胞懸濁液およびフローサイトメトリーを妨害するため、可能な限り脱落膜の限りを削除します。滑らかな連続的な剥離動きが最良の結果を達成することをお勧めします。 </li><li> two器官の間の接合部での胎盤から卵黄嚢を切り​​取って、そっと胎盤から卵黄嚢と卵黄血管を引き出します。注意は、特に初期の胚で、胎盤の絨毛膜板を混乱しないように注意してください。 </li><li>注意深くピンセットと相互に臍帯と胎盤の絨毛膜板を保持し、離れて胎盤から臍帯と接続されている胚を引き出します。 </li><li>単一細胞懸濁液の調製中に凝集細胞を最小限にするために胎盤の端に余分な巨大な細胞組織を削除します。 </li><li> PBS 5パーセントFCSとし、氷の上で、そのような15 mlのファルコンチューブのような適当な容器に胎盤を収集する。解剖全ての胎盤に対して、この手順を実行します。 </li><li>この時点で、あなたはそのような体外培養や移植のように、フローサイトメトリー、機能アッセイのための単一細胞懸濁液の調製に進むことができます、または組織の固定および最終的な免疫組織化学のために埋め込み固定冷凍ブロック用に全体胎盤を準備。 </li></ol> 2。胎盤組織の単細胞懸濁液の調製<ol><li>我々は現在、FACS分析のための胎盤から単一の細胞懸濁液を生成する方法を紹介します。 </li><li>胎盤からの単細胞懸濁液を調製するために、機械的および酵素的解離が必要です。最初に10％FCS、1％ペニシリン/ストレプトマイシン溶液をPBSに0.1％コラゲナーゼ溶液を調製。 </li><li>あなたがいくつあるかに応じて、胎盤にコラゲナーゼ溶液の適切なボリュームを追加します。倍胎盤のボリュームと2〜5ミリリットルの間であるボリュームをお勧めします。 </li><li>機械的に針3回を通じてコラゲナーゼ溶液と胎盤を継代により組織を混乱させるために5 mlのシリンジに装着した16 G針を使用してください。 18 - G針を繰り返します。 </li><li> 45分間37℃、5％CO 2インキュベーター内のサンプルを置きます。 </li><li>コラゲナーゼ20 - G針を介して、通過細胞溶液、および37に、さらに45分間インキュベート℃で45分間のインキュベーション後</li><li>それぞれの針と22 - Gと25 Gの針を介してコラゲナーゼ、通過細胞溶液で1.5時間、総インキュベーション後の3回。 </li><li>新しい15 mlのファルコンチューブに接続されている50μmのフィルターを通してサンプルをフィルタリングする、4℃でPBS 5パーセントFCSと遠心機で洗う℃で5分間300℃× gで</li><li>この時点で、我々は、FACS分析または機能性アッセイに適した単一の細胞懸濁液を持っている。 </li></ol> 3。凍結切片用組織の固定<ol><li>すぐにそれぞれの胚から胎盤を分離した後、それらは寒い1X PBSに配置されます。通常は96ウェルプレートは、若い胎盤（E8.5 - 11.5）に適していますが、古い胎盤で、E12.5と古いが、24ウェルプレートの方が便利かもしれません。 </li><li>それぞれの胎盤がよく新鮮な融解4パーセントparaformaldahydeまたはPFAで満たされた新たに転送されます。 E12.5胎盤の場合、組織は手で新しいウェルに移動されますが、E10.5のために、ピペットで慎重にPBSを除去し、PFAを追加することができます。それは、胎盤が完全に組織の年齢やサイズに応じて、4℃で2〜4時間浸漬されていることが重要です。 </li><li> 30％ショ糖に組織を移し、あるいは組織が非常に小さく、壊れやすい場合は、PFAを吸引し、直接ショ糖を加える。この段階では、胎盤は、溶液中でフローティングされます。組織は、4℃で一晩滞在しましょう​​℃に</li><li> 30％ショ糖で一晩ステップの後、組織は最終的に適切な凍結保存を示すウェルの底に沈むはず。ショ糖溶液の半分を削除して、OCTでボリュームを交換し、℃で1-2時間を4に戻って組織を置く。これは、OCTの浸透を容易にします。 </li><li>それを埋め込むことができる、その後、1時間に100％OCTに組織を移す。 </li></ol> 4。固定組織の埋め込み<ol><li>適切な組織の識別（ショーの例の金型）のラベルは、プラスチック金型。 </li><li> OCTのソリューションからティッシュを引き出して、下に向け臍帯側と円盤状の胎盤を向きや慎重に確実に圧力をリリースする前に、前後に刃を移動するために確実にきれいなカミソリの刃でスライスして半分に胎盤を切るさらにカット。胎盤は、後に胎盤造血幹細胞を可視化するために最善prientationを達成するために半分にカットされます。 </li><li>金型の底面と接触する面取り部と金型の下部に片足胎盤の半分。残りの半分のための新しい金型を繰り返します。 E10.5胎盤の場合は、両方の半分は、同じテクニックを使用して、同じ金型に配置されることがあります。 </li><li>金型にOCTを注ぐときに、それは正しい方向に胎盤を維持することが困難な場合があります。鉗子で組織の半分を保持することによって場所に胎盤を固定します。徐々に金型内にOCTのソリューションを注ぎ、気泡を生成することは避けてください。 E10.5胎盤の場合は、鉗子によって1つの組織の半分をホールドし、鉗子の外側に対して、他の組織の半分を置きます。ゆっくりとしてE12.5胎盤で説明したOCTのソリューションを注ぐ。 </li><li>金型がOCTでいっぱいになると、すぐにドライアイスの上に金型を配置、OCTは組織を凍結白いフラッシュを向けるだろう</li><li> DRIERITEを含む小さなビニール袋に金型を配置し、直ちに-80℃の冷凍庫で保管してください。 </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

このプロトコールに記載される実験手順は、胎盤組織と造血幹細胞や前駆細胞の分離と可視化が可能になります。胎児の発育を通して胎盤や他の胎児の造血臓器における造血幹細胞の予想される細胞の収量と数に関する包括的な概要については、我々はスゲカスらを参照してください。 2005年。胎盤で造血幹細胞および他の造血細胞の開発のローカライズのために我々はローズ、らを参照してください。 2008

材料のリスト ： 100mMのグルコース 10mMのNaN3 15 mlコニカルチューブ（BD） 16 G、18 G、20 G、22 G、25 G針（BD） 30％のショ糖液 35ミリメートルのペトリ皿（BD） 4％パラホルムアルデヒド 50 - mのセルのフィルタ（Celltrics） 5 mlのシリンジ（BD） アルカリホスファターゼ基質キットI（ベクトル赤、ベクトル） アルカリフォスファターゼ基質キットIII（ベクトル青、ベクトル） 抗生物質（ペニシリン/ストレプトマイシン、P / S）ソリューション コラゲナーゼI型（シグマ） Cryomold使い捨てビニール片金型（10mm x 10mmの× 5mmの、組織- Tek社） サイトケラチン一次抗体（DakoCytomation） 解剖ピンセット、ステンレス鋼やチタン、5番または55 DRIERITE ウシ胎児血清（FBS） Fisherbrandプラスチック顕微鏡スライドメーラー（フィッシャー） グルコースオキシダーゼ1000u/ml レバミゾール（ベクター） マウントメディア、水性ベースのマウンティングメディウム（VectamountTM AQ） 正常ウマ血清（ベクトル） 最適な切削温度（OCT、ティッシュ- Tek社） ペルオキシダーゼ基質キットDAB（ベクトル） リン酸塩は、カルシウム/マグネシウム（WのCa2 + / Mg2 +を）と緩衝生理食塩水（PBS） リン酸塩は、カルシウム/マグネシウム（W / OのCa2 + / Mg2 +は）なしで緩衝生理食塩水（PBS） プラスチック製のスライドボックス（100スライドを保持する） プラスチックチューブ（Nalgene 180 PVC無毒オートクレーブ可能） ポリプロピレン丸底試験管5 mlの（12x75ミリメートル、BDファルコン） プロテイナーゼK 20mg/ml溶液（Amresco） 研究プロダクツインターナショナルコーポレーションスーパーパップペンHT *スライドマーカー（2,5 mmの、フィッシャー） 組織培養U字型（丸底）治療を96ウェルまたは24ウェルプレート（BDファルコン） Tween20を、SigmaUltra（シグマ） チラミド増幅キット（Invitrogen社） ベクタステインABCアルカリホスファターゼスタンダードキット（ベクター） ベクタステインABC標準エリートキット（ベクター）

isolation and analysis of hematopoietic stem cells from the placenta

造血幹細胞（HSC）は、自己更新し、個々の生涯を通して血液系統のすべての細胞型を生成する機能を持っている。すべての造血幹細胞は、それらのプールサイズは自己複製細胞分裂によって維持され、その後、胚発生時に出現する。多くの解剖学的部位が胎児の造血中に参加するとHSCの開発の過程を調節する造血幹細胞との重要な発達のイベントの解剖学的起源を特定することはややこしくなっている。最近、我々は造血幹細胞がユニークな微環境のニッチ（スゲカス、ら、2005、ロードス、ら2008）で生成し、展開されている主要な造血器官として胎盤を同定した。その結果、胎盤は、彼らの出現と初期の展開時の造血幹細胞の重要な源です。 この記事では、我々はそれぞれ、造血幹細胞および胎盤におけるHSCプールの大きさのピークの開始の発達段階に対応するE10.5からマウス胎盤の分離およびE12.5胚の解剖の技術を、示す。さらに、我々はフローサイトメトリーまたは機能アッセイで使用するための単一細胞懸濁液中に胎盤組織の酵素的および機械的解離のために最適化されたプロトコルを提示する。我々は、胎盤の単細胞懸濁液のためのコラゲナーゼの使用は、造血幹細胞の十分な収量を示すことを発見した。胎盤からHSC収量に影響を与える重要な要因は、前に機械的解離の度合い、及び酵素処理、の期間です。 我々はまた彼らの正確な細胞のニッチにおける免疫組織化学によって開発造血幹細胞の可視化のための固定凍結胎盤組織切片の調製のためのプロトコルを提供しています。造血特異的な抗原はパラフィン包埋切片の準備中に保持されていないとして、我々は日常的に胎盤造血幹細胞および前駆細胞をローカライズするための固定凍結切片を使用してください。

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

我々は、開発中の主要な造血器官として胎盤を同定した。我々はその造血幹細胞（HSC）が生成され、ユニークな微環境のニッチの胎盤で展開されている両方とも発見。ここで、我々はマウス胎盤における造血幹細胞の分離と可視化に必要な実験手法を説明します。

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

isolation-and-analysis-of-hematopoietic-stem-cells-from-the-placenta

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Cell Biology

Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

20.3K ビュー.  University of California, Los Angeles. 我々は、開発中の主要な造血器官として胎盤を同定した。我々はその造血幹細胞（HSC）が生成され、ユニークな微環境のニッチの胎盤で展開されている両方とも発見。ここで、我々はマウス胎盤における造血幹細胞の分離と可視化に必要な実験手法を説明します。

ビデオ: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

造血幹細胞の分離と移植（造血幹細胞）

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

マウスの眼から網膜幹細胞の単離

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

マウス胚のレトロウイルス感染は、造血分化の解析のための細胞由来の胚様体幹細胞

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNAシーケンシング解析によりEML造血前駆細胞における自己再生および分化を調節する重要な要因の同定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...