PWDは、ウエスタン大学のジェラルドM.キダーにヘルスリサーチフェローシップ（CIHR）とCIHR助成のカナダの協会によってサポートされています。著者はまた、ここで提示されたプロトコルの開発を支援するためのゲルフ大学でJulang李を承認したいと思います。

1。メディアの準備<ol><li>実験が行われる細胞培養インキュベーターで緩めキャップを使用し、場所の前にメディア2-3時間を準備します。現在のプロトコルで使用されるメディア： <ol><li> 1×B27、40 ngの/ mlのbFGFを、および20 ngの/ mlのEGFを補ったDMEM/F12から成る細胞培地を食い止める準備。 </li><li> 0.05 IUのFSH、0.03 IU LH、3 mg / mlのBSA、5μL/ mlのITS、0.23 mMのピルビン酸ナトリウム、1 mg / mlのフェツイン、および1 ngの/ mlのEGFを補ったM199から成る生殖細胞分化培地を準備します。全ての添加剤から構成されるが、FSHを省く基本培地は、LHおよびEGF℃で最大一週間のために準備し、4℃で保存することができます。 </li></ol></li></ol> 2。細胞培養の準備を食い止める<ol><li>以前に12を説明したように生まれたばかりの仔マウスから幹細胞を分離します。 インビトロ生殖細胞分化のための通路2-4で幹細胞を利用する。の効率OLC形成を直接開始幹細胞の質に関連している。それは、すぐに人口が通路2付近で、一般的になっている清潔で健康な表示される分化を開始するのが最善です。一節過去さらに培養幹細胞2悪影響OLC形成効率を（未発表結果）に影響します。 </li><li>従来のサブ培養幹細胞を分化を開始する48時間。すべて中断球状細胞凝集体を取り外し、血清ピペットを用いて、培養皿からメディアを費やし、、15 mlチューブで開催。それだけでサスペンド球状の細胞の集合体ではなく自発的に差別されているお皿の底に付着した細胞を回収することが重要です。 </li><li>ペレットを5分間500 xgで細胞。、上清を捨て、新鮮な幹細胞培地を500μlに再懸濁し、37℃に温め優しく部分の細胞を解離する凝集体をピペット。積極的に細胞をピペッティングしないでくださいこのようなsが細胞生存率が低下します。低アタッチメント10センチ細胞培養皿に新鮮予め温めておいた幹細胞培地の9.5ミリリットルに部分的に解離された細胞を洗浄します。 </li><li>分化の開始前に48時間37℃、5％CO 2細胞培養インキュベーターに細胞を返す。 </li></ol> 3。 OLC分化<ol><li>を15mlチューブに培養皿と場所から幹細胞を除去する血清用ピペットを使用して、接続されているすべてのセルを残す。ペレットを500μlの滅菌PBSで500×gで再懸濁しの細胞。活発なピペット広いを用いて細胞は10〜20セルより大きくない塊に1,000μlの先端を産んだ。定期的に解離の際、細胞の小さな量を削除し、集約された細胞が10〜20個々の細胞の塊である確認のため白色光顕微鏡下で確認してください。解離にわたり減少、細胞の生存率になります。 </li><li>解離細胞は9.5 mlのPBSを加え、1を追加細胞計数のために血球計数器にこの細胞懸濁液5μlの。 5分間500×gでペレットは、細胞。 </li><li> 1.32X10 6細胞/分化培地におけるmlの濃度で細胞を再懸濁する。 </li><li> 500μlのウェルあたりに平底24ウェルサスペンション皿を追加することにより、プレートの細胞を。 </li><li> 48時間、5％CO 2で37℃細胞培養インキュベーターに24ウェルディッシュ°Cを置きます。それに応じてラベル1.5mlチューブで各ウェル場所から250μlの培地を除去。各ウェルに250μlの新鮮な分化培地を追加します。 5分間500 xgで過ごした培地を遠心分離。し、上清を捨てる。 50μlの新鮮な分化培地における任意のペレット化した細胞を再懸濁しと分化プレートにうまく対応する文化に戻ります。 </li><li>すべての48時間は、分化の12日目に半分の培地を変更してください。 </li></ol>

<ol>
	<li>van den Hurk, R., Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+mammalian+oocytes+and+their+growth,+differentiation+and+maturation+within+ovarian+follicles.">Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.</a> Theriogenology. 63, 1717-1751 (2005).</li><li>Molyneaux, K. A., Stallock, J., Schaible, K., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+living+mouse+germ+cell+migration.">Time-lapse analysis of living mouse germ cell migration.</a> Dev. Biol. 240, 488-498 (2001).</li><li>Lawson, K. A., Hage, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+origin+of+primordial+germ+cells+in+the+mouse.">Clonal analysis of the origin of primordial germ cells in the mouse.</a> Ciba Found Symp. 182, 68-91 (1994).</li><li>Ginsburg, M., Snow, M. H., McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cells+in+the+mouse+embryo+during+gastrulation.">Primordial germ cells in the mouse embryo during gastrulation.</a> Development. 110, 521-528 (1990).</li><li>Hubner, K., Fuhrmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocytes+from+mouse+embryonic+stem+cells.">Derivation of oocytes from mouse embryonic stem cells.</a> Science. 300, 1251-1256 (2003).</li><li>Toyooka, Y., Tsunekawa, N., Akasu, R., Noce, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+can+form+germ+cells+in+vitro.">Embryonic stem cells can form germ cells in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 11457-11462 (2003).</li><li>Wang, L., Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-like+Cells+Induced+from+Mouse+Spermatogonial+Stem+Cells.">Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells.</a> Cell Biosci. 2, 27 (2012).</li><li>Dyce, P. W., Wen, L., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+germline+potential+of+stem+cells+derived+from+fetal+porcine+skin.">In vitro germline potential of stem cells derived from fetal porcine skin.</a> Nat. Cell Biol. 8, 384-390 (2006).</li><li>Danner, S., Kajahn, J., Geismann, C., Klink, E., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocyte-like+cells+from+a+clonal+pancreatic+stem+cell+line.">Derivation of oocyte-like cells from a clonal pancreatic stem cell line.</a> Mol. Hum. Reprod. 13, 11-20 (2007).</li><li>Cheng, X., Chen, S., Yu, X., Zheng, P., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP15+gene+is+activated+during+human+amniotic+fluid+stem+cell+differentiation+into+oocyte-like+cells.">BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells.</a> DNA Cell Biol. 31, 1198-1204 (2012).</li><li>Song, S. H., Kumar, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+porcine+multipotent+stem/stromal+cells+derived+from+skin,+adipose,+and+ovarian+tissues+and+their+differentiation+in+vitro+into+putative+oocyte-like+cells.">Characterization of porcine multipotent stem/stromal cells derived from skin, adipose, and ovarian tissues and their differentiation in vitro into putative oocyte-like cells.</a> Stem Cells Dev. 20, 1359-1370 (2011).</li><li>Dyce, P. W., Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+germ+line+potential+of+stem+cells+derived+from+newborn+mouse+skin.">In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin.</a> PLoS One. 6, e20339 (2011).</li><li>Linher, K., Dyce, P., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cell-like+cells+differentiated+in+vitro+from+skin-derived+stem+cells.">Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells.</a> PLoS One. 4, e8263 (2009).</li><li>Hayashi, K., Ogushi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offspring+from+Oocytes+Derived+from+in+Vitro+Primordial+Germ+Cell-Like+Cells+in+Mice.">Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-Like Cells in Mice.</a> Science. , (2012).</li></ol>

利害の衝突は宣言されていない。

官能OLCsを最新の状態にすると、in vitroアッセイを使用して完全に開発されていない。最近、林らによる研究は、in vitroで形成されており、さらなる開発14 インビボで導入細胞PGC-等から子孫を産生することができた。それらはインビボで移植後に回収し、細胞のようPGCを形成することができたES細胞または人工多能性細胞で始まるが、成熟受精、および...

初期胚発生時には、配偶子形成は性腺尾根の始原生殖細胞（PGC）の形成、移行、および最終的に植民地化を含む一連の段階を介して行われます。この時間の間のPGCがますます成熟配偶子1に増殖および分化を受ける。始原生殖細胞は胚の後腸に尿膜のベースから、最終的には最終的には性腺尾根に定着背壁に沿って移動しているという事実には、2を勉強するには、それらを非常に困難になります。分野における進歩にもかかわらず、PGCの形成および分化を理解しようとする研究は、その限られた数、位置、渡り鳥自然3,4によって妨げてきた。 近年、胚性幹細胞は、 インビトロ 5,6 の生殖細胞を形成する電位を有することが示されている。同様に、いくつかの体性幹細胞は、生殖細胞のfollのを形成する可能性を有することが示されている体外培養7-11 のおかげ。最近生まれたばかりのマウスの皮膚から単離された細胞を幹生殖細胞様細胞と初期段階の卵母細胞12にin vitroで分化させた。分化中のOct4を発現する幹細胞のサブセットが増加し、構造が類似している卵丘 - 卵母細胞複合体を形成した。卵母細胞様細胞（OLCs）培養物から採取し、天然の卵母細胞と比較することができる。 40-45程度を測定するこの培養方法のOLCsを使用して得られるようGdf9b、VASA、そしてDAZLなどの卵母細胞に発現する類似のマーカー。現在までに生じOLCsが成熟することができないまま、12を受精する。 OLCsは、これらのOLCsを形成するために使用されるプロトコルは、依然として内の初期段階の生殖細胞の形成と発展を研究するためのアッセイとして有用性を有することができる機能することができないままであるが、インビトロ系で閉じた。そうからOLCsの形成を議論初版発行マチック幹細胞は、幹細胞源8として胎児のブタ皮膚を利用した。分化誘導時に早期に形成されたPGC様細胞は、OCT4としてOLCsと急行などPGCマーカーにVASA、STELLA、C-KIT、及びDAZL 13の前駆体であることが示されたその研究に拡大。このデータは、in vitroでの生殖細胞の形成と分化を研究するために、このシステムを利用する可能性をサポートしています。新生児マウスの皮膚からOLCsを形成するために利用プロトコルはこのビデオの記事で実証される。このプロトコルは、in vitroモデルにおけるそれらの増殖および分化に重要な要素を明らかにするための手段を提供することができる生殖細胞の発達を研究するために提供する。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>PHG0311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>bFGF</td>
			<td>Invitrogen</td>
			<td>PHG0266</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ITS</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-6003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Invitrogen</td>
			<td>11360-070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FSH</td>
			<td>Sioux Biochemicals</td>
			<td>915</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LH</td>
			<td>Sioux Biochemicals</td>
			<td>925</td>
			<td></td>
		 </tr>
		 <tr>
			<td>M199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>24 well plates</td>
			<td>Nunc</td>
			<td>144530</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 cm dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		 </tr>
		 <tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352095</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

differentiation of newborn mouse skin derived stem cells into germ-like cells in vitro

生殖細胞の形成と分化を研究することは、伝統的に低い細胞数と途上胚奥深くその場所のために非常に困難であった。 試験管ベースのシステムで 、 &quot;クローズド&quot;の可用性は、配偶子形成の我々の理解のための貴重な証明することができた。新生児マウス皮膚から単離された体性幹細胞から卵​​母細胞様細胞（OLCs）の形成が実証されており、このビデオプロトコルで可視化することができる。結果OLCsはそのようなのOct4、ヴァーサ、BMP15、そしてSCP3として卵母細胞と一致し、様々なマーカーを発現する。しかし、彼らは成熟や減数分裂を完了するために、障害のために受精を受けることができないままです。このプロトコルは、より成熟した配偶子に生殖細胞の初期段階の形成と分化を研究するために有用であるシステムを提供します。初期の分化の間のOct4（電位生殖細胞様細胞）を発現する細胞の数をfしかしながら、現在、約5％に達するOLCsのormationは比較的非効率的なままです。特別な注意を出発細胞集団の健康と早いパッセージであることを確認するために取られるべきであるにもかかわらずプロトコルは比較的単純です。

最初に細胞が培養皿の底に付着すると広がる。分化小さな浮遊OCT4陽性細胞に42から72時間後に形成し、増殖する（ 図1A）。まもなくこれらの細胞の可視化後大半が消え、付着した細胞は、培養底に密集コロニー凝集体を形成します。数日の後、これらの凝集体は、培養表面からデタッチします。これらの凝集体のいくつかにおいて大きなセル（ 図1bおよび図1c）...

Watch this Scientific Journal Video about Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro at JoVE.com

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

近年、インビトロ培養法において用い生殖細胞の形成は、体性幹細胞のいくつかの異なるタイプを使用して実証されている。この記事では、視覚的に早期の卵母細胞様細胞に新生マウス由来幹細胞の分化を紹介します。

生殖様細胞に新生マウス皮膚由来幹細胞の分化<em&gt;インビトロ</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

differentiation-newborn-mouse-skin-derived-stem-cells-into-germ-like

Research

JoVE Journal

Biology

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

9.2K ビュー.  The University of Western Ontario. 近年、インビトロ培養法において用い生殖細胞の形成は、体性幹細胞のいくつかの異なるタイプを使用して実証されている。この記事では、視覚的に早期の卵母細胞様細胞に新生マウス由来幹細胞の分化を紹介します。

ビデオ: Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

ハンギングドロップ法を用いたマウス胚性幹（MES）細胞のin vitroにおける分化

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

生物学

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

運動ニューロンにマウス胚性幹細胞の効率的な分化

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...