PWD Batı Üniversitesi&#39;nde Gerald M. Kidder için Sağlık Araştırma Bursu (CIHR) ve CIHR hibe Kanadalı bir Enstitüleri tarafından desteklenmektedir. Yazar ayrıca Burada sunulan protokol gelişimi ile yardım için Guelph Üniversitesi&#39;nde Julang Li kabul etmek istiyorum.

1. Medya Hazırlık <ol><li> Deney yer alacak hücre kültürü inkübatör bir gevşetti kapağı ile kullanım ve yer önceden medya 2-3 saat hazırlayın. Bu protokolde kullanılan Medya: <ol><li> 1 x B27, 40 ng / ml bFGF ve 20 ng / ml EGF ile desteklenmiş DMEM/F12 oluşan hücre orta kök hazırlayın. </li><li> 0.05 IU FSH, 0.03 IU LH, 3 mg / ml BSA, 5 ul / mL, 0.23 mM sodyum piruvat, 1 mg / ml fetuin, ve 1 ng / ml EGF ile takviye edilmiş M199 oluşan germ hücre farklılaşması orta hazırlayın. Tüm katkı maddelerinden oluşan ama FSH kullanılmadan bir baz vasatı, LH ve EGF ° C ila bir hafta için hazırlanmış ve 4 &#39;de saklanabilir. </li></ol></li></ol> 2. Hücre Kültürü Hazırlanması Kök <ol><li> Daha önce 12 açıklandığı gibi yeni doğan fare yavrular kök hücre izole edin. In vitro germ hücreli farklılaşmasında için geçiş 2-4 kök hücre yararlanın. VerimiOLC oluşumunu doğrudan ana kök hücrelerinin kalitesi ile ilgilidir. Bu en kısa sürede nüfusun geçit yaklaşık 2 genel olarak olan temiz ve sağlıklı göründüğü gibi farklılaşma başlamak en iyisidir. Ayrıca kültür kök hücreler son geçit 2 olumsuz OLC oluşumu verimliliği (yayınlanmamış) etkiler. </li><li> Önce alt-kültürü kök hücreler farklılaşma başlamadan 48 saat. Tüm askıya küresel hücre agrega ve harcanan medya kültür çanak, bir serolojik pipet kullanarak, ve bir 15 ml tüp içinde yer çıkarın. Sadece askıya küresel hücrelerin agrega değil, kendiliğinden ayırıyoruz çanak alt bağlı hücreleri toplamak için önemlidir. </li><li> 5 dakika boyunca 500 xg&#39;de Pelet hücreleri., Supernatant ve taze kök hücre, 500 ul ortam içinde tekrar süspansiyon 37 ° C ye ısıtıldı Yavaşça kısmen hücreleri ayırmak için agrega pipetle. Agresif hücre PipetiBu kadar s hücre canlılığı azaltacaktır. Düşük bir bağlanma 10 cm hücre kültürü kabı, taze, önceden ısıtılmış kök hücre orta 9.5 mi içine kısmen ayrışmış hücreleri yıkayın. </li><li> Farklılaşmanın başlamasından önce, 48 saat boyunca 37 ° C,% 5 CO2 inkübatör hücre kültürü hücreleri dönün. </li></ol> 3. OLC Farklılaşma <ol><li> Serolojik bir pipet kullanarak 15 ml tüp içinde kültür çanak ve yerden kök hücreleri çıkarmak, takılı olan hücreler geride bırakmak. 500 ul steril PBS içinde 500 xg ve tekrar süspansiyon de Pelet hücreleri. Güçlü pipet geniş kullanarak hücrelerin 10-20 hücreleri daha büyük değil kümeleri içine 1.000 ul ucu delik. Periyodik ayrışma sırasında, hücre küçük bir miktar kaldırmak ve toplu hücreler 10-20 tek tek hücrelerin kümeleri olan onaylamak için beyaz bir ışık mikroskobu altında kontrol edin. Ayrışma boyunca düşük hücre canlılığı ile sonuçlanacaktır. </li><li> Ayrışmış hücreleri için 9.5 ml PBS ekleyin ve 1 ekleyinHücre sayımı için bir hemasitometre bu hücre süspansiyonu 5 ul. 5 dakika boyunca 500 xg&#39;de Pelet hücreleri. </li><li> 1.32X10 6 hücre / farklılaşma ortamda ml &#39;lik bir konsantrasyonda, hücre yeniden askıya. </li><li> Düz dipli içine de ortalama 24 iyi süspansiyon çanak 500 ul ekleyerek plaka hücreleri. </li><li> 48 saat boyunca% 5 CO2 içinde 37 ° C&#39;de hücre kültürü inkübatöründe içine 24 ° C de çanak yerleştirin. Bir buna etiketli 1.5 ml tüp her iyi ve yerden 250 ul orta çıkarın. Her bir kuyunun 250 ul taze farklılaşma orta ekleyin. 5 dakika boyunca 500 x g&#39;de santrifüj harcanan aracı. ve supernatant atın. 50 ul taze farklılaşma ortamda herhangi bir pelet hücreleri tekrar süspansiyon ve farklılaşma plaka üzerinde de ilgili kültür dönmek. </li><li> Her 48 saat farklılaşma gün 12 yarım orta değişiklere. </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>

Çıkar çatışması ilan etti.

Fonksiyonel OLCS Bugüne kadar in vitro tamamen kullanılarak geliştirilmiştir edilmemiştir. Son Hayashi ve arkadaşları tarafından yapılan bir çalışmada. In vitro oluşan ve daha da geliştirilmesi 14 in vivo transfer hücreleri İKK-gibi gelen yavrular üretmek mümkün oldu. Onlar in vivo nakli sonrasında kurtarıldı, hücreleri gibi PGC oluşturmak başardık ES hücreleri veya uyarılmış pluripotent hücreler ile başlayan, olgunlaşmış döllenm...

Erken embriyo sırasında, gametogenez primordial germ hücreli (PGC) oluşumu, göç ve gonadal sırtlar sonunda kolonizasyon dahil olmak üzere aşamalarında bir dizi oluşur. Bu süre zarfında PGCs giderek daha olgun gamet 1 içine çoğalması ve farklılaşması tabi. PGCs embriyo hindgut içine allantoisi en tabanından ve son olarak sonunda gonadal sırtlar kolonize dorsal duvar boyunca göç gerçeğini 2 çalışma onları son derece zor hale getirir. Alanında ilerlemelere rağmen, PGC oluşumu ve farklılaşma anlamaya çalışırken çalışmalar sınırlı sayısı, yeri ve göçmen doğa 3,4 engellemektedir edilmiştir. Son yıllarda, embriyonik kök hücrelerinin in vitro 5,6 germ hücreleri oluşturmak için potansiyele sahip olduğu gösterilmiştir. Benzer şekilde, birçok somatik kök hücreleri de germ hücreleri FOLL oluşturmak için potansiyele sahip olduğu gösterilmiştirin vitro kültür 7-11 nedeniyle. Son zamanlarda yeni doğan fare deri izole kök hücre germ-benzeri hücreler ve erken evre oosit 12 in vitro ayırt edildi. Farklılaşma sırasında Oct4 ifade eden kök hücrelerin alt artmış ve yapılar benzeyen kümülüs-oosit kompleksleri oluşturulmuştur. Oosit-benzeri hücreler (OLCS) kültürlerden aldı ve doğal oosit ile karşılaştırılabilir. 40-45 mikron ölçüm Bu kültür yöntemi OLCS kullanılarak elde edilir böyle Gdf9b, VASA ve DAZL olarak oosit için ifade benzer işaretleri. Bugüne kadar ortaya çıkan OLCS 12 olgun veya döllenmiş olması mümkün kalır. OLCS bu OLCS oluşturmak için kullanılan protokol hala içinde daha erken bir aşamada germ hücrelerinin oluşumu ve gelişimini incelemek için bir test olarak yardımcı olabilir çalışması için mümkün kalmasına rağmen bir in vitro sistemde kapalı. Kadar gelen OLCS oluşumunu tartışırken orijinal yayınmatic kök hücreler bir kök hücre kaynağı 8 olarak fetal domuz deri kullanılmaktadır. Bağlı farklılaşma sırasında erken oluşan İKK-benzeri hücreler, Oct4 olarak OLCS ve ekspres bu İKK işaretleri için VASA, STELLA, C-KIT ve DAZL 13 öncüleri olduğu gösterilmiştir olduğunu çalışmaya genişletilmesi. Bu, in vitro olarak germ hücre oluşumu ve farklılaşma incelemek için bu sistemi kullanan potansiyel destekler. Yeni doğan fare deriden OLCS oluşturmak için kullanılan protokol Bu video makalede gösterilecektir. Bu protokol kendi çoğalması ve farklılaşması için faktörlerin önemli aydınlatmak için bir araç sağlayabilir germ hücre gelişimi incelemek için bir in vitro model sunuyor.

<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

Germ hücre oluşumu ve farklılaşması incelenmesi geleneksel olarak düşük hücre sayıları ve gelişmekte olan embriyoların içinde derin kendi konumu nedeniyle çok zor olmuştur. In vitro tabanlı sistem bir &quot;kapalı&quot; durumu gametogenez anlayışımız için çok değerli olabilecek. Yeni doğan fare deri izole somatik kök hücreler, gelen oosit-benzeri hücreler (OLCS) oluşumu gösterilmiştir ve bu video protokolde görüntülenebilir. Elde edilen OLCS gibi Oct4 Vasa, Bmp15 ve SCP3 gibi oosit ile tutarlı çeşitli işaretleri ifade eder. Ancak, olgunlaşma ya da mayoz tamamlamak için bir hatası nedeniyle gübreleme geçmesi mümkün kalır. Bu protokol daha olgun gamet içine germ hücrelerinin erken oluşumu ve farklılaşma eğitim için yararlı bir sistem sağlayacaktır. Erken farklılaşması sırasında Oct4 eksprese eden hücrelerin sayısına (potansiyel germ hücreleri gibi), ancak şu anda, F ~% 5 ulaşırOLCS bir ormation nispeten verimsiz kalır. Özel bakım başlangıç ​​hücre popülasyonu sağlıklı ve erken bir geçit olduğundan emin olmak için alınması gereken rağmen protokol oldukça yalındır.

Başlangıçta hücrelerin kültür çanak altına eklemek ve yaymak. Farklılaşma içine 42-72 saatte küçük askıya Oct4 pozitif hücreler oluşturmak ve (Şekil 1A) çoğalırlar. Kısa bir süre bu hücrelerin görselleştirme sonra çoğunluğu kaybolur ve bağlı hücrelerin kültür altındaki yoğun koloni agrega oluşturacaktır. Birkaç gün sonra bu agrega kültür yüzeyinden ayırır. Bu agrega bazılarında bir büyük hücreli (Şekil 1b ve 1c) görül...

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

Son yıllarda, in vitro kültür yöntemler kullanılarak germ hücre oluşumunu somatik kök hücrelerin birkaç farklı türde kullanılarak gösterilmiştir. Bu makale, görsel erken oosit benzeri hücrelere yeni doğan fare edilen kök hücrelerin farklılaşma sunacak.

Germ benzeri hücreler içine Yenidoğan Fare Cilt Türetilmiş Kök Hücre Farklılaşma<em&gt; In vitro</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 Görüntüleme.  The University of Western Ontario. Son yıllarda, in vitro kültür yöntemler kullanılarak germ hücre oluşumunu somatik kök hücrelerin birkaç farklı türde kullanılarak gösterilmiştir. Bu makale, görsel erken oosit benzeri hücrelere yeni doğan fare edilen kök hücrelerin farklılaşma sunacak.

Video: 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.

Asma Bırak Yöntemiyle Fare Embriyonik Kök (MES) Hücreler in vitro Farklılaşma

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 ...

Biyoloji

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.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

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

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 Retroviral Vector kullanarak İsteyerek Pluripotent Kök Hücrelerinin (iPSCs) içine İnsan somatik hücreleri yeniden programlama

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.

Motor Nöronlar içine Fare Embriyonik Kök Hücre Verimli Farklılaşma

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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

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

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.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Rhesus Macaque Adipoz Türevi Kök Hücrelerin İzolasyonu, Proliferasyonu ve Farklılaşması

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...