PWD è supportato da una Canadian Institutes of Health Research fellowship (CIHR) e una borsa di CIHR di Gerald M. Kidder alla Western University. L&#39;autore desidera inoltre ringraziare Julang Li presso l&#39;Università di Guelph per un aiuto con lo sviluppo del protocollo presentato qui.

1. Media Preparazione <ol><li> Preparare i media 2-3 ore in anticipo di utilizzo e luogo con un tappo allentato nella cultura incubatore cella in cui l&#39;esperimento avrà luogo. Supporti utilizzati nel presente protocollo: <ol><li> Preparare staminali mezzo cella costituita dal DMEM/F12 addizionato con 1 x B27, 40 ng / ml bFGF, e 20 ng / ml EGF. </li><li> Preparare cellule germinali terreno di differenziamento composto M199 addizionato con 0,05 UI di FSH, LH 0,03 IU, 3 mg / ml BSA, 5 microlitri / ml ITS, 0,23 mM di piruvato di sodio, 1 mg / ml Fetuina, e 1 ng / ml EGF. Un mezzo base costituita da tutti gli additivi ma omettendo FSH, LH e EGF può essere preparata e conservata a 4 ° C per una settimana. </li></ol></li></ol> 2. Stem Cell Culture Preparazione <ol><li> Isolate le cellule staminali da cuccioli appena nati del mouse come descritto in precedenza 12. Utilizzare le cellule staminali al passaggio 2-4 per la differenziazione in vitro delle cellule germinali. L&#39;efficienza diFormazione OLC è direttamente correlata alla qualità delle cellule staminali iniziali. È meglio iniziare la differenziazione non appena la popolazione appare sana e pulita che è generalmente intorno passaggio 2. Ulteriormente coltivando le cellule staminali passato passaggio 2 influisce negativamente sull&#39;efficienza formazione OLC (risultati non pubblicati). </li><li> 48 hr prima di iniziare differenziazione sub-coltura delle cellule staminali. Rimuovere tutti gli aggregati sferici sospesi cellulari e media spesi dal piatto di coltura, utilizzando una pipetta sierologica, e posto in una provetta da 15 ml. È importante raccogliere solo i sospese aggregati sferici di cellule e non le cellule attaccate al fondo del piatto che si differenzia spontaneamente. </li><li> Agglomerare le cellule a 500 xg per 5 min., Scartare il surnatante e risospendere in 500 ml di mezzi di cellule staminali fresche riscaldato a 37 ° C. Pipettare delicatamente gli aggregati di dissociare parzialmente le cellule. Non aggressivo pipettare la cellas per evitare di ridurre la vitalità cellulare. Lavare le cellule parzialmente dissociato in 9,5 ml di fresco pre-riscaldato medio di cellule staminali su un 10 centimetri piatto di coltura cellulare a basso attaccamento. </li><li> Riportare le cellule ad una ° C, 5% CO 2 incubatore di coltura cellulare 37 per 48 ore prima di iniziare differenziazione. </li></ol> 3. Differenziazione OLC <ol><li> Usando una pipetta sierologica rimuovere le cellule staminali dal piatto di coltura e posto in una provetta da 15, lasciarsi alle spalle tutte le celle collegate. Agglomerare le cellule a 500 xg e risospendere in 500 microlitri di PBS sterile. Pipetta vigoroso le cellule utilizzando un ampio foro 1000 punta microlitri in ciuffi non più grandi di 10-20 cellule. Periodicamente rimuovere una piccola quantità di cellule, durante la dissociazione, e verificare sotto un microscopio a luce bianca per confermare le cellule sono aggregati in gruppi di 10-20 cellule individuali. Oltre dissociazione provocherà vitalità cellulare ridotta. </li><li> Aggiungere 9,5 ml di PBS per le cellule dissociate e aggiungere 15 microlitri di sospensione cellulare ad un emocitometro per la conta cellulare. Agglomerare le cellule a 500 xg per 5 min. </li><li> Risospendere le cellule ad una concentrazione di 1.32X10 6 cellule / ml in terreno di differenziamento. </li><li> Piastra le cellule con l&#39;aggiunta di 500 microlitri per pozzetto in un fondo piatto 24 piatto sospensione bene. </li><li> Posizionare il piatto ben 24 in una coltura cellulare incubatore a 37 ° C in 5% CO 2 per 48 ore. Togliere 250 medie microlitri di ogni bene e mettere in un marcato corrispondentemente 1.5 tubo ml. Aggiungere 250 microlitri terreno di differenziamento fresco in ciascun pozzetto. Centrifugare il medium speso a 500 xg per 5 min. e scartare il surnatante. Risospendere le cellule in pellet in 50 microlitri terreno di differenziamento fresco e ritornare alla cultura corrispondente pozzetto della piastra differenziazione. </li><li> Ogni 48 ore cambia la metà del mezzo fino al giorno 12 di differenziazione. </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>

Nessun conflitto di interessi dichiarati.

Ad oggi oLc funzionali non sono stati sviluppati utilizzando una completamente in vitro. Recentemente uno studio condotto da Hayashi et al. Era in grado di produrre prole da PGC-come le cellule formati in vitro e trasferimento in vivo per un ulteriore sviluppo 14. Partendo da cellule ES o cellule pluripotenti indotte sono stati in grado di formare PGC come le cellule che, una volta recuperati a seguito di trapianto in vivo sono stati in grado di essere maturato, fe...

Durante l&#39;embriogenesi precoce, gametogenesi avviene attraverso una serie di tappe tra cui cellule germinali primordiali (PGC) formazione, migrazione, e, infine, la colonizzazione delle creste delle gonadi. Durante questo periodo PGC subiscono proliferazione e differenziazione in sempre gameti più mature 1. Il fatto che il PGC migrano dalla base del allantoide nella hindgut embrione e infine lungo la parete dorsale finalmente colonizzando le creste delle gonadi li rende estremamente difficile da studiare 2. Nonostante i progressi nel campo, gli studi che tentano di comprendere la formazione PGC e differenziazione sono stati ostacolati dal loro numero limitato, la posizione e la natura migratoria 3,4. Negli ultimi anni le cellule staminali embrionali hanno dimostrato di avere il potenziale per formare le cellule germinali in vitro 5,6. Analogamente numerosi cellule staminali somatiche hanno anche dimostrato di avere il potenziale di formare cellule germinali Follgrazie in coltura in vitro 7-11. Recentemente staminali cellule isolate dalla cute neonato topo sono state differenziate in vitro in cellule germinali-simili e primi ovociti stage 12. Durante la differenziazione del sottoinsieme delle cellule staminali che esprimono Oct4 aumentata e le strutture che assomigliano complessi cumulo-ovocita si sono formati. L&#39;ovocita-come le cellule (oLc) possono essere ritirati dalle culture e confrontati con ovociti naturali. Utilizzando questo metodo di coltura oLc di misura 40-45 micron si ottengono che esprimono i marcatori simili a ovociti come Gdf9b, VASA, e DAZL. Ad oggi le oLc risultanti rimangono incapaci di maturare o essere fecondati 12. Sebbene i oLc rimangono incapaci di funzionare il protocollo utilizzato per formare questi oLc può ancora avere utilità come un saggio per studiare la formazione e lo sviluppo delle cellule germinali fase precedente all&#39;interno di un sistema chiuso in vitro. La pubblicazione originale discutere la formazione di oLc da cosìcellule staminali matic utilizzati pelle porcina fetale come fonte di cellule staminali 8. Ampliando tale studio le cellule PGC-come si formano presto durante la differenziazione indotta hanno dimostrato di essere i precursori per la oLc ed esprimere tali marcatori PGC come OCT4, VASA, STELLA, C-KIT, e DAZL 13. Questi dati supportano la possibilità di utilizzare questo sistema per studiare la formazione di cellule germinali e differenziazione in vitro. Il protocollo utilizzato per formare oLc dalla pelle appena nato del mouse sarà dimostrato in questo articolo il video. Questo protocollo offre un modello in vitro per lo studio dello sviluppo delle cellule germinali che possono fornire un mezzo per delucidare i fattori importanti per la loro proliferazione e differenziazione.

<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

Studiare la formazione delle cellule germinali e la differenziazione è sempre stato molto difficile a causa di un basso numero di cellule e la loro posizione nel profondo di sviluppo degli embrioni. La disponibilità di un &quot;chiuso&quot; nel sistema basato vitro potrebbe rivelarsi prezioso per la nostra comprensione della gametogenesi. La formazione di ovociti-come le cellule (oLc) da cellule staminali somatiche, isolate dalla pelle neonato topo, è stata dimostrata e può essere visualizzato in questo protocollo video. Le oLc risultanti esprimono vari marker coerenti con gli ovociti come Oct4, Vasa, BMP15 e SCP3. Tuttavia, rimangono in grado di sottoporsi a maturazione o la fecondazione a causa di un mancato completamento della meiosi. Questo protocollo fornirà un sistema che è utile per studiare la formazione fase iniziale e la differenziazione delle cellule germinali in gameti più mature. Durante precoce differenziazione del numero di cellule che esprimono Oct4 (potenziali cellule germinali-simili) raggiunge ~ 5%, tuttavia attualmente il formazione delle oLc rimane relativamente inefficiente. Il protocollo è relativamente semplice ma particolare cura deve essere presa per assicurare la popolazione di cellule di partenza è sano e ad un passaggio precoce.

Inizialmente le cellule attribuiscono alla cultura piatto fondo e sparsi. A 42-72 ore in differenziazione piccoli sospesi OCT4 cellule positive formano e proliferano (Figura 1A). Poco dopo la visualizzazione di queste cellule maggioranza scompare e le cellule attaccate formerà aggregati colonia dense sul fondo cultura. A seguito di un paio di giorni questi aggregati si staccano dalla superficie cultura. In alcuni di questi aggregati può osservare una grande cella (Figura 1b e ...

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

Negli ultimi anni la formazione di cellule germinali utilizzando metodi in coltura in vitro è stata dimostrata utilizzando diversi tipi di cellule staminali somatiche. Questo articolo presenta visivamente la differenziazione delle cellule staminali derivate topo neonato per primi cellule ovocita-like.

Differenziazione del neonato mouse pelle cellule staminali derivate in cellule germinali-simili<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 Views.  The University of Western Ontario. Negli ultimi anni la formazione di cellule germinali utilizzando metodi in coltura in vitro è stata dimostrata utilizzando diversi tipi di cellule staminali somatiche. Questo articolo presenta visivamente la differenziazione delle cellule staminali derivate topo neonato per primi cellule ovocita-like.

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.

La differenziazione in vitro di staminali embrionali di topo (MES) Cellule con il metodo goccia

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 ...

Ricerca

Biologia

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.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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.

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con GFP

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.

Differenziazione efficiente di cellule staminali embrionali di topo in neuroni motori

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

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.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

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-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

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.

Isolamento, proliferazione e differenziamento di cellule staminali derivate dall'adiposio del macaco rhesus

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