Ringraziamo Laura Clarke per la sua preziosa assistenza. Questo lavoro è supportato da NCE: Rete delle cellule staminali, CIHR e NIH.

1. Preparare le soluzioni Dissecting e soluzioni enzimatiche <ol><li> Rendere il Artificiale fluido cerebro-spinale (ACSF) e il siero senza supporti in anticipo e conservare in frigorifero. </li><li> Pesare l&#39;acido Kynurenic (0,2 mg / ml) e scioglierlo in 10 ml di Hilo ACSF a 37 ° C a bagno-maria in anticipo dal momento che non si dissolvono rapidamente. </li><li> Pesare la tripsina (1,33 mg / mL) e ialuronidasi (0,67 mg / mL) e posto in un tubo di 15 ml e la tiene a -20 ° C fino al momento dell&#39;uso. Aggiungere la soluzione di acido Kynurenic a questo tubo e filtro con un filtro di 22 micron siringa immediatamente prima dell&#39;uso. </li><li> Pesare inibitore della tripsina (Ovamucoid: 1 mg / mL) e si dissolvono in caldo senza siero Media e filtrare con filtro 22μm siringa. </li><li> Fare dei mass media placcatura: Siero senza supporti contenenti FGF2 (10 ng / ml) ed eparina (2 mg / mL). </li></ol> 2. Isolare cellule staminali della retina dalla Eye mouse <ol><li> Isolamento delle cellule staminali della retina viene eseguita in un locale specifico sterile dedicata agli esperimenti coltura principale. Prima di iniziare questa procedura, impostare il microscopio da dissezione e la luce fredda di origine e quindi tracciare gli strumenti sterili dissezione. Ogni cappa è uno sterilizzatore a caldo tallone per la sterilizzazione degli strumenti tra i passaggi. </li><li> Noi isolare le cellule staminali della retina da occhi che sono stati posti immediatamente in una piastra di Petri sterile contenente artificiale fluido cerebro-spinale (ACSF) dopo la loro rimozione dai topi sacrificati in base a protocolli animali approvati etica. </li><li> Sotto il microscopio da dissezione, pulire l&#39;occhio con le pinze: liberarsi dei capelli e il tessuto connettivo che è attaccato alla cornea / confine sclerale. Poi il trasferimento l&#39;occhio ad un nuovo piatto con ACSF. </li><li> Mentre dolcemente tenendo gli occhi fissi con una pinza dentata, utilizzare angolato micro-dissezione forbici per rimuovere i muscoli oculari. Togliere il nervo ottico anche se è ancora attaccato l&#39;occhio. Cercate di non schiacciare l&#39;occhio durante questo processo. Trasferire gli occhi ad un nuovo piatto con ACSF. </li><li> Uso successivo curve micro-dissezione forbici per tagliare l&#39;occhio a metà: cominciare dal nervo ottico e tagliare il centro della cornea, riuniti di nuovo al nervo ottico, dove è stato avviato il taglio. È l&#39;ideale se i due pezzi di occhi sono più o meno equivalente di dimensioni perché questo farà il prossimo passo più facile. </li><li> Tenendo le cornee con due pinze, buccia delicatamente l&#39;occhio due metà a parte. Rimuovere ed eliminare l&#39;obiettivo, viscere, e la retina neurale dai gusci degli occhi. Trasferire i gusci di un nuovo piatto con ACSF. </li><li> Ora siamo pronti a isolare l&#39;epitelio ciliare. Per iniziare, orientare la conchiglia oculare in modo che la cornea è sulla destra e l&#39;epitelio pigmentato retinico (RPE) si trova sulla sinistra. Delicatamente il guscio pin occhio verso il basso con pinze dritto sul lato RPE. </li><li> Quindi, usare un bisturi per tagliare la cornea e iride lontano dal epitelio ciliare dell&#39;occhio spingendo delicatamente verso il basso il bisturi e non con movimenti di taglio. </li><li> Dopo di che, tagliare l&#39;epitelio ciliare lontano dal RPE. Usa non seghettato pinze per trasferire la striscia di epitelio ciliare ad un nuovo 35-mm piatto contenente ACSF. </li><li> Allo stesso modo, isolare l&#39;epitelio ciliare dal guscio altro occhio. </li><li> Una volta che entrambe le strisce ciglia epiteliali sono stati raccolti, trasferire le strisce in un piatto da 35 mm contenente 2 ml di dispasi. Posizionare il piatto con le strisce in un incubatore a 37 ° C per 10 minuti. </li><li> Dopo 10 minuti, trasferire le strisce dal dispasi in un piatto contenente 2 ml di tripsina filtrata, ialuronidasi, e Acido Kynurenic. Porre la capsula a 37 ° C per 10 minuti. </li><li> Ora ritorno al microscopio da dissezione. Mentre si tiene la sclera giù con pinza dritta, utilizzare il fondo della curva non seghettato pinze per raschiare delicatamente l&#39;epitelio ciliare lontano dalla sclera. </li><li> Rimuovere la sclera dal piatto. Tutto ciò che dovrebbe essere lasciato nel piatto ora sono le cellule di interesse e la soluzione enzimatica. </li><li> Utilizzando un incendio lucidato cotone collegato pipetta, trasferire questa soluzione in un tubo di 14 ml. Triturare questa soluzione 30 volte di spezzare le cellule epiteliali delicatamente forzando la soluzione dentro e fuori della pipetta. </li><li> Centrifugare la provetta per 5 minuti a 1500 giri. Quando la centrifugazione è fatto, togliere il tubo dalla centrifuga con attenzione in quanto le cellule sono inclini a staccarsi dal fondo della provetta in questa fase. </li><li> Aspirare delicatamente la maggior parte delle surnatante utilizzando un incendio lucidato pipetta, e si aggiunge 1 ml di inibitore della tripsina nel siero senza supporti alle cellule. Utilizzare un piccolo pozzo di cotone collegato pipetta per triturare il campione di circa 50 volte fino a quando si tratta di una cella singola sospensione. </li><li> Centrifugare la provetta per 5 minuti a 1500 RPM. </li><li> Rimuovere il surnatante e sostituirlo con 1 ml di vostro mezzo di placcatura. Triturare delicatamente con un incendio lucidato pipetta di vetro per risospendere le cellule. </li><li> Dopo che il conteggioING l&#39;, le cellule piastra loro alla densità desiderata in un 24-pozzetti. Noi di solito tavola 10 cellule / mL, compilando tutti i pozzetti prima media e quindi aggiungendo la sospensione cellulare per ottenere un volume finale di 500 l di media. </li><li> Posizionare la piastra a 37 ° C CO 2 incubatore dove non verrà spostato fino a contare le sfere che si presentano dopo 7 giorni. </li></ol> 3. Risultati di isolamento di cellule staminali della retina <ol><li> Quando questa procedura viene eseguita con successo, il sezionato cellule epiteliali ciliare dovrebbe essere simile a questo dopo essere stato dissociato e placcato a bassa densità (Figura 1). </li><li> Dopo 7 giorni di cultura, la retina sfere di cellule staminali che si presentano sono contati. Una sfera dovrebbe essere superiore a 75 micron di diametro e fluttuante per essere contato come una cellula staminale sfera derivati. (Figura 2). Tuttavia, alcune cellule avranno limitato sferoidi proliferazione e la forma che non soddisfano i criteri dimensioni e non verranno conteggiati come sfere di cellule staminali derivate; un esempio di uno sferoide è mostrato qui (Figura 2). </li></ol> 4. Rappresentante Risultati <img src="/files/ftp_upload/2209/2209fig1.jpg" alt="Figura 1" /> Figura 1. <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="Figura 2" /> Figura 2.

<ol>
	<li>Tropepe, V., Coles, B. L., Chiasson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+in+the+adult+mammalian+eye.">Retinal stem cells in the adult mammalian eye.</a> Science. 287, 2032-2036 (2000).</li><li>Ahmad, I., Tang, L., Pham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+progenitors+in+the+adult+mammalian+eye.">Identification of neural progenitors in the adult mammalian eye.</a> Biochem Biophys Res Commun. 270, 517-521 (2000).</li><li>Coles, B. L., Angenieux, B., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+isolation+and+the+characterization+of+human+retinal+stem+cells.">Facile isolation and the characterization of human retinal stem cells.</a> Proc Natl Acad Sci U S A. 101, 15772-15777 (2004).</li><li>Cicero, S. A., Johnson, D., Reyntjens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proc Natl Acad Sci U S A. 106, 6685-6690 (2009).</li><li>Lamba, D., Karl, M., Reh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+cell+replacement:+a+view+from+the+eye.">Neural regeneration and cell replacement: a view from the eye.</a> Cell Stem Cell. 2 (6), 538-549 (2008).</li><li>Coles, B. L., Horsford, D. J., McInnes, R. R., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+retinal+progenitor+cells+leads+to+an+increase+in+the+retinal+stem+cell+population+in+vivo.">Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo.</a> Eur J Neurosci. 23, 75-82 (2006).</li><li>Ang&#233;nieux, B., Schorderet, F. D., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+is+a+neuronal+differentiation+factor+for+retinal+stem+cells+in+vitro.">Epidermal growth factor is a neuronal differentiation factor for retinal stem cells in vitro.</a> Stem Cells. 24 (3), 696-706 (2006).</li><li>Xu, S., Sunderland, M. E., Coles, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proliferation+and+expansion+of+retinal+stem+cells+require+functional+Pax6.">The proliferation and expansion of retinal stem cells require functional Pax6.</a> Dev Biol. 304, 713-721 (2007).</li><li>Canola, K., Ang&#233;nieux, B., Tekaya, M., Quiambao, A., Naash, M. I., Munier, F. L., Schorderet, D. F., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+transplanted+into+models+of+late+stages+of+retinitis+pigmentosa+preferentially+adopt+a+glial+or+a+retinal+ganglion+cell+fate.">Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.</a> Invest Ophthalmol Vis Sci. 48 (1), 446-4454 (2007).</li><li>Canola, K., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cells+committed+towards+the+photoreceptor+fate+for+retinal+transplantation.">Generation of cells committed towards the photoreceptor fate for retinal transplantation.</a> Neuroreport. 18 (9), 851-855 (2007).</li><li>Djojosubroto, M. W., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells:+promising+candidates+for+retina+transplantation.">Retinal stem cells: promising candidates for retina transplantation.</a> Cell Tissue Res. 331 (1), 347-357 (2008).</li><li>Inoue, T., Coles, B. L., Dorval, K., Bremner, R., Bessho, Y., Kageyama, R., Hino, S., Matsuoka, M., Craft, C. M., McInnes, R. R., Tremblay, F., Prusky, G. T., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+functional+photoreceptor+differentiation+from+adult+human+retinal+stem+cells.">Maximizing functional photoreceptor differentiation from adult human retinal stem cells.</a> Stem Cells. 28 (3), 489-500 (2010).</li><li>Tropepe, V., Sibilia, M., Ciruna, B. G., Rossant, J., Wagner, E. F., Kooy, D. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.+Dev+Biol.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol.</a> , 208-201 (1999).</li><li>Coles-Takabe, B. L., Brain, I., Purpura, K. A., Karpowicz, P., Zandstra, P. W., Morshead, C. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo descrive come isolare le cellule staminali della retina dalla epitelio ciliare del mouse occhio 1-3. La metodologia per isolare le strisce di epitelio ciliare può variare, tuttavia gli enzimi e la metodologia per il raggiungimento di una singola cella di sospensione sono stati ottimizzati in questo protocollo. E &#39;anche importante che le strisce di epitelio ciliare che sono stati isolati non contengono grandi quantità di RPE o di cornea da quando questi sembrano avere un impatto negativo sul numero totale delle sfere che possono essere isolate da ogni occhio. Inoltre, il siero senza mezzi di comunicazione che facciamo che è stato originariamente formulato per il cervello neurosfere 13, è l&#39;ideale per la coltivazione di cellule staminali della retina sfere. Si dovrebbe anche fare in modo che le cellule non sono disturbati dopo che sono stati placcati e posti nell&#39;incubatrice in modo che le sfere in crescita non aggregato insieme 14. Una volta che le cellule staminali della retina hanno formato sfere clonale, possono essere calcolate per ottenere un numero potenziale di cellule staminali per occhio. Le sfere allora può quindi essere dissociato in cellule singole e diversi passaggi per accertare le capacità di auto-rinnovamento o differenziate in diversi tipi di cellule della retina neurale e RPE utilizzando diverse combinazioni di fattori di crescita e / o proteine.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
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<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
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<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
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<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
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<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
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<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
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<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
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<td>VWR/BD </td>
<td>CACB354235</td>
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<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
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<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
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<td>Kynurenic Acid</td>
<td>&nbsp;</td>
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<td>K3375</td>
<td>1000 IU</td>
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<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
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<td>35 mm dishes</td>
<td>&nbsp;</td>
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<td>CA354235</td>
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<td>24-well plate</td>
<td>&nbsp;</td>
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<td>CA73521-004</td>
<td>&nbsp;</td>
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<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
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<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
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<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
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<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
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<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
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isolation of retinal stem cells from the mouse eye

Il topo adulto cellule staminali della retina (RSC) è una rara cellula quiescente trovati all&#39;interno dell&#39;epitelio ciliare (CE) dell&#39;occhio dei mammiferi 1,2,3. La marcatura CE è costituito da strati interni e pigmentate non pigmentate cellula esterna, e le colonie clonale RSC che nascono da una singola cellula pigmentata dalla CE sono costituiti da due cellule pigmentate e non pigmentate, che possono essere differenziati per formare tutte le tipi di cellule della retina neurale e la RPE. Vi è una certa polemica sul fatto che tutte le cellule all&#39;interno delle sfere contengono tutti almeno alcuni pigmenti 4; ma le cellule sono ancora in grado di formare i diversi tipi di cellule trovati all&#39;interno della retina neurale 1-3. In alcune specie, come gli anfibi e pesci, i loro occhi sono in grado di rigenerazione dopo una lesione 5, tuttavia, l&#39;occhio dei mammiferi non mostra tali proprietà rigenerative. Cerchiamo di identificare le cellule staminali in vivo e per capire i meccanismi che mantengono le cellule staminali della retina dei mammiferi quiescente 6-8, anche dopo l&#39;infortunio così come servirsi di esse come una potenziale fonte di cellule per riparare i modelli fisici o genetici di lesioni agli occhi attraverso il trapianto di 9-12. Qui si descrive come isolare le cellule ciliari epiteliali dall&#39;occhio del mouse e farle crescere in coltura in modo da formare la retina sfere clonale di cellule staminali. Poiché non ci sono marcatori conosciuti delle cellule staminali in vivo, queste sfere sono l&#39;unico modo conosciuto per identificare in modo prospettico la popolazione di cellule staminali all&#39;interno del epitelio ciliare dell&#39;occhio.

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

In questo video ci dimostra come isolare le cellule staminali della retina dalla epitelio ciliare dell&#39;occhio del mouse e farle crescere in coltura per formare clonale sfere della retina. Le sfere che sono isolati possiedono le caratteristiche cardine delle cellule staminali: l&#39;auto-rinnovamento e multipotenzialità.

Isolamento di cellule staminali della retina dalla Eye mouse

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

isolation-of-retinal-stem-cells-from-the-mouse-eye

Research

JoVE Journal

Biology

24.6K Views.  University of Toronto. In questo video ci dimostra come isolare le cellule staminali della retina dalla epitelio ciliare dell'occhio del mouse e farle crescere in coltura per formare clonale sfere della retina. Le sfere che sono isolati possiedono le caratteristiche cardine delle cellule staminali: l'auto-rinnovamento e multipotenzialità.

Video: Isolation of Retinal Stem Cells from the Mouse Eye

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e analisi delle cellule staminali ematopoietiche dalla placenta

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

Ricerca

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento di cellule staminali del mouse ghiandole salivari

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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 ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Colture cellulari primarie dall'epitelio retinico del pigmento del Mouse

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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

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

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

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo adulto

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

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissezione e coltura efficienti delle cellule epiteliali del pigmento retino del topo primario

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolamento delle cellule primarie dell'epitelio pigmentato retinico del topo

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...