Nous remercions Laura Clarke pour son aide inestimable. Ce travail est soutenu par des RCE: Réseau de cellules souches, les IRSC et les NIH.

1. Préparer les solutions de dissection et de solutions enzymatiques <ol><li> Faites le liquide céphalo-rachidien artificiel (ACSF) et les médias sans sérum à l&#39;avance et réfrigérer. </li><li> Peser l&#39;acide kynurénique (0,2 mg / ml) et dissoudre dans 10 ml de hilo ACSF au bain-marie à 37 ° C à l&#39;avance car il ne se dissolvent pas facilement. </li><li> Peser la trypsine (1,33 mg / mL) et de hyaluronidase (0,67 mg / mL) et le lieu dans un tube de 15 ml et le conserver à -20 ° C jusqu&#39;à ce que nécessaire. Ajouter la solution d&#39;acide Kynurenic à ce tube et le filtre à l&#39;aide d&#39;un filtre 22 um seringue juste avant l&#39;utilisation. </li><li> Peser inhibiteur de la trypsine (Ovamucoid: 1 mg / mL) et dissoudre dans chaude milieu sans sérum et le filtre en utilisant un filtre seringue 22μm. </li><li> Faire les médias placage: un milieu sans sérum contenant FGF2 (10 ng / ml) et l&#39;héparine (2 pg / ml). </li></ol> 2. Isoler des cellules souches rétiniennes de l&#39;Oeil de la souris <ol><li> L&#39;isolement des cellules souches rétiniennes est effectuée dans un local spécifique dédié aux expériences stériles culture primaire. Avant de commencer cette procédure, mis en place le microscope à dissection froide et source de lumière, puis disposer les instruments stériles dissection. Chaque hotte est un stérilisateur à chaud de perles pour stériliser les instruments entre les étapes. </li><li> Nous allons isoler les cellules souches rétiniennes dans les yeux qui ont été immédiatement placés dans une boîte de Pétri stérile contenant artificielle liquide céphalo-rachidien (ACSF) après leur retrait de souris sacrifiées en fonction de protocoles approuvés éthique animale. </li><li> Sous la loupe binoculaire, nettoyer l&#39;œil avec une pince: se débarrasser de la chevelure et le tissu conjonctif qui est attaché à la cornée / frontière scléral. Puis transfert à l&#39;œil d&#39;un nouveau plat avec l&#39;ACSF. </li><li> Tout en douceur en maintenant l&#39;œil fixe avec des pinces dentelées, l&#39;utilisation de micro-dissection angle de ciseaux pour retirer les muscles oculaires. Retirez le nerf optique ainsi si elle est toujours attachée à l&#39;œil. Essayez de ne pas écraser l&#39;œil au cours de ce processus. Transfert yeux pour un nouveau plat avec l&#39;ACSF. </li><li> Suivant l&#39;utilisation de micro-dissection courbes des ciseaux pour couper l&#39;œil dans la moitié: commencer au nerf optique et couper par le milieu de la cornée, réunion de retour au nerf optique où la coupe a été lancé. Il est idéal si les deux morceaux de l&#39;œil sont à peu près équivalente en taille car cela rendra plus facile la prochaine étape. </li><li> Tenir les cornées l&#39;aide de deux pinces, retirez délicatement l&#39;œil les deux moitiés de l&#39;autre. Retirez et jetez les lentilles, les viscères, et la rétine neurale à partir de coquilles oeil. Transférer les coquilles dans un plat avec les nouveaux ACSF. </li><li> Maintenant nous sommes prêts à isoler l&#39;épithélium ciliaire. Pour commencer, orienter la coquille des yeux afin que la cornée est sur votre droite et l&#39;épithélium pigmentaire rétinien (EPR) est sur la gauche. Délicatement la coquille broches oeil vers le bas avec une pince sur le côté droit du RPP. </li><li> Ensuite, utiliser un scalpel pour couper la cornée et l&#39;iris loin de l&#39;épithélium ciliaire de l&#39;œil en appuyant doucement sur le bistouri plutôt que d&#39;utiliser mouvements de sciage. </li><li> Après cela, couper l&#39;épithélium ciliaire loin de l&#39;EPR. Utilisez une pince non dentelé de transférer la bande de l&#39;épithélium ciliaire à une nouvelle de 35 mm plat contenant ACSF. </li><li> De la même façon, isoler l&#39;épithélium ciliaire de la coquille autre œil. </li><li> Une fois les deux bandes ciliaires épithéliales ont été recueillies, le transfert des bandes dans un plat de 35 mm contenant 2 ml de dispase. Placer le plat avec les bandes dans un incubateur à 37 ° C pendant 10 minutes. </li><li> Après 10 minutes, transférer les bandes de la dispase dans un plat contenant 2 ml de trypsine filtré, hyaluronidase, et l&#39;acide kynurénique. Placer le plat à 37 ° C pendant 10 minutes. </li><li> Revenez maintenant à la loupe binoculaire. Tout en tenant le bas de la sclère avec une pince droite, utiliser le bas de la incurvé non dentelé des forceps pour gratter délicatement l&#39;épithélium ciliaire loin de la sclérotique. </li><li> Retirer la sclérotique de l&#39;antenne. Tout ce qui doit être laissé dans le plat sont maintenant les cellules d&#39;intérêt et de la solution enzymatique. </li><li> Utiliser un feu poli coton branchée pipette, transférer cette solution dans un tube de 14 ml. Triturer cette solution 30 fois pour briser les cellules épithéliales en douceur forçant la solution dans et hors de la pipette. </li><li> Centrifuger le tube pendant 5 minutes à 1500 RPM. Lorsque la centrifugation est terminée, retirer le tube de la centrifugeuse avec soin, car les cellules sont enclins à se détacher du fond du tube à ce stade. </li><li> Doucement aspirer la majorité des surnageant avec une pipette polie au feu, puis ajoutez 1 ml d&#39;inhibiteur de la trypsine dans un milieu sans sérum pour les cellules. Utilisez un petit coton branchée forage pipette de triturer l&#39;échantillon d&#39;environ 50 fois jusqu&#39;à ce qu&#39;elle soit une suspension à cellule unique. </li><li> Centrifuger le tube à nouveau pendant 5 minutes à 1500 RPM. </li><li> Enlever le surnageant et le remplacer par 1 mL de votre milieu d&#39;étalement. Triturer doucement à l&#39;aide d&#39;une pipette en verre poli au feu pour remettre les CELLS. </li><li> Après comptage des cellules, plaque eux à la densité désirée dans une plaque de 24 puits. En général, nous plaque 10 cellules / uL en remplissant chaque puits d&#39;abord avec support et en ajoutant ensuite la suspension de cellules pour obtenir un volume final de 500 pl de médias. </li><li> Placer la plaque dans un 37 ° C CO 2 incubateur où il ne sera pas déplacé jusqu&#39;à ce que vous comptez les sphères qui surviennent après 7 jours. </li></ol> 3. Résultats pour isoler des cellules souches rétiniennes <ol><li> Lorsque cette procédure est effectuée avec succès, le disséqué ciliaire des cellules épithéliales devrait ressembler à ceci après avoir été dissocié et étalées à faible densité (figure 1). </li><li> Après 7 jours de culture, les sphères de cellules souches rétiniennes qui se posent sont comptés. Une sphère doit être plus de 75 um de diamètre et flottant à être compté comme une cellule souche dérivée sphère. (Figure 2). Toutefois, certaines cellules ont limité la prolifération et la forme de sphéroïdes qui ne satisfait pas au critère de taille et ne seront pas comptés comme des sphères de cellules souches dérivées; un exemple d&#39;un tel sphéroïde est montré ici (figure 2). </li></ol> 4. Les résultats représentatifs <img src="/files/ftp_upload/2209/2209fig1.jpg" alt="Figure 1" / > Figure 1. <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="Figure 2" / > Figure 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>

Ce protocole décrit comment isoler des cellules souches rétiniennes de l&#39;épithélium ciliaire de l&#39;oeil de souris 1-3. La méthodologie utilisée pour isoler les bandes de l&#39;épithélium ciliaire peut varier, cependant les enzymes et la méthodologie permettant d&#39;atteindre une suspension à cellule unique ont été optimisés dans ce protocole. Il est également important que les bandes de l&#39;épithélium ciliaire qui ont été isolés ne contiennent de grandes quantités de RPE ou cornée puisque ceux-ci semblent avoir un impact négatif sur le nombre total des sphères qui peuvent être isolés de chaque œil. En outre, les milieux sans sérum que nous faisons qui a été initialement formulé pour le cerveau neurosphères 13, est idéal pour la culture de cellules souches rétiniennes sphères. On devrait également s&#39;assurer que les cellules ne sont pas perturbés après avoir été plaqué et placés dans l&#39;incubateur afin que les sphères de plus en plus ne s&#39;agrègent pas ainsi 14. Une fois les cellules souches rétiniennes ont formé des sphères clonale, elles peuvent être comptées pour obtenir un nombre potentiel de cellules souches par œil. Les sphères, puis peuvent ensuite être dissociés en cellules individuelles et passages pour vérifier les capacités d&#39;auto-renouvellement ou différenciée selon les types de cellules différentes de la rétine neuronale et RPE en utilisant différentes combinaisons de facteurs de croissance et / ou des protéines.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
</tr>
<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>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
</tr>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
<tr>
<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
<tr>
<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
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<tr>
<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
</tr>
<tr>
<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<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

Les souches de souris adultes des cellules rétiniennes (RSC) est une cellule quiescente rares trouvées dans l&#39;épithélium ciliaire (CE) de l&#39;œil des mammifères 1,2,3. Le CE est composé de non-pigmentées intérieure et pigmentées des couches externes des cellules, et les colonies clonales SRC qui découlent d&#39;une seule cellule pigmentée de la CE sont constitués de deux cellules pigmentées et non pigmentées qui peuvent être différenciés pour former tous les types de cellules de la rétine neuronale et le RPP. Il ya une certaine controverse quant à savoir si toutes les cellules au sein des sphères contiennent tous au moins quelques pigments 4, mais les cellules sont encore susceptibles de former les différents types de cellules trouvées au sein de la rétine neurale 1-3. Chez certaines espèces, comme les amphibiens et les poissons, leurs yeux sont capables de régénération après une blessure 5, toutefois, les yeux des mammifères montre pas de telles propriétés régénératrices. Nous cherchons à identifier les cellules souches in vivo et de comprendre les mécanismes qui maintiennent les cellules souches rétiniennes de mammifères repos 6-8, même après des blessures ainsi que de les utiliser comme une source potentielle de cellules pour aider à la réparation des modèles physiques ou génétiques des lésions oculaires par la transplantation 9-12. Nous décrivons ici comment isoler les cellules épithéliales ciliaires de l&#39;oeil de la souris et les faire grandir dans la culture afin de former la rétine sphères clonale de cellules souches. Comme il n&#39;existe pas de marqueurs connus de la cellule souche in vivo, ces sphères sont le seul moyen connu d&#39;identification prospective de la population de cellules souches au sein de l&#39;épithélium ciliaire de l&#39;œil.

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

Dans cette vidéo, nous allons démontrer comment isoler des cellules souches rétiniennes de l&#39;épithélium ciliaire de l&#39;oeil de la souris et les faire grandir dans la culture pour former clonale sphères de la rétine. Les sphères qui sont isolées possèdent les propriétés cardinales des cellules souches: l&#39;auto-renouvellement et multipotentiality.

L&#39;isolement des cellules souches rétiniennes de l&#39;Oeil de la souris

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

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JoVE Journal

Biology

24.6K vues.  University of Toronto. Dans cette vidéo, nous allons démontrer comment isoler des cellules souches rétiniennes de l'épithélium ciliaire de l'oeil de la souris et les faire grandir dans la culture pour former clonale sphères de la rétine. Les sphères qui sont isolées possèdent les propriétés cardinales des cellules souches: l'auto-renouvellement et multipotentiality.

Vidéo: 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.

Isolement et analyse de cellules souches hématopoïétiques à partir du 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. ...

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Biologie

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;isolement et la dérivation de cellules germinales embryonnaires de souris

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.

L&#39;isolement des cellules souches de la souris salivaires Gland

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

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-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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.

Cultures de cellules primaires de l’épithélium pigmentaire rétinien de souris

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.

Isolement, caractérisation et différenciation des cellules souches cardiaques du coeur de souris adulte

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.

Dissection efficace et culture des cellules épithéliales rétiniennes primaires de pigment de souris

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.

Isolement de cellules épithéliales pigmentées rétiniennes primaires de souris

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