Ce travail a été financé par une nouvelle Faculty Award II du California Institute of Regenerative Medicine (RN2-00921-1), et un prix financée par les NIH National de la Recherche (F32-HL104924)

1. Préparation des mangeoires embryonnaires de souris <ol><li> La FAE utilisé dans la co-culture des CSEh doivent être préalablement traités par irradiation ou mitomycine-C pour inhiber les cellules en cours de division de. </li><li> Deux heures avant le semis, MEF, manteau chaque plasma de 60 mm boîte de culture traitée avec 2 ml de 0,05% de gélatine. </li><li> Décongeler une fiole de traité MEF et la plaque à ~ 20.000 cellules / cm 2. </li><li> Permettre aux cellules d&#39;adhérer nuit. </li></ol> 2. Co-ensemencement CSEh sur les MEF <ol><li> De nouvelles colonies peut être initiée à partir de cultures cryoconservés ou repiquage des cultures actuelles sur de nouveaux MEF boîtes revêtues, comme ci-dessous. </li><li> Laver la plaque contenant les colonies de CSEh grandes pour être repiquées 1 fois avec 3 ml de PBS 1x réchauffé. </li><li> Ajouter 3 ml de collagénase IV / solution de PBS (1 mg / ml) et incuber pendant 5-10 min à 37 ° C. (Remarque: Les cellules ne deviennent distincts ou ballon comme on le voit par la trypsine). </li><li> Bien que les cellules sont encore encollagénase, l&#39;utilisation d&#39;un bord d&#39;un embout de pipette 5 ml, de créer un motif de grille sur les colonies, les casser en petits amas cellulaires. </li><li> Utiliser l&#39;extrémité de la pointe de pipette (tenue de façon perpendiculaire à la plaque de culture) pour racler le fond de la plaque de culture, déloger toutes les cellules de la surface. </li><li> Recueillir les cellules dans un tube Falcon et de spin au (1000 rpm) pendant 5 min. </li><li> Après centrifugation, aspirer le milieu, laissant le culot cellulaire dans le tube Falcon. </li><li> Aspirer le milieu MEF MEF de l&#39;plaquée la veille. </li><li> Re-plaque les cellules à un ratio de 1:3 de la plaque d&#39;origine, et se nourrissent avec 3 ml de milieu de CSEh </li></ol> 3. Appauvrissant la FAE de la co-culture Remarque: l&#39;homme cultures ESC doit être à haute densité, généralement entre 10 et 14 jours de culture. <ol><li> Placez l&#39;extrémité d&#39;une pipette d&#39;aspiration sur le bord de la plaque et permettre l&#39;aspiration pour tirer doucement le bord de la feuille MEF. </li><li> ReMove les médias du plat et laissez la pointe d&#39;attraper la feuille de cellules et déplacer lentement la pointe d&#39;une manière arquée au-dessus de la plaque. Remarque: La feuille peut obstruer à la pointe de la pipette, mais ce n&#39;est pas problématique, car elle ne doit pas empêcher l&#39;utilisateur de tirer manuellement la feuille de la surface de la plaque de culture. Si les cellules sont collées à la fin de l&#39;aspirateur, on peut utiliser la partie supérieure de la boîte de culture à rompre la feuille de cellules pour l&#39;aspiration terminée. </li><li> Remplacer immédiatement moyenne CSEh, et mettre la plaque arrière dans l&#39;incubateur. </li></ol> 4. Les résultats représentatifs Le résultat final de la procédure de suppression MEF produit de petites colonies de CSEh non perturbées (figure 1D) pouvant subir une expansion significative en très grandes colonies (figure 2), tout en maintenant l&#39;expression des décideurs pluripotence SSEA-4, oct ¾, et Tra 1-81 (Figure 3). Figure 1. La morphologie des colonies ESC Avant et après le retrait du MEF. Embryonnaires humaines colonies de cellules souches à un jour) 1 et B) le jour 2 après le semis sur des boîtes de culture. C) haute densité Embryonnaires humaines colonies de cellules souches (H7) entouré par MEF. Les deux colonies de CSEh sont présentées en pointillés rouges. D) Après le retrait du MEF en utilisant la technique d&#39;aspiration décrits, on se retrouve avec une colonie isolée CSEh avec très peu MEF entourant la colonie intacte. <img alt="Figure 2" src="/files/ftp_upload/3951/3951fig2.jpg" /> Figure 2. Expansion Colony ESC 10 jours après le retrait du MEF. La colonie CSEh d&#39;origine immédiatement après l&#39;épuisement du MEF (à gauche) est autorisé à augmenter en une colonie très importante (à droite) approassociés quelque 800 um, 10x. Noter que la colonie originale est reproduite au même grossissement (10x) comme la colonie composite. <img alt="Figure 3" src="/files/ftp_upload/3951/3951fig3.jpg" /> Figure 3. Immunocoloration des colonies de CSEh MEF appauvries. Identification par immunofluorescence des colonies jusqu&#39;à 10 jours après l&#39;épuisement du MEF montre que les colonies maintenir leur expression de marqueurs de pluripotence comme évidente ici octobre ¾, SSEA-4 et Tra-1-81 coloration. En outre, ces colonies de CSEh ne présentent pas de différenciation vers un destin mésodermique, indiqué par l&#39;absence de Flk-1. A, E, I et M) sont des images de transmission de la lumière des colonies immunoflouscently tachés, 20, 10, 10 et 2x, . respectivement B, F, J et N) montrent les noyaux -. cellules DAPI colorés de colonies par immunofluorescence tachés, 20, 10, 10 et 2x, respectivement CD) montrent l&#39;expression dehumaine marqueur de cellules souches SSEA-4 uniquement, et l&#39;image composite, 20x. GH) montrent l&#39;expression de cellules souches humaines marqueur octobre ¾ seulement, et l&#39;image composite, KL 10x.) montrent l&#39;expression de marqueurs des cellules souches humaines Tra 1-81 seulement , et l&#39;image composite, 10x. MP) La dernière ligne si les images représentent une colonie décrivant les frontières lisses typiques vus à ce grossissement, 2x, et ces colonies n&#39;étaient pas expressément O) Flk-1, un marqueur précoce de la différenciation du mésoderme, ni ne ils P) contient aucun fibroblastes, comme indiqué par l&#39;absence de coloration DDR2. Vidéo supplémentaire. Retrait fibroblastes par aspiration de feuilles. <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">Cliquez ici pour voir le film</a> .

<ol>
	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <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+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</li><li>Thomson, J. A. <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+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La méthode présentée dans ce manuscrit offre une alternative rapide et moins coûteux à éliminer les cellules nourricières de fibroblastes humains à partir de cultures de cellules embryonnaires. La suppression réussie de fibroblastes est subordonnée à l&#39;existence d&#39;une monocouche serrée confluente de ces cellules qui se développe en plus des cultures de cellules souches. Après 7-10 jours, les colonies de CSEh croissance va pousser les fibroblastes nourriciers vers l&#39;extérieur, générant une mo...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Marqueur fibroblastes </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> Marqueur noyau cellulaire </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> CSE humaines Marker </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> CSE humaines Marker </td></tr><tr><td> Flk-1 </td><td> BD Pharma </td><td> 555307 </td><td> Marqueur de différenciation précoce </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> CSE humaines Marker </td></tr></tbody></table> Tableau 1. Tableau des raisons spécifiquesgents utilisés pour l&#39;immunomarquage CSEh.

rapid fibroblast removal from high density human embryonic stem cell cultures

Souris fibroblastes embryonnaires (MEF) ont été utilisés pour établir des cellules souches embryonnaires humaines (CSEh) après l&#39;isolement des cultures blastocyste 1. Ce système d&#39;alimentation maintient CSEh à partir de subir une différenciation spontanée lors de l&#39;expansion cellulaire. Cependant, cette méthode de co-culture demande beaucoup de travail, nécessite un personnel hautement qualifié, et la pureté des rendements CSEh faible 4. De nombreux laboratoires ont tenté de minimiser le nombre de cellules nourricières dans les cultures de CSEh (c.-à matrice incorporant boîtes revêtues ou d&#39;autres types de cellules nourricières 5-8). Ces systèmes de culture modifiés ont montré une certaine promesse, mais n&#39;ont pas supplanté la méthode standard pour la culture des CSEh avec la mitomycine C traités par les fibroblastes de souris embyronic afin de retarder indésirables différenciation spontanée des cultures de CSEh. Par conséquent, les cellules nourricières utilisés dans l&#39;expansion de CSEh doivent être retirés au cours d&#39;expériences de différenciation. Bien que plusieurs techniques sont disponibles pour la purification de la coopération CSEhlonies (FACS, MACS, ou l&#39;utilisation de vecteurs résistants aux médicaments) de mangeoires, ces techniques demandent beaucoup de travail, coûteux et / ou destructive à l&#39;CSEh. L&#39;objectif de ce projet était d&#39;inventer une méthode de purification qui permet la récolte d&#39;une population pure de CSEh. Nous avons observé que dans une culture confluente CSEh, la population MEF peut être enlevé en utilisant une aspiration simple et rapide de la feuille MEF. Cette suppression est tributaire de plusieurs facteurs, y compris latéral de cellule à cellule de liaison de MEF qui ont une faible affinité de liaison à la boîte de culture styrène, et la capacité des colonies de cellules souches à pousser les fibroblastes vers l&#39;extérieur lors de la génération de leurs propres &quot; niche ». Le CSEh ont ensuite été examinés pour SSEA-4, Oct3 / 4 et Tra 1-81 expression jusqu&#39;à 10 jours après le retrait du MEF pour assurer le maintien de la pluripotence. En outre, les colonies de CSEh ont été en mesure de poursuivre sa croissance dans de grandes formations après le retrait du MEF, offrant un niveau supplémentaire de l&#39;expansion sur les CSEh.

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Malgré les efforts en cours pour les cultures de transition vers d&#39;alimentation sans conditions, la dérivation et la culture de cellules souches embryonnaires humaines (CSEh) restent largement tributaires des co-cultures avec mangeoires embryonnaires de souris (MEF). Ici, nous montrons une nouvelle méthodologie pour éliminer rapidement départs à partir de cultures de CSEh avant l&#39;expérimentation.

Suppression des fibroblastes rapide de haute densité embryonnaires humaines cultures de cellules souches

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

rapid-fibroblast-removal-from-high-density-human-embryonic-stem-cell

Research

JoVE Journal

Biology

11.7K vues.  University of California, Merced. Malgré les efforts en cours pour les cultures de transition vers d'alimentation sans conditions, la dérivation et la culture de cellules souches embryonnaires humaines (CSEh) restent largement tributaires des co-cultures avec mangeoires embryonnaires de souris (MEF). Ici, nous montrons une nouvelle méthodologie pour éliminer rapidement départs à partir de cultures de CSEh avant l'expérimentation.

Vidéo: Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES repiquage souches embryonnaires humaines lignées cellulaires à la trypsine

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Recherche

Biologie

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Souris épidermique Neural Stem Cell Crest (EPI-NCSC) cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Préparation du Pooled lysat plaquettaire humain (pHPL) comme un supplément efficace pour animaux sans sérum humain cultures de cellules souches

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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.

La différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

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

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.