Le protocole pour la génération des cellules B CD40 humains activés à partir des PBMC est divisé en deux parties: la partie A montre la préparation du ligand CD40 exprimant cellules NIH/3T3, qui sera utilisée comme plaque de cellules nourricières lié. Partie B décrit les réelles CD40-B de la culture. A. Préparation de cellules nourricières (tCD40L NIH/3T3) Le NIH/3T3 tCD40L est une adhérente lignée murine de cellules fibroblastes, qui ne devrait jamais devenir complètement confluentes. Les cellules sont donc découpé deux fois par semaine. La culture sur plus de six semaines n&#39;est pas recommandée. <ol><li> Retirer ancien milieu de la culture primaire avec une pipette stérile et laver les cellules avec 10 ml de PBS 1x. Aspirer le PBS après le lavage. </li><li> Ajouter 4 ml de trypsine / EDTA dans un flacon de 75 cm2 pendant 5-10 minutes à 37 ° C. Utilisez tapotant doucement pour détacher les cellules. </li><li> Ajouter 10 ml de milieu de type sauvage et pivotant doucement. </li><li> Transférer la suspension cellulaire dans un tube de 50 ml avec une pipette stérile et le spin des cellules vers le bas à 225 xg pendant 5 min. </li><li> Retirer le surnageant et remettre le culot dans 10 mL de milieu de type sauvage. Comptez le nombre de cellules d&#39;une aliquote de la suspension cellulaire et préparer trois tubes de 50 ml avec le nombre approprié de cellules: <ol><li> 1,5 x 10 6 cellules pour repiquage </li><li> 0,2 x 10 6 cellules / puits pour l&#39;irradiation utilisée pour la culture de cellules CD40-B </li><li> restant à geler (si nécessaire). </li></ol></li><li> Faites tourner les cellules vers le bas à 225 xg pendant 5 min. </li><li> Enlever le surnageant. <ol><li> Pour repiquage: resuspendre 1,5 x 10 6 cellules dans 10 ml de type moyenne sauvages dans un flacon de 75 cm2 de culture de cellules (1,5 densité cellulaire x 10 5 cellules / mL), ajouter G-418 [0,7 mg / ml] et incuber les cellules à 37 ° C avec 5% de CO 2. Fractionner les cellules deux fois par semaine. </li><li> Pour la culture de cellules CD40-B: Vous avez besoin de 1,2 x 10 6 cellules pour une plaque de 6 puits. Resuspendre les cellules dans un milieu de type sauvage à une densité de 0,1 x 10 6 cellules / ml et les irradier à 78 Gy. Planche 2 mL de la suspension cellulaire dans chaque puits et les incuber à 37 ° C avec 5% de CO 2. Utilisez cette assiettes préparées pour la stimulation des lymphocytes B lors tCD40L cellules NIH/3T3 sont adhérentes (au moins 4 heures: vérifier l&#39;adhérence avec le microscope, ne pas attendre plus de 24 h pour commencer la stimulation des lymphocytes B). (Continuer avec B.) </li></ol></li></ol> B. CD 40-B de culture cellulaire I. Préparation des PBMC pour CD40-stimulation (jour 0): S&#39;il vous plaît noter: avant de continuer de s&#39;assurer que les cellules nourricières sont adhérentes. Toujours ajouter de nouvelles solutions de l&#39;interleukine-4 et la cyclosporine A au milieu de croissance immédiatement avant utilisation. <ol><li> Prenez PBMC, frais ou décongelés de façon appropriée. Resuspendre PBMC deux fois dans 50 ml de PBS 1x pour les laver et de spin-down première fois à 265 xg pendant 7 min et une seconde fois à 190 xg pendant 7 min pour éliminer d&#39;autres cellules. Rejeter le surnageant et remettre en suspension les cellules dans 20 ml de PBS. Déterminer le nombre de cellules dans une aliquote de la suspension cellulaire. </li><li> Isoler quantité requise de cellules à 225 xg pendant 5 min. Pour une plaque à 6 puits 4 x 10 6 cellules / puits sont nécessaires, donc 24 x 10 6 cellules par plaque. </li><li> Enlever le surnageant et remettre en suspension les PBMC à 1 x 10 6 cellules / ml dans le CD40-B milieu de culture fraîchement supplémenté avec 50 U / ml d&#39;interleukine-4 comme un facteur de croissance et de 0,63 g / mL cyclosporine A pour prévenir la excroissance de cellules T (Compte tenu de la concentration se référer à un milieu de culture ml!). </li><li> Retirer le surnageant de 6 puits plaque pré-incubées avec des cellules NIH/3T3 tCD40L. </li><li> Laver les adhérents tCD40L cellules NIH/3T3 avec 2 ml de PBS par puits d&#39;abord et dans une seconde étape avec 2 ml de milieu B CD40-linge. </li><li> Ajouter délicatement 4 ml de suspension PBMC (1 x 10 6 cellules / ml) dans chaque puits de la plaque de 6 puits. </li><li> Incuber les cellules à 37 ° C avec 5% de CO 2. </li><li> Au jour 7, les cellules reculture (Continuez à 3.2.). </li></ol> II. Remise en culture des cellules B CD40 (7 jours, puis tous les 3-4 jours): <ol><li> La récolte des grappes CD40 des cellules B à partir de 6 puits plaque par remise en suspension avec une pipette de 10 ml et de les mettre dans un tube de 50 ml. </li><li> Isoler à 225 xg pendant 7 minutes et remplacer complètement le surnageant avec CD40-B milieu de lavage. Alors que le comptage des cellules du montant d&#39;une aliquote, tourner les cellules vers le bas à 225 xg pendant 5 minutes. Resuspendre les cellules CD40-CD40-B en B milieu de culture à une concentration de 1 x 10 6 cellules / ml. </li><li> Ajouter de nouvelles solutions de l&#39;interleukine-4 à une concentration de 50 U / ml et 0,63 mg / ml de cyclosporine A dans le milieu. </li><li> Retirer le surnageant dans une plaque de 6 puits de pré-incubées avec des cellules NIH/3T3 tCD40L. </li><li> Laver les cellules adhérentes avec 2 ml de PBS par puits d&#39;abord et dans une seconde étape avec 2 ml de milieu B CD40-linge. </li><li&gt; Ajouter délicatement 4 ml (1 x 10 6 cellules / ml) de suspension CD40-B pour chaque puits de la plaque à 6 puits. </li><li> Incuber les plaques à 37 ° C avec 5% de CO 2. </li><li> Repiquer les cellules à nouveau tous les 3-4 jours pour finir avec très pure des cellules B CD40-activé après un total de 14 jours. </li></ol> C. Dépannage - Que faire si les cellules CD40-B ne poussent pas? <ol><li> Avez-vous vérifié l&#39;expression du ligand CD40 des cellules nourricières? </li><li> La plaque lié cellules nourricières utilisé pour la stimulation ne doit pas être âgés de plus de 24h? </li><li> Est-ce une contamination par des mycoplasmes possibles? </li><li> Était la solution interleukine-4 utilisé pour la supplémentation fraîchement dégelé et avait l&#39;activité biologique approprié? </li><li> Cyclosporine A a été ajoutée dans la concentration correcte? </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Figure 1" /> Figure 1. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">cliquez ici</a> pour une version agrandie de la figure 1. <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Figure 2" /> Figure 2. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">cliquez ici</a> pour une version agrandie de la figure 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

A. Préparation des médias: <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> Cellules nourricières type de support sauvages </td><td colspan="2"> Milieu de sélection de cellules Feeder </td></tr><tr><td> Réactifs </td><td> Concentration </td><td> Réactifs </td><td> Concentration </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutamine </td><td> 365 pg / ml </td><td> L-Glutamine </td><td> 365 pg / ml </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> HEPES </td><td> 10 mM </td></tr><tr><td> Gentamycine </td><td> 15 ug / ml </td><td> Gentamycine </td><td> 15 ug / ml </td></tr><tr><td></td><td></td><td> G-418 </td><td> 0,7 mg / ml </td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> CD40-B milieu de lavage </td><td colspan="2"> CD40-B milieu de culture </td></tr><tr><td> Réactifs </td><td> Concentration </td><td> Réactifs </td><td> Concentration </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L-Glutamine </td><td> 584 pg / ml </td><td> L-Glutamine </td><td> 584 pg / ml </td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 mM </td><td> HEPES </td><td> 25 mM </td></tr></tbody><tbody><tr><td> Gentamycine </td><td> 15 ug / ml </td><td> Gentamycine </td><td> 15 ug / ml </td></tr></tbody><tbody><tr><td> </td><td> </td><td> rh transferrine </td><td> 50 ug / ml </td></tr></tbody><tbody><tr><td> </td><td> </td><td> L&#39;insuline rh </td><td> 5 ug / ml </td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB-Humanserum </td><td> 10% </td></tr></tbody></table> B. Réactifs Divers: <table cellspacing="0" border="1"><tbody><tr><td> Réactifs </td><td> Société </td><td> Ordonnance n </td></tr><tr></tr><tr><td> DMEM / Ham F12 </td><td> AAP </td><td> Pas de chat: E15-813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> REF 21980-032 </td></tr><tr><td> Dulbecco PBS (10x) </td><td> AAP </td><td> Cat No H15-011 </td></tr><tr><td> AB-sérum humain </td><td> Invitrogen </td><td> Cat No 34005100 </td></tr><tr><td> holo-transferrine humaine </td><td> Sigma </td><td> Aucun chat T0665 </td></tr><tr><td> Insuline humaine </td><td> Sigma </td><td> Aucun chat I2643 </td></tr><tr><td> Gencin ® </td><td> Sélectionnez Delta </td><td> Art Pas 7395800 </td></tr><tr><td> Interleukine-4 de l&#39;homme </td><td> Immunotools </td><td> Cat No 11130045 </td></tr><tr><td> Cyclosporine A </td><td> Novartis </td><td> Cat No 0078-0109-01 NDC </td></tr><tr><td> FBS </td><td> LONZA </td><td> Aucun chat DE14-802C </td></tr><tr><td> Tampon HEPES </td><td> AAP </td><td> Cat No S11-001 </td></tr><tr><td> G-418 Sulfate </td><td> AAP </td><td> Aucun chat P02-012 </td></tr><tr><td> Trypsine / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table> C. Fournitures diverses: <table cellspacing="0" border="1"><tbody><tr><td> Réactifs </td><td> Société </td><td> Ordonnance n </td></tr><tr></tr><tr><td> Stérile pipette </td><td> Sarstedt </td><td></td></tr><tr><td> 6-même la plaque </td><td> NUNC </td><td> Cat No 140675 </td></tr><tr><td> 50mL tube conique </td><td> BD Falcon ™ </td><td> Cat No 352070 </td></tr><tr><td> Flacon de culture tissulaire </td><td> SARSTEDT </td><td> Aucun chat 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

CD40-activé des cellules B (CD40-cellules B) ont été identifiés comme une source alternative de l&#39;immuno-stimulation des cellules présentatrices d&#39;antigènes (APC) pour l&#39;immunothérapie du cancer 1-3. Comparé aux cellules dendritiques (CD), la meilleure APC caractérisée, les cellules CD40-B ont plusieurs propriétés biologiques distinctes et techniques. Similaires aux PED, les cellules B montrent une augmentation de l&#39;expression du CMH et des molécules de co-stimulation (Fig. 1b), présentent une forte capacité migratoire et la présentation des antigènes présents efficacement aux cellules T, après stimulation par un ligand interleukine-4 et CD40 (CD40L). Cependant, contrairement aux CD immatures ou matures, les cellules CD40-B expriment la triade lymphatiques pleine noeud homing composé de CD62L, CCR7/CXCR4, et la fonction des leucocytes antigènes-1 (LFA1, CD11a/CD18), nécessaires à tête chercheuse d&#39;organes lymphoïdes secondaires (Fig.1a) 3. Cellules CD40-B peuvent être générés sans difficultés à partir de très petites quantités de sang périphérique qui peut être élargi in vitro à de très grandes quantités de très-pur-CD40 des cellules B (&gt; 10 9 cellules par patient) provenant de donneurs sains ainsi que le cancer patients (Fig.1c, d) 1,4. Dans ce protocole, nous montrons comment obtenir pleinement activé CD40-B à partir des cellules PBMC humaines. Molécules clés pour la culture cellulaire sont CD40-ligand, l&#39;interleukine-4 (IL-4) et la cyclosporine A. (CSA), qui sont reconstituées dans un cycle de culture de 3-4 jours Aux fins de laboratoire CD40-stimulation est fournie par des cellules exprimant NIH/3T3 humaine recombinante ligand CD40 (tCD40L NIH/3T3) 5. Pour éviter la contamination par des cellules non transfectées, l&#39;expression du ligand CD40 humaine sur les transfectants doit être vérifié régulièrement (Fig.2). Après 14 jours de CD40-B cultures cellulaires consistent de plus de 95% de cellules B pur et une expansion des cellules CD40-B de plus de 65 jours est souvent possible sans aucune perte de fonction 1, 4. Cellules CD40-B efficacement relever, traiter et présenter les antigènes aux cellules T 6. Ils ne sont pas seulement naϊve écoute, mais également d&#39;élargir les cellules T mémoire 7,8. CD40-activé des cellules B peut être utilisé pour étudier activation des cellules B, la différenciation et la fonction. Par ailleurs, ils représentent un outil prometteur pour la vaccination thérapeutique ou préventive contre les tumeurs 9.

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

Dans cette vidéo, nous présentons les<em&gt; Ex vivo</emGénération&gt; et l&#39;expansion des cellules B CD40-activées (CD40-B) à partir de cellules mononucléées du sang périphérique (PBMC) par stimulation par CD40 ligand et l&#39;interleukine-4.

Génération de l&#39;homme CD40-B activés cellules

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

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Biology

15.1K vues.  University Hospital of Cologne, Department I of Internal Medicine. Dans cette vidéo, nous présentons les Ex vivo et l'expansion des cellules B CD40-activées (CD40-B) à partir de cellules mononucléées du sang périphérique (PBMC) par stimulation par CD40 ligand et l'interleukine-4.

Vidéo: Generation of Human CD40-activated B cells

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Préparation Étaler chromosomique des cellules souches embryonnaires humaines pour le caryotype

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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Biologie

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes humains avec les lentivirus TF Stemgent Homme Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Génération robuste des hépatocytes comme des cellules embryonnaires humaines à partir populations de cellules souches

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caractérisation phénotypique et fonctionnelle des cellules endothéliales formant colonie dérivés du sang de cordon ombilical humain

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Génération de l&#39;homme cellules souches pluripotentes induites à partir de sang périphérique en utilisant le vecteur lentiviral STEMCCA

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Génération d&#39;dispersée présomitique mésoderme cultures cellulaires pour l&#39;imagerie de la segmentation Horloge poisson zèbre dans des cellules individuelles

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Génération de cardiomyocytes humains: Un protocole de différenciation des induits des cellules souches pluripotentes humaines chargeur sans

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Génération et de la Culture du sang excroissance des cellules endothéliales de sang périphérique humain

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Haut-débit CRISPR Vector Construction et caractérisation de l&#39;ADN Modifications par génération de tomate Poilu Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...