Das Protokoll für die Erzeugung von menschlichen CD40-aktivierte B-Zellen aus PBMC ist in zwei Teile gegliedert: Teil A zeigt die Herstellung von CD40-Liganden exprimieren NIH/3T3-Zellen, die als Platten-gebundenen Feeder-Zellen verwendet werden. Teil B beschreibt die eigentliche CD40-B Kultur. A. Herstellung von Feeder-Zellen (tCD40L NIH/3T3) Die tCD40L NIH/3T3 ist ein Anhänger murine Fibroblasten-Zelllinie, die nie sollte vollständig konfluent. Die Zellen sind daher zweimal pro Woche aufgeteilt. Die Kultivierung von mehr als 6 Wochen wird nicht empfohlen. <ol><li> Entfernen Sie alte Medium aus dem Primär-Kultur mit einer sterilen Pipette und waschen Sie die Zellen mit 10 ml 1x PBS. Saugen Sie das PBS nach dem Waschen. </li><li> 4 mL Trypsin / EDTA in einer 75 cm 2-Kolben für 5-10 Minuten bei 37 ° C. Verwenden leichtes Klopfen auf die Zellen zu lösen. </li><li> Fügen Sie 10 ml Wildtyp-mittel-und Schwenk-sanft. </li><li> Übertragen Sie die Zellsuspension in ein 50 ml-Tube mit einer sterilen Pipette und drehen Sie die Zellen nach unten bei 225 xg für 5 min. </li><li> Entfernen Sie den Überstand und resuspendieren Sie das Pellet in 10 ml Wildtyp-Medium. Zählen Sie die Zellzahl eines Aliquots der Zellsuspension und bereiten drei 50 ml-Röhrchen mit der entsprechenden Anzahl von Zellen: <ol><li> 1,5 x 10 6 Zellen zur Subkultivierung </li><li> 0,2 x 10 6 Zellen / well für die Bestrahlung von CD40-B-Zell-Kultur verwendet </li><li> Rest einfrieren (falls erforderlich). </li></ol></li><li> Drehen Sie die Zellen nach unten bei 225 xg für 5 min. </li><li> Entfernen Sie den Überstand. <ol><li> Für Subkultivierung: resuspendieren 1,5 x 10 6 Zellen in 10 mL Wildtyp-Medium in einem 75 cm 2 Zellkulturflasche (Zelldichte 1,5 x 10 5 Zellen / ml), fügen G-418 [0,7 mg / mL] und inkubieren Sie die Zellen bei 37 ° C mit 5% CO 2. Split die Zellen zweimal pro Woche. </li><li> Für die CD40-B-Zell-Kultur: Sie müssen 1,2 x 10 6 Zellen für eine 6-Well-Platte. Resuspendieren der Zellen in Wildtyp-Medium mit einer Dichte von 0,1 x 10 6 Zellen / ml und bestrahlen sie bei 78 Gy. Plate 2 mL der Zellsuspension in jede Vertiefung und inkubieren sie bei 37 ° C mit 5% CO 2. Mit diesem vorbereiteten Platten für B-Zell-Stimulation, wenn tCD40L NIH/3T3-Zellen Anhänger sind (mindestens 4 Stunden: check Einhaltung mit dem Mikroskop; warten Sie nicht länger als 24 h auf B-Zell-Stimulation beginnen). (Fahren Sie mit B.) </li></ol></li></ol> B. CD 40-B-Zell-Kultur I. Vorbereitung der PBMCs für CD40-Stimulation (Tag 0): Bitte beachten Sie: Bevor Sie fortfahren vergewissern, dass Feeder-Zellen adhärent sind. Fügen Sie immer frische Lösungen von Interleukin-4 und Cyclosporin A, um das Wachstum mittelfristig unmittelbar vor dem Gebrauch. <ol><li> Nehmen PBMCs, entweder frisch oder in geeigneter Weise aufgetaut. Resuspendieren PBMCs zweimal in 50 ml 1x PBS zu waschen und Spin-down ersten Mal bei 265 xg für 7 min und ein zweites Mal bei 190 xg für 7 min zu anderen Zellen zu entfernen. Überstand verwerfen und die Zellen in 20 ml PBS. Bestimmen Sie die Anzahl der Zellen in einem Aliquot der Zellsuspension. </li><li> Spin down benötigte Menge an Zellen bei 225 xg für 5 min. Für eine 6-Well-Platte 4 x 10 6 Zellen / well benötigt werden, also 24 x 10 6 Zellen pro Platte. </li><li> Entfernen Sie den Überstand und resuspendieren Sie die PBMC mit 1 x 10 6 Zellen / ml in CD40-B Kulturmedium frisch mit 50 U / ml Interleukin-4 ergänzt als Wachstumsfaktor und 0,63 ug / ml Cyclosporin A Auswuchs der T-Zellen zu verhindern (Angesichts Konzentrationen beziehen sich auf ein ml Kulturmedium!). </li><li> Entfernen Sie den Überstand aus 6-Well-Platte vorinkubiert mit tCD40L NIH/3T3-Zellen. </li><li> Waschen Sie die Anhänger tCD40L NIH/3T3-Zellen mit 2 mL PBS pro Vertiefung ersten und in einem zweiten Schritt mit 2 ml CD40-B Waschmedium. </li><li> Vorsichtig 4 ml PBMC Suspension (1 x 10 6 Zellen / ml) in jede Vertiefung der 6-Well-Platte. </li><li> Inkubieren Sie die Zellen bei 37 ° C mit 5% CO 2. </li><li> Am Tag 7 reculture der Zellen (Fahren Sie mit 3.2.). </li></ol> II. Rekultivierung von CD40-B-Zellen (Tag 7 und dann alle 3-4 Tage): <ol><li> Ernte-Clustern von CD40-B-Zellen aus 6-Well-Platte durch Resuspendieren mit einer 10 mL Pipette und sie gemeinsam in eine 50 ml Tube. </li><li> Spin down bei 225 xg für 7 Minuten und vollständig ersetzen den Überstand mit CD40-B Waschmedium. Während des Zählens der Zelle Betrag eines aliquoten, drehen Sie das Zellen nach unten bei 225 xg für 5 Minuten. Resuspendieren CD40-B-Zellen in CD40-B Kulturmedium in einer Konzentration von 1 x 10 6 Zellen / ml. </li><li> Fügen Sie frische Lösungen von Interleukin-4 in einer Konzentration von 50 U / ml und 0,63 pg / mL Cyclosporin A auf das Medium. </li><li> Entfernen Sie den Überstand aus einer 6-well-Platte mit tCD40L NIH/3T3-Zellen vorinkubiert. </li><li> Waschen Sie die adhärenten Zellen mit 2 ml PBS pro Vertiefung ersten und in einem zweiten Schritt mit 2 ml CD40-B Waschmedium. </li><li&gt; vorsichtig 4 ml (1 x 10 6 Zellen / ml) von CD40-B Suspension in jede Vertiefung der 6-Well-Platte. </li><li> Die Platten bei 37 ° C mit 5% CO 2. </li><li> Subculture Zellen wieder alle 3-4 Tage bis zum Ende mit hochreinem humanen CD40-aktivierte B-Zellen nach insgesamt 14 Tagen. </li></ol> C. Trouble-Shooting - Was passiert, wenn CD40-B-Zellen nicht wachsen? <ol><li> Haben Sie überprüft, für die CD40-Ligand Expression von Feeder-Zellen? </li><li> Die Platte gebundene Nährzellen der Stimulation dürfen nicht älter 24 Stunden? </li><li> Ist eine Kontamination mit Mykoplasmen möglich? </li><li> War der Interleukin-4-Lösung zur Ergänzung eingesetzt frisch aufgetaut und hatte die entsprechende biologische Aktivität? </li><li> War Cyclosporin A aufgenommen in der richtigen Konzentration? </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Abbildung 1" /> Abbildung 1. Bitte <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">klicken Sie hier</a> für eine größere Version der Abbildung 1. <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Abbildung 2" /> Abbildung 2. Bitte <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">klicken Sie hier</a> für eine größere Version der Abbildung 2 dargestellt.

<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. Herstellung der Medien: <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> Feeder Zelle Wildtyp-Medium </td><td colspan="2"> Feeder Zelle Selektionsmedium </td></tr><tr><td> Reagens </td><td> Konzentration </td><td> Reagens </td><td> Konzentration </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutamin </td><td> 365 pg / mL </td><td> L-Glutamin </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> Gentamycin </td><td> 15 ug / mL </td><td> Gentamycin </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 Waschmedium </td><td colspan="2"> CD40-B Kulturmedium </td></tr><tr><td> Reagens </td><td> Konzentration </td><td> Reagens </td><td> Konzentration </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L-Glutamin </td><td> 584 pg / mL </td><td> L-Glutamin </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> Gentamycin </td><td> 15 ug / mL </td><td> Gentamycin </td><td> 15 ug / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> rh Transferrin </td><td> 50 ug / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> rh Insulin </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. Sonstige Reagenzien: <table cellspacing="0" border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Bestell-Nr </td></tr><tr></tr><tr><td> DMEM / Ham &#39;s F12 </td><td> PAA </td><td> Cat No: 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> PAA </td><td> Cat No H15-011 </td></tr><tr><td> AB-Humanserum </td><td> Invitrogen </td><td> Cat No 34005100 </td></tr><tr><td> Holo-Transferrin menschlichen </td><td> Sigma </td><td> Cat No T0665 </td></tr><tr><td> Insulin human </td><td> Sigma </td><td> Cat No I2643 </td></tr><tr><td> Gencin ® </td><td> Delta Select </td><td> Art.-Nr. 7395800 </td></tr><tr><td> Rekombinanten humanen Interleukin-4 </td><td> Immunotools </td><td> Cat No 11130045 </td></tr><tr><td> Cyclosporin A </td><td> Novartis </td><td> Cat No NDC 0078-0109-01 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Cat No DE14-802C </td></tr><tr><td> HEPES-Puffer </td><td> PAA </td><td> Cat No S11-001 </td></tr><tr><td> G-418 Sulfat </td><td> PAA </td><td> Cat No P02-012 </td></tr><tr><td> Trypsin / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table> C. Sonstiges Zubehör: <table cellspacing="0" border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Bestell-Nr </td></tr><tr></tr><tr><td> Sterile Pipettenspitzen </td><td> Sarstedt </td><td></td></tr><tr><td> 6-Well-Platte </td><td> NUNC </td><td> Cat No 140675 </td></tr><tr><td> 50 ml konischen Rohr </td><td> BD Falcon ™ </td><td> Cat No 352070 </td></tr><tr><td> Zellkulturflasche </td><td> SARSTEDT </td><td> Cat No 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

CD40-aktivierte B-Zellen (CD40-B-Zellen) wurden als eine alternative Quelle für immunstimulierende Antigen-präsentierende Zellen (APC) für die Immuntherapie von Krebs 3.1 identifiziert worden. Im Vergleich zu Dendritische Zellen (DCs), den am besten charakterisierten APC, CD40-B-Zellen haben mehrere verschiedene biologische und technische Eigenschaften. Ähnlich wie DCs, B-Zellen eine erhöhte Expression von MHC-und kostimulatorischen Molekülen (Abb. 1b) zeigen, weisen eine starke Wanderungsbewegungen Kapazität und präsentieren Antigen-Präsentation effizient auf T-Zellen nach Stimulation mit Interleukin-4 und CD40-Ligand (CD40L). Doch im Gegensatz zu unreifen oder reifen DCs, Express-CD40-B-Zellen die volle Lymphknoten Homing Triade bestehend aus CD62L, CCR7/CXCR4 und Leukozyten-Antigen-Funktion-1 (LFA1, CD11a/CD18), die für Homing zu sekundären lymphatischen Organen (Abb. 1a) 3. CD40-B-Zellen können ohne Schwierigkeiten aus sehr geringen Mengen im peripheren Blut, die weiter in vitro werden, um sehr große Mengen an hochreinen CD40-B-Zellen (&gt; 10 9 Zellen pro Patient) von gesunden Spendern sowie Krebs kann erweitert erzeugt werden Patienten (Fig.1c, d) 1,4. In diesem Protokoll haben wir demonstrieren, wie voll aktiviert CD40-B-Zellen aus humanen PBMC erhalten. Key-Moleküle für die Zellkultur sind CD40-Ligand, Interleukin-4 (IL-4) und Cyclosporin A (CsA), die in einer 3-4 Tage alten Kultur-Zyklus wieder aufgefüllt werden. Für Laborzwecke CD40-Stimulation wird durch NIH/3T3-Zellen exprimierenden rekombinanten humanen CD40-Liganden (tCD40L NIH/3T3) 5 zur Verfügung gestellt. Zur Vermeidung von Kontaminationen mit nicht-transfizierten Zellen ist die Expression des humanen CD40-Liganden auf die Transfektanten regelmäßig überprüft werden (Abb.2). Nach 14 Tagen CD40-B-Zell-Kulturen bestehen aus mehr als 95% reinen B-Zellen sowie eine Ausweitung des CD40-B-Zellen über 65 Tage ist oft möglich, ohne Verlust der Funktion 1, 4. CD40-B-Zellen effizient aufnehmen, verarbeiten und präsentieren Antigene an T Zellen 6. Sie haben nicht nur prime naϊve, sondern auch zu erweitern Memory T-Zellen 7,8. CD40-aktivierte B-Zellen können verwendet werden, um B-Zell-Aktivierung, Differenzierung und Funktion zu untersuchen. Darüber hinaus stellen sie ein vielversprechendes Werkzeug für therapeutische oder präventive Impfung gegen Tumoren 9.

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

Generation of Human CD40-activated B cells

In diesem Video stellen wir Ihnen die<em&gt; Ex vivo</em&gt; Erzeugung und Erweiterung des menschlichen CD40-aktivierten B-Zellen (CD40-B) aus peripheren mononukleären Blutzellen (PBMC) durch Stimulation mit CD40-Ligand und Interleukin-4.

Generation of Human CD40-aktivierten B-Zellen

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

Research

JoVE Journal

Biology

15.1K Aufrufe.  University Hospital of Cologne, Department I of Internal Medicine. In diesem Video stellen wir Ihnen die Ex vivo Erzeugung und Erweiterung des menschlichen CD40-aktivierten B-Zellen (CD40-B) aus peripheren mononukleären Blutzellen (PBMC) durch Stimulation mit CD40-Ligand und Interleukin-4.

Serie: 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.

Chromosomale Spread-Vorbereitung von humanen embryonalen Stammzellen für die Karyotypisierung

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

Forschung

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.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung menschlichen Fibroblasten mit dem Stemgent Menschliche 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 ...

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.

Robuste Generierung von Leber-ähnlichen Zellen aus humanen embryonalen Stammzell-Populationen

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.

Phänotypische und funktionelle Charakterisierung von Endothelial Kolonie-bildenden Zellen aus humanem Nabelschnurblut

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.

Generation of Human induzierten pluripotenten Stammzellen aus dem peripheren Blut mit dem STEMCCA Lentivirusvektor

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.

Generierung von Dispersed präsomitischen Mesoderm Zellkulturen für Imaging der Zebrafisch-Segmentierung Uhr in Einzelzellen

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.

Generation of Human Cardiomyocytes: Eine Differenzierung Protokoll von Feeder-freien menschlichen induzierten pluripotenten Stammzellen

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.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit 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.

Generation und Kultur der Blut Outgrowth Endothelzellen aus menschlichen peripheren Blut-

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.

High-throughput CRISPR Vector Konstruktion und Charakterisierung von DNA-Modifikationen durch Erzeugung von Tomaten Hairy Roots

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