Il protocollo per la generazione di umani CD40 delle cellule B attivate da PBMC è diviso in due parti: la parte A dimostra la preparazione di ligando CD40 esprimere NIH/3T3 celle, che sarà usato come piatto legato cellule nutrici. Parte B descrive l&#39;attuale B CD40-cultura. A. Preparazione di cellule di alimentazione (tCD40L NIH/3T3) Il NIH/3T3 tCD40L è un aderente murino linea cellulare di fibroblasti, che non dovrebbe mai diventare completamente confluenti. Le cellule sono quindi suddivisi due volte alla settimana. Coltura per più di 6 settimane non è raccomandato. <ol><li> Rimuovere medio vecchio dalla coltura primaria con una pipetta sterile e lavare le cellule con 10 ml di PBS 1x. Aspirare il PBS dopo il lavaggio. </li><li> Aggiungere 4 ml di tripsina / EDTA in un centimetro 75 2 fiasco per 5-10 minuti a 37 ° C. Usa picchiettando leggermente per staccare le cellule. </li><li> Aggiungere 10 ml di medium di tipo selvatico e girevole con delicatezza. </li><li> Trasferire la sospensione cellulare in un tubo da 50 ml con una pipetta sterile e girare le celle in basso a 225 xg per 5 min. </li><li> Rimuovere il supernatante e risospendere il pellet in 10 ml di medium di tipo selvatico. Contare il numero di cellulare di una aliquota della sospensione cellulare e preparare tre da 50 mL con il numero appropriato di cellule: <ol><li> 1,5 x 10 6 cellule per subcolture </li><li> 0,2 x 10 6 cellule / pozzetto di irradiazione utilizzate nel CD40-B colture cellulari </li><li> resto di congelare (se necessario). </li></ol></li><li> Spin le cellule verso il basso a 225 xg per 5 min. </li><li> Rimuovere il surnatante. <ol><li> Per subcolture: risospendere 1,5 x 10 6 cellule in 10 ml di tipo medio selvatici in un CM 75 2 fiasco coltura cellulare (cellule densità di 1,5 x 10 5 cellule / ml), aggiungere G-418 [0,7 mg / ml] e incubare le cellule a 37 ° C con 5% di CO 2. Dividere le celle due volte alla settimana. </li><li> Per la B CD40-coltura cellulare: Avete bisogno di 1,2 x 10 6 cellule per un 6-pozzetti. Risospendere le cellule in terreno di tipo selvatico ad una densità di 0,1 x 10 6 cellule / ml e irradiare li a 78 Gy. Tavola 2 ml di sospensione cellulare in ciascun pozzetto e incubare loro a 37 ° C con 5% di CO 2. Utilizzare questa piastre preparate per la stimolazione delle cellule B quando tCD40L NIH/3T3 cellule sono aderenti (almeno 4 ore: controllare l&#39;aderenza con il microscopio, non aspettare più di 24 ore per iniziare la stimolazione delle cellule B). (Continua con B.) </li></ol></li></ol> B. CD 40-B di coltura cellulare I. Preparazione di PBMC per CD40-stimolazione (giorno 0): Attenzione: prima di continuare a constatare che le cellule feeder siano aderenti. Aggiungere sempre soluzioni fresche di interleuchina-4 e ciclosporina A per il terreno di coltura immediatamente prima dell&#39;uso. <ol><li> Prendete PBMC, fresco o opportunamente scongelati. Risospendere PBMC due volte in 50 mL di PBS 1x di lavarli e spin down prima volta a 265 xg per 7 min e una seconda volta a xg 190 per 7 minuti per eliminare altre cellule. Gettare il surnatante e risospendere le cellule in 20 ml di PBS. Determinare il numero di cellule in una parte aliquota della sospensione cellulare. </li><li> Spin down quantità necessaria di cellule a 225 xg per 5 min. Per un 6-pozzetti 4 x 10 6 cellule / pozzetto sono necessari, quindi 24 x 10 6 cellule per piastra. </li><li> Rimuovere il supernatante e risospendere il PBMC con 1 x 10 6 cellule / ml in B CD40-terreno di coltura fresco integrato con 50 U / mL di interleuchina-4 come un fattore di crescita e di 0,63 mg / ml ciclosporina A per prevenire conseguenza di cellule T (concentrazioni indicati si riferiscono a un mezzo ml di cultura!). </li><li> Rimuovere il surnatante da 6 pozzetti di pre-incubazione piatto con tCD40L NIH/3T3 cellule. </li><li> Lavare l&#39;aderente tCD40L NIH/3T3 cellule con 2 ml di PBS per primo pozzo e in una seconda fase con 2 ml di CD40-B medio di lavaggio. </li><li> Delicatamente aggiungere 4 ml di sospensione PBMC (1 x 10 6 cellule / ml) in ciascun pozzetto della piastra ben 6. </li><li> Incubare le cellule a 37 ° C con 5% di CO 2. </li><li> Il giorno 7, reculture le cellule (Continua al punto 3.2.). </li></ol> II. Ricoltivazione di cellule CD40-B (7 ° giorno e poi ogni 3-4 giorni): <ol><li> Cluster raccolta di cellule CD40-B da 6 pozzetti da risospendere con una pipetta da 10 mL e la piscina in un tubo da 50 ml. </li><li> Spin down a 225 xg per 7 minuti e sostituire completamente il sopranatante con CD40-B medio di lavaggio. Mentre conta la quantità delle cellule di una aliquota, girare la celle in basso a 225 xg per 5 minuti. Risospendere le cellule B CD40-CD40-B in terreno di coltura ad una concentrazione di 1 x 10 6 cellule / ml. </li><li> Aggiungi soluzioni fresche di interleuchina-4 in una concentrazione di 50 U / mL e 0,63 mcg / mL ciclosporina A per la media. </li><li> Rimuovere il surnatante da un 6-pozzetti pre-incubate con tCD40L NIH/3T3 cellule. </li><li> Lavare le cellule aderenti con 2 ml di PBS per primo pozzo e in una seconda fase con 2 ml di CD40-B medio di lavaggio. </li><li&gt; delicatamente aggiungere 4 ml (1 x 10 6 cellule / ml) di sospensione CD40-B in ciascun pozzetto della piastra ben 6. </li><li> Incubare le piastre a 37 ° C con 5% di CO 2. </li><li> Cellule sottocultura di nuovo ogni 3-4 giorni per finire con elevata purezza umano CD40 delle cellule B attivate dopo un totale di 14 giorni. </li></ol> C. Soluzione dei problemi - Che cosa succede se le cellule CD40-B non crescono? <ol><li> Hai controllato di espressione ligando CD40 delle cellule alimentatore? </li><li> La piastra-bound cellule alimentatore utilizzato per la stimolazione non deve essere più di 24 ore? </li><li> È una possibile contaminazione con micoplasma? </li><li> È stata la interleuchina-4 soluzione utilizzata per la supplementazione appena scongelato e aveva l&#39;attività biologica appropriata? </li><li> È stato aggiunto ciclosporina A nella concentrazione giusta? </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. Si prega di <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">cliccare qui</a> per una versione più grande della figura 1. <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Figura 2" /> Figura 2. Si prega di <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">cliccare qui</a> per una versione più grande della figura 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. Preparazione dei supporti: <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> Alimentatore cellule wild type media </td><td colspan="2"> Alimentatore cellulare medio selezione </td></tr><tr><td> Reagente </td><td> Concentrazione </td><td> Reagente </td><td> Concentrazione </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutammina </td><td> 365 mg / ml </td><td> L-Glutammina </td><td> 365 mg / 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> Gentamicina </td><td> 15 mg / ml </td><td> Gentamicina </td><td> 15 mg / 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 lavaggio medio </td><td colspan="2"> CD40-B terreno di coltura </td></tr><tr><td> Reagente </td><td> Concentrazione </td><td> Reagente </td><td> Concentrazione </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L-Glutammina </td><td> 584 mg / ml </td><td> L-Glutammina </td><td> 584 mg / 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> Gentamicina </td><td> 15 mg / ml </td><td> Gentamicina </td><td> 15 mg / ml </td></tr></tbody><tbody><tr><td> </td><td> </td><td> rh transferrina </td><td> 50 mg / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> L&#39;insulina rh </td><td> 5 mcg / ml </td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB-Humanserum </td><td> 10% </td></tr></tbody></table> B. Varie Reagenti: <table cellspacing="0" border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Il decreto n. </td></tr><tr></tr><tr><td> DMEM / Ham 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> Nessun gatto H15-011 </td></tr><tr><td> AB-siero umano </td><td> Invitrogen </td><td> Nessun gatto 34005100 </td></tr><tr><td> olo-transferrina umana </td><td> Sigma </td><td> Nessun gatto T0665 </td></tr><tr><td> Insulina umana </td><td> Sigma </td><td> Nessun gatto I2643 </td></tr><tr><td> Gencin ® </td><td> Delta Select </td><td> Art. n. 7395800 </td></tr><tr><td> Ricombinante umano interleuchina-4 </td><td> Immunotools </td><td> Nessun gatto 11130045 </td></tr><tr><td> Ciclosporina A </td><td> Novartis </td><td> Nessun gatto NDC 0078-0109-01 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Nessun gatto DE14-802C </td></tr><tr><td> HEPES Buffer </td><td> PAA </td><td> Nessun gatto S11-001 </td></tr><tr><td> G-418 Solfato </td><td> PAA </td><td> Nessun gatto P02-012 </td></tr><tr><td> Tripsina / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table> C. Varie Forniture: <table cellspacing="0" border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Il decreto n. </td></tr><tr></tr><tr><td> Puntali sterili </td><td> Sarstedt </td><td></td></tr><tr><td> 6 pozzetti </td><td> NUNC </td><td> Nessun gatto 140675 </td></tr><tr><td> 50mL tubo conico </td><td> BD Falcon ™ </td><td> Nessun gatto 352070 </td></tr><tr><td> Tessuto cultura fiasco </td><td> Sarstedt </td><td> Nessun gatto 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

CD40-attivate le cellule B (linfociti B CD40-) sono stati identificati come fonte alternativa di immuno-stimolatorie cellule presentanti l&#39;antigene (APC) per l&#39;immunoterapia del cancro 1-3. Rispetto alle cellule dendritiche (DC), il miglior APC caratterizzato, le cellule CD40-B hanno diverse proprietà distinte biologici e tecnici. Simile alla DC, le cellule B mostrano un aumento dell&#39;espressione di MHC e molecole co-stimolatorie (Fig.1b), mostrano una forte capacità migratoria e presentazione dell&#39;antigene presente in modo efficiente alle cellule T, dopo stimolazione con il ligando dell&#39;interleuchina-4 e CD40 (CD40L). Tuttavia, a differenza di DC immature o mature, le cellule B CD40-esprimono la piena triade nodo homing linfa composto da CD62L, CCR7/CXCR4, e la funzione dei leucociti antigene-1 (LFA1, CD11a/CD18), necessaria per homing di organi linfatici secondari (Fig.1a) 3. Cellule CD40-B può essere generato senza difficoltà da piccolissime quantità di sangue periferico che può essere ulteriormente espanse in vitro di grandi quantità di altamente puri-CD40 delle cellule B (&gt; 10 9 cellule per paziente) da donatori sani e come il cancro pazienti (Fig.1c, d) 1,4. In questo protocollo dimostriamo come ottenere pienamente attivato le cellule B CD40-da PBMC umane. Molecole chiave per la coltura cellulare sono ligando CD40, l&#39;interleuchina-4 (IL-4) e ciclosporina A (CsA), che viene rifornito in un ciclo di 3-4 cultura giorno. Per scopi di laboratorio CD40-stimolazione è fornito da NIH/3T3 cellule che esprimono CD40 ligando ricombinante umano (tCD40L NIH/3T3) 5. Per evitare la contaminazione con le cellule non transfettate, espressione del ligando CD40 umano sulla trasfettate deve essere controllata regolarmente (Fig.2). Dopo 14 giorni CD40-B colture cellulari costituiti da oltre il 95% delle cellule B puro e una espansione del CD40 delle cellule B con più di 65 giorni è spesso possibile senza alcuna perdita di funzione 1, 4. Le cellule B CD40-in modo efficiente raccogliere, trasformare e antigeni presenti alle cellule T 6. Non solo naϊve primo, ma anche di espandere le cellule T di memoria 7,8. CD40-attivate le cellule B possono essere utilizzati per studiare l&#39;attivazione delle cellule B, la differenziazione e la funzione. Inoltre, essi rappresentano uno strumento promettente per la vaccinazione terapeutica o preventiva contro i tumori 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 questo video vi presentiamo il<em&gt; Ex vivo</em&gt; Generazione ed espansione dei diritti umani CD40 delle cellule B attivate (CD40-B) dalle cellule mononucleari del sangue periferico (PBMC) attraverso la stimolazione con il ligando CD40 e l&#39;interleuchina-4.

Generazione di Human CD40 delle cellule B attivate

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 Views.  University Hospital of Cologne, Department I of Internal Medicine. In questo video vi presentiamo il Ex vivo Generazione ed espansione dei diritti umani CD40 delle cellule B attivate (CD40-B) dalle cellule mononucleari del sangue periferico (PBMC) attraverso la stimolazione con il ligando CD40 e l'interleuchina-4.

Video: 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.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

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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Biologia

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.

Generazione di cellule staminali pluripotenti indotte da fibroblasti umani Riprogrammare con il Stemgent Lentivirus umani TF 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.

Generazione robusta di epatociti-come le cellule da popolazioni di cellule staminali embrionali

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.

Caratterizzazione fenotipica e funzionale delle cellule endoteliali Colony Forming derivato dal sangue del cordone ombelicale umano

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.

Generazione di cellule pluripotenti indotte umane staminali da sangue periferico mediante il STEMCCA lentivirale Vector

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.

Generazione di Dispersi Presomitic mesoderma colture cellulari, per Immagini del Zebrafish segmentazione Orologio in celle singole

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.

Generazione di cardiomiociti umani: Un protocollo di differenziazione da alimentatore-Free umani cellule staminali pluripotenti indotte

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.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con 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.

Generazione e Cultura Sangue escrescenza endoteliali cellule da sangue periferico umano

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 vettore Costruzione e caratterizzazione di modifiche del DNA da Generazione di Roots pomodoro Peloso

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