Questo lavoro è stato finanziato da un premio II Facoltà di nuovo dal California Institute of Regenerative Medicine (RN2-00921-1), e un NIH-finanziato Premio Nazionale delle Ricerche (F32-HL104924)

1. Preparazione di Alimentatori embrionali di topo <ol><li> Il MEFs utilizzato nella co-coltura delle hESC devono essere preventivamente trattati mediante irradiazione o mitomicina-C per inibire le cellule dalla fase di divisione. </li><li> Due ore prima della semina MEF, cappotto ogni plasma 60 mm trattato piastra di coltura con 2 ml di 0,05% gelatina. </li><li> Scongelare una fiala di trattato MEF e piastra a ~ 20.000 cellule / cm 2. </li><li> Consentire alle cellule di aderire durante la notte. </li></ol> 2. Co-semina hESC sul MEF <ol><li> Nuove colonie possono essere avviate da colture crioconservati o passaging culture attuali sui nuovi MEF rivestite piatti, come indicato di seguito. </li><li> Lavare la piastra contenente le colonie hESC grandi da diversi passaggi 1 volta con 3 ml di caldo 1x PBS. </li><li> Aggiungere 3 ml di collagenasi IV / PBS soluzione (1 mg / ml), e incubare per 5-10 minuti a 37 ° C. (Nota: Le cellule non diventerà distinto o palla come visto con tripsina). </li><li> Mentre le cellule sono ancora nellacollagenasi, utilizzare un bordo di una punta di pipetta da 5 ml, creare un modello di griglia sulle colonie, il loro inserimento in ciuffi piccole cellule. </li><li> Utilizzare l&#39;estremità della punta di pipetta (tenuto in maniera perpendicolare alla piastra di coltura) a raschiare il fondo della piastra di coltura, rimuovendo tutte le cellule dalla superficie. </li><li> Raccogliere le cellule in un tubo Falcon e centrifugare a (1000 rpm) per 5 min. </li><li> Dopo centrifugazione, aspirare il terreno, lasciando il pellet di cellule nel tubo Falcon. </li><li> Aspirare il mezzo MEF dal placcato MEF il giorno prima. </li><li> Re-piastra le cellule in un rapporto 1:3 dalla piastra originale, e mangimi con 3 ml di terreno hESC </li></ol> 3. L&#39;esaurimento MEF dal Co-cultura Nota: le culture umane CES deve essere ad alta densità, di solito tra 10 e 14 giorni di coltura. <ol><li> Posizionare la punta di una pipetta aspirante sul bordo della piastra e consentire l&#39;aspirazione di tirare delicatamente il bordo del foglio MEF. </li><li> RTogliete il supporto dal piatto, e che questo venga a prendere il foglio di cella e spostare lentamente la punta in modo arcuato sopra la piastra. Nota: Il foglio può intasare la punta della pipetta, ma questo non è un problema in quanto non deve impedire all&#39;utente di tirare manualmente il foglio dalla superficie della piastra di coltura. Se le cellule sono bloccati alla fine dell&#39;aspiratore, è possibile utilizzare la parte superiore del piatto di coltura per rompere il foglio cella per la completa aspirazione. </li><li> Sostituire media hESC immediatamente, e mettere la piastra posteriore nell&#39;incubatore. </li></ol> 4. Risultati rappresentativi Il risultato finale del processo di rimozione MEF produce piccole colonie hESC inalterate (Figura 1D) in grado di sottoporsi a una forte espansione in colonie molto grandi (Figura 2), pur mantenendo l&#39;espressione dei produttori di pluripotenza SSE-4 ottobre, ¾, e TRA 1-81 (Figura 3). Figura 1. ESC Colony Morfologia Prima e dopo la rimozione del MEF. Umani embrionali colonie di cellule staminali in A) giorno 1 e B) 2 ° giorno dopo la semina su piastre di coltura. C) ad alta densità Colonie umane embrionali cellule staminali (H7) circondato da MEF. Le due colonie hESC sono descritti nella trattini rossi. D) Dopo la rimozione MEF utilizzando la tecnica di aspirazione descritta, ci ritroviamo con una colonia di cellule staminali embrionali umane isolato con pochissimi MEF che circonda la colonia intatto. <img alt="Figura 2" src="/files/ftp_upload/3951/3951fig2.jpg" /> Figura 2. Colony espansione ESC 10 giorni dopo la rimozione MEF. La colonia hESC originale immediatamente dopo l&#39;esaurimento MEF (a sinistra) è consentito di espandersi in una colonia molto grande (a destra) approximately 800 micron, 10x. Si noti che la colonia originale immaginata allo stesso ingrandimento (10x) come colonia composito. <img alt="Figura 3" src="/files/ftp_upload/3951/3951fig3.jpg" /> Figura 3. Immunostaining del MEF-impoverito colonie hESC. Identificazione immunofluorescente delle colonie fino a 10 giorni dopo l&#39;esaurimento MEF indica che le colonie mantenere la loro espressione di marcatori di pluripotenza, come evidente da ottobre ¾, SSE-4, e Tra-1-81 colorazione. Inoltre, queste colonie hESC non presentano differenziazione verso un destino mesodermica, indicato dall&#39;assenza di Flk-1. A, E, I e M) sono immagini di trasmissione della luce delle colonie immunoflouscently macchiati, 20, 10, 10 e 2x, . rispettivamente B, F, J e N) mostrano i nuclei -. cellule DAPI colorate delle colonie immunofluorescently macchiati, 20, 10, 10 e 2x, rispettivamente CD) mostrano l&#39;espressione dimarcatore di cellule staminali umane SSE-4 solo, e l&#39;immagine composita, 20x. GH) mostrano l&#39;espressione di cellule staminali umane marcatore ottobre ¾ solo, e l&#39;immagine composita, KL 10x.) mostrano l&#39;espressione del marcatore di cellule staminali umane Tra 1-81 solo , e l&#39;immagine composita, 10x. MP) L&#39;ultima riga se le immagini ritraggono una colonia raffigurante i confini tipici lisce visto in questo ingrandimento, 2x, e queste colonie non ha espresso O) Flk-1, un marker precoce di differenziazione mesoderma, né essi P) contiene fibroblasti come dimostra l&#39;assenza di colorazione DDR2. Video supplementare. Rimozione fibroblasti da aspirazione Foglio. <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">Clicca qui per visualizzare filmati</a> .

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

Nessun conflitto di interessi dichiarati.

Il metodo presentato in questo manoscritto offre un&#39;alternativa rapida e meno costosa per eliminare cellule di alimentazione fibroblasti umani da colture di cellule embrionali. Un&#39;efficace rimozione dei fibroblasti dipende dall&#39;esistenza di un monostrato confluente strettamente di queste cellule che si sviluppa in colture di cellule staminali più lunghi. Dopo 7-10 giorni, le colonie hESC crescenti spingerà i fibroblasti feeder in una direzione verso l&#39;esterno, generando un monostrato di fibroblasti sem...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Fibroblast Marker </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> Nucleo cellulare Marker </td></tr><tr><td> SSE-4 </td><td> Millipore </td><td> MAB4304 </td><td> Umano ESC Marker </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> Umano ESC Marker </td></tr><tr><td> Flk-1 </td><td> BD Pharma </td><td> 555307 </td><td> Differenziazione marker precoce </td></tr><tr><td> Tradi-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> Umano ESC Marker </td></tr></tbody></table> Tabella 1. Tabella di motivi specificisignori utilizzato per la immunocolorazione cellule staminali embrionali umane.

rapid fibroblast removal from high density human embryonic stem cell cultures

Mouse fibroblasti embrionali (MEF) sono stati usati per creare cellule staminali embrionali umane (hESC) colture dopo l&#39;isolamento blastocisti 1. Questo sistema di alimentazione mantiene hESCs dal subire differenziazione spontanea durante l&#39;espansione delle cellule. Tuttavia, questo metodo di co-cultura è un lavoro dispendioso, richiede personale altamente qualificato, e la purezza hESC rendimenti bassi 4. Molti laboratori hanno tentato di minimizzare il numero di cellule feeder in colture hESC (cioè incorporando matrice rivestite piatti o altri tipi di cellule alimentatore 5-8). Questi sistemi di coltura modificati hanno mostrato qualche promessa, ma non hanno soppiantato il metodo standard per hESC coltura con fibroblasti di topo mitomicina C trattati con embyronic al fine di ritardare indesiderato differenziazione spontanea delle culture hESC. Pertanto, le cellule di alimentazione utilizzati in espansione hESC devono essere rimosse durante esperimenti di differenziazione. Sebbene diverse tecniche sono disponibili per purificare il co hESClonies (FACS, MACS, o l&#39;uso di vettori resistenti ai farmaci) da alimentatori, queste tecniche sono alta intensità di manodopera, costoso e / o distruttiva per la cellule staminali embrionali umane. Lo scopo di questo progetto è stato quello di inventare un metodo di purificazione che consente la raccolta di una popolazione pura di hESC. Abbiamo osservato che in una cultura hESC confluenti, la popolazione MEF può essere rimosso con una semplice e rapida aspirazione del foglio MEF. Questa rimozione dipende da diversi fattori, tra laterale cellula-cellula legame di MEFs che hanno una bassa affinità di legame alla piastra di coltura stirene, e la capacità di colonie di cellule staminali per spingere i fibroblasti verso l&#39;esterno durante la generazione della propria &quot; di nicchia &quot;. Il hESC sono stati poi esaminati per SSE-4, Oct3 / 4 e Tra 1-81 espressione fino a 10 giorni dopo la rimozione MEF per garantire il mantenimento della pluripotenza. Inoltre, le colonie hESC sono stati in grado di continuare a crescere in grandi formazioni da dopo la rimozione del MEF, che fornisce un ulteriore livello di espansione cellule staminali embrionali umane.

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

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Nonostante gli sforzi volti a culture di transizione a feeder liberi condizioni, la derivazione e la cultura di cellule staminali embrionali umane (hESC) rimangono in gran parte dipendente dal co-colture con alimentatori embrionali di topo (MEF). Qui vi mostriamo una nuova metodologia per la rapida rimozione di alimentatori da culture hESC prima della sperimentazione.

Fibroblast rimozione veloce da alta densità Culture staminali embrionali umane

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

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JoVE Journal

Biology

11.7K Views.  University of California, Merced. Nonostante gli sforzi volti a culture di transizione a feeder liberi condizioni, la derivazione e la cultura di cellule staminali embrionali umane (hESC) rimangono in gran parte dipendente dal co-colture con alimentatori embrionali di topo (MEF). Qui vi mostriamo una nuova metodologia per la rapida rimozione di alimentatori da culture hESC prima della sperimentazione.

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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

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

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

RIFLESSI Passaging sulle cellule staminali embrionali umane-linee con tripsina

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

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Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

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


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

Derivazione di cellule staminali embrionali umane da Immunosurgery

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

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

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

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

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

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

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

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

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

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

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

Preparazione del pool lisato piastrinico umani (pHPL) come supplemento efficiente per animali siero-Free Culture sulle cellule staminali umane

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

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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

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

Video Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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

Establishing Primary Adult Fibroblast Cultures From Rodents

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

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

Stabilire colture primarie di fibroblasti adulti dai roditori

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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

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