Dr. Rountree riconosce il sostegno corrente dal Miracle Network per bambini, Istituto Superiore di Sanità, K08DK080928 e R03DK088013, e l&#39;American Cancer Society Research Scholar Award, MGO-11651. Dr. Rountree riconosce che questa procedura è stato inizialmente sviluppato e raffinato, mentre finanziato dal Programma di Sviluppo Pediatric Scientist (NICHD assegnazione di sovvenzioni K12-HD00850).

<ol>
	<li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD133-expressing+murine+liver+oval+cell+population+with+bilineage+potential.">A CD133-expressing murine liver oval cell population with bilineage potential.</a> Stem Cells. 25, 2419-2429 (2007).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++liver+cancer+stem+cells+from+methionine+adenosyl+transferase+1A-deficient+mice+demonstrate+resistance+to+transforming+growth+factor+(TGF)-beta-induced+apoptosis.">CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis.</a> Hepatology. 49, 1277-1286 (2009).</li><li>Rountree, C. B., Ding, W., He, L., Stiles, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+CD133-expressing+liver+cancer+stem+cells+in+liver-specific+phosphatase+and+tensin+homolog+deleted+on+chromosome+10-deleted+mice.">Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice.</a> Stem Cells. 27, 290-299 (2009).</li><li>Rountree, C. B., Senadheera, S., Mato, J. M., Crooks, G. M., Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+liver+cancer+stem+cells+during+aging+in+methionine+adenosyltransferase+1A-deficient+mice.">Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice.</a> Hepatology. 47, 1288-1297 (2008).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J. Cell. Physiol. 213, 286-300 (2007).</li><li>Fausto, N., Campbell, J. S., Riehle, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Hepatology. 43, 45-53 (2006).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+Stem+Cells.+III.+Emergent+themes+of+liver+stem+cell+biology:+niche,+quiescence,+self-renewal,+and+plasticity.">Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 290, 189-193 (2006).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+liver+repopulating+capacity+of+murine+oval+cells.">The origin and liver repopulating capacity of murine oval cells.</a> Proc Natl Acad Sci U. S. A. 100, 11881-11888 (2003).</li><li>Greenbaum, L. E., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stem+cells+in+liver+repair+and+fibrosis.">The role of stem cells in liver repair and fibrosis.</a> Int. J. Biochem. Cell Biol. 43, 222-229 (2011).</li><li>Choi, S. S., Omenetti, A., Syn, W. K., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Hedgehog+signaling+in+fibrogenic+liver+repair.">The role of Hedgehog signaling in fibrogenic liver repair.</a> Int. J. Biochem. Cell. Biol. 43, 238-244 (2011).</li><li>Khurana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scopolamine+treatment+and+muscarinic+receptor+subtype-3+gene+ablation+augment+azoxymethane-induced+murine+liver+injury.">Scopolamine treatment and muscarinic receptor subtype-3 gene ablation augment azoxymethane-induced murine liver injury.</a> J. Pharmacol. Exp. Ther. 333, 639-649 (2010).</li><li>Sell, S., Leffert, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cancer+stem+cells.">Liver cancer stem cells.</a> J. Clin. Oncol. 26, 2800-2805 (2008).</li><li>Lee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+prognostic+subtype+of+human+hepatocellular+carcinoma+derived+from+hepatic+progenitor+cells.">A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.</a> Nat. Med. 12, 410-416 (2006).</li><li>Sell, S., Dunsford, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+stem+cell+origin+of+hepatocellular+carcinoma+and+cholangiocarcinoma.">Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma.</a> Am. J. Pathol. 134, 1347-1363 (1989).</li><li>Amin, R., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+tgf-Beta+in+hepatic+carcinogenesis.">Liver stem cells and tgf-Beta in hepatic carcinogenesis.</a> Gastrointest Cancer Res. 2, 27-30 (2008).</li><li>Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor/stem+cells+give+rise+to+liver+cancer+due+to+aberrant+TGF-beta+and+IL-6+signaling.">Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.</a> Proc Natl Acad Sci. 105, 2445-2450 (2008).</li><li>Kitisin, K., Pishvaian, M. J., Johnson, L. B., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+molecular+signaling+pathways+in+hepatocellular+carcinoma.">Liver stem cells and molecular signaling pathways in hepatocellular carcinoma.</a> Gastrointest Cancer Res. 1, 13-21 (2007).</li><li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+fails+to+differentiate+into+liver+epithelium+during+murine+development+and+regeneration.">Bone marrow fails to differentiate into liver epithelium during murine development and regeneration.</a> Hepatology. 45, 1250-1260 (2007).</li><li>Shimano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+oval+cells+have+the+side+population+phenotype+defined+by+expression+of+ATP-binding+cassette+transporter+ABCG2/BCRP1.">Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1.</a> Am J Pathol. 163, 3-9 (2003).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+isolation+and+clonal+identification+of+self-renewing+bipotent+hepatic+progenitor+cells+in+adult+mouse+liver.">Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.</a> Hepatology. , (2008).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+and+characterization+of+self-renewing+pluripotent+stem+cells+in+the+developing+liver.">Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.</a> J Cell Biol. 156, 173-184 (2002).</li><li>Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpCAM-positive+hepatocellular+carcinoma+cells+are+tumor-initiating+cells+with+stem/progenitor+cell+features.">EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.</a> Gastroenterology. 136, 1012-1024 (2009).</li><li>Yamashita, T., Budhu, A., Forgues, M., Wang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hepatic+stem+cell+marker+EpCAM+by+Wnt-beta-catenin+signaling+in+hepatocellular+carcinoma.">Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.</a> Cancer Res. 67, 10831-10839 (2007).</li><li>Furuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li><li>Kordes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++hepatic+stellate+cells+are+progenitor+cells.">CD133+ hepatic stellate cells are progenitor cells.</a> Biochem Biophys Res Commun. 352, 410-417 (2007).</li><li>Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Growth+Factor+10+is+critical+for+liver+growth+during+embryogenesis+and+controls+of+hepatoblast+survival+via+b-catenin+activation.">Fibroblast Growth Factor 10 is critical for liver growth during embryogenesis and controls of hepatoblast survival via b-catenin activation.</a> Hepatology. , (2007).</li><li>Turner, 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+hepatic+stem+cell+and+maturational+liver+lineage+biology.">Human hepatic stem cell and maturational liver lineage biology.</a> Hepatology. 53, 1035-1045 (2011).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Galicia, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+hepatic+tumor+progenitor+cells+in+Pten-null+mice+requires+liver+injury+and+is+reversed+by+loss+of+AKT2.">Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.</a> Gastroenterology. 139, 2170-2182 (2010).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+of+murine+liver+tumor+cells+promotes+invasion.">Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion.</a> Hepatology. 52, 945-953 (2010).</li></ol>

Dr. Rountree riceve il sostegno alla ricerca da Farmaceutica Bayer. Drs. Ding e Dang Crooks e la signora e la signora Vankirk non hanno informazioni da segnalare.

A differenza del sistema emopoietico, in cui le cellule staminali ematopoietiche sono responsabili per il mantenimento di un sistema di differenziazione cellulare che replacesnormal fisiologico turn-over dei leucociti, i globuli rossi e piastrine, cellule staminali del fegato, o un adulto le cellule progenitrici epatiche, non partecipano al fegato normale omeostasi. 5,6 Dopo lesioni epatiche acute o epatectomia parziale, epatociti, come epitelio fegato differenziata, sottoposti a vari cicli di proliferazione per sostituire la massa epatica perso. 5,6 Solo durante lesioni croniche sono le cellule staminali del fegato osservati a proliferare. 1,5 -11 Queste adulte, le cellule staminali organo specifiche proposte per differenziare in epatociti e colangiociti sia. 1,5-8 interessante notare che la stragrande maggioranza di cancro del fegato si sviluppa sullo sfondo di lesioni croniche, e quindi le cellule staminali del fegato sono anche ipotizzato di essere i precursori di un sottoinsieme di cancro al fegato. 2-4,12-17 Una delle sfide principali per il lavoro delle cellule staminali nel fegato è l&#39;isolamento delle popolazioni di cellule rare per l&#39;analisi funzionale. Per esempio, la stragrande maggioranza delle cellule nel fegato sono epatociti, che sono significativamente più grandi colangiociti e altri più piccoli non le cellule parenchimali. Rompendo il fegato intero in compartimenti cellulari (epatociti grande - frazione parenchimale e celle più piccole - non parenchimali frazione), e inoltre la selezione di cellule CD45 negative (non le cellule emopoietiche), siamo in grado di arricchire la popolazione di cellule staminali a partire da più di 600 volte. 1,18-21 Da notare che siamo in alcun modo indicare che la popolazione CD133 + è un puro al 100% della popolazione di cellule staminali, ma rappresenta chiaramente una popolazione eterogenea di cellule, con lineage diversi e potenziali ripopolamento. Uno dei limiti principali del campo è definizioni di cellule staminali e cellule progenitrici. Usiamo il termine &quot;cellula staminale&quot; più ampiamente in questo lavoro, ma nella definizione rigorosa, CD133 + non-parenchimali cellule rappresentano un bi-lignaggio popolazione progenitrice. Dato lo stato di staminali progenitrici di ricerca corrente e nel fegato, la presente relazione fornisce un punto di partenza per gli investigatori che sono interessati a questo campo. Come marcatori emergono nuovi, come EpCAM, 22,23 o fattori di trascrizione, come SOX9, 24 possono essere incorporati. Per esempio, abbiamo riscontrato un tasso piuttosto elevato di sovrapposizione tra carcinomi delle cellule delle cellule CD133 + e +. In questo rapporto, abbiamo un processo di dettaglio per l&#39;isolamento di cellule staminali dal fegato di topo normale e cronica modelli di danno epatico, che dimostrano un aumento arginare la proliferazione delle cellule del fegato. Questo metodo presenta chiari vantaggi rispetto immunoistochimica standard di surgelati o formalina fegato fissi studi funzionali utilizzando cellule vive può essere eseguita dopo l&#39;isolamento. 1,3,4 L&#39;obiettivo specifico di questo processo è quello di isolare una popolazione relativamente puro di cellule staminali del fegato che può clonale essere espanse in vitro. Il limite principale di questa procedura è che la maggior parte delle cellule isolate non saranno possibili, dopo la citometria a flusso (vedere la sezione Problemi di ripresa). Questo è il risultato di ore necessarie per preparare le cellule, e le numerose procedure necessarie per perfezionare la popolazione prima dell&#39;isolamento. Se il fegato viene digerito in sospensione singola cella per l&#39;immediata analisi FACS, la differenza di dimensioni tra epatociti e le altre non le cellule parenchimali renderà efficace cancello FACS creazione impossibile. Se il non-parenchimali cellule epatiche sono utilizzati, senza eliminazione delle cellule ematopoietiche, c&#39;è il rischio che un numero significativo di cellule CD133 + possono essere di origine ematopoietiche possono contaminare la frazione. Inoltre, il trattamento delle cellule attraverso il filtro Miltenyi, solo le cellule singole e gruppi molto piccoli di cellule vengono raccolti. Questo assicura che il campione non intasare l&#39;ago FACS di aspirazione. Alternative per questa procedura comprendono la modifica di qualsiasi indicatore alternativo superficie cellulare, come CD49f, EpCAM, o una combinazione di marcatori, come il CD133 + + EpCAM Una seconda relazione pubblicata utilizza un gradiente di densità per isolare le cellule staminali del fegato di un non-parenchimale frazione. Questa procedura richiede un Ultra-centrifuga (8000 xg), aggiunge un tempo significativo alla procedura, e nella nostra esperienza, ha ridotto significativamente pre-FACS resa cellulari e post-FACS vitalità 8. Esperimenti futuri includono una più ampia gamma di analisi di espressione genica, compresi i geni del fegato fetale, come HNF3, HNF4α, e l&#39;aggiunta αfp.In, Western blot e immunocitochimica di proteine ​​espresse possono essere utilizzate per confermare la RT-PCR risultati di cellule in coltura. Un problema da tenere presente è opera recente che hanno identificato l&#39;espressione CD133 sulle cellule stellate epatiche. 25 Ora di routine schermo i nostri campioni per i marcatori di cellule stellate (vedi sezione Problemi Shooting), e non hanno identificati contaminazione significativa nelle nostre frazioni. Questo può essere correlato a diverse tecniche di digestione e di isolamento delle cellule del fegato. 2,3,26 Sulla base del fatto che la maggior parte delle cellule non saranno possibili, immediatamente dopo l&#39;isolamento FACS, si raccomanda che le cellule essere placcato, sia come massa cellule CD133 + o singole cellule, prima dell&#39;uso degli animali. 5-7 giorni in vitro a migliorare significativamente i risultati delle analisi del tumore. Inoltre, visto i rigori di isolamento singola cella, questa deve essere condotta solo una volta colonie può essere espansa da CD133 + grosso cellule isolate. Una discussione più approfondita delle diverse condizioni di coltura e le proteine ​​impalcatura che può essere utilizzata per arginare la cultura del fegato e cellule progenitrici è stato ben caratterizzato da Lola Reid. Questo lavoro fornisce condizioni alternative e modifiche, che gli investigatori possono incorporare nei propri programmi di ricerca, una volta tecniche di isolamento di base sono masterizzati. 27,28 Dr. Lavoro Reid fornisce anche un&#39;analisi più dettagliata della biologia lignaggio e la maturazione tra cellule staminali epatiche e precursori obbligati. In termini di analisi del tumore in vivo, abbiamo avuto successo usando appena cellule isolate e le cellule CD133 + espansione clonale delle cellule in vitro, utilizzando principalmente 1x10 6 cellule. Ci siamo focalizzati ontwo ceppi genetici di danno epatico cronico, il MAT1a - / - e PTEN specifici fegato - / - topi, e abbiamo utilizzato sia topi nudi e topi wild-type come ospiti per la crescita del tumore. Nella nostra esperienza, CD133 + cellule staminali del fegato solo formare tumori se sono isolati da modelli significativi danni al fegato che sono pre-maligne. Si noti che i tumori formati da cellule CD133 + generale hanno sia carcinoma epatocellulare e le caratteristiche colangiocarcinoma, suggerendo una cellula o di cellule staminali progenitrici di origine ai tumori. 2-4,29 Follow-up dopo i tumori sono documentati comprende di serie l&#39;analisi patologica del tessuto tumorale (colorazione H &amp; E) e la colorazione immunoistochimica. Inoltre, i tumori possono essere tritati e digerito per l&#39;analisi FACS o ri-cultura. 2-4,30 In conclusione, abbiamo una procedura dettagliata per l&#39;isolamento, l&#39;espansione e la caratterizzazione di base di cellule CD133 + staminali del fegato e CD133 + cellule staminali del cancro. Risoluzione dei problemi: CD45 contaminazione: Per la Fase 3, se vi è la contaminazione del CD45 + cellule, che può essere valutata aggiungendo un CD45-FITC Ab prima analisi FACS e isolamento, controllare che il anticorpo CD45 microbead non è scaduto e che il filtrato è stato raccolto solo mentre il filtro è stato nel supporto magnetico. Qualsiasi filtrato raccolto, mentre il filtro non è nel supporto magnetico conterrà le cellule CD45 +. Basso numero di cellule: Per il fegato illeso, il numero totale di CD133 + non parenchimali isolato può essere inferiore a 10.000. Queste cellule sono rari nel fegato di riposo. Per un modello di lesione cronica, come ad esempio la dieta DDC 0,1%, il numero aumenterà notevolmente a 100.000 cellule. Se il numero totale di cellule è significativamente al di sotto di questi numeri, una questione da considerare è la colorazione FACS Ab. Verificare che l&#39;Ab CD133 non è scaduto, come colorazione poveri si tradurrà in una resa scadente. Inoltre, si consiglia di eseguire una analisi FACS del fegato non-parenchimali cellule per determinare la popolazione relativa prima di tentare l&#39;isolamento FACS. Vitalità cellulare basso dopo FACS: Una delle questioni di redditività può essere correlato al modo in cui le cellule sono trattati e in quale tempo. Idealmente, l&#39;intera procedura di isolamento delle cellule, punti 1-4, deve essere condotta senza ritardi tra i passaggi e completato nella stessa giornata. Qualsiasi ritardo significativo tra i passaggi 1-4 ridurrà di molto la vitalità cellulare. Una seconda questione relative alla vitalità cellulare può essere correlata alla pressione guaina utilizzata per l&#39;isolamento FACS. Si consiglia di utilizzare una pressione guaina inferiore. Infine, una volta che le cellule sono isolate, devono essere immediatamente placcato, in quanto l&#39;eventuale archiviazione significativo sul ghiaccio dopo l&#39;ordinamento anche ridurre la vitalità. CD133 + non parenchimali eterogeneità: Recenti rapporti indicano che le cellule stellate epatiche può anche avere CD133 espressione e di avere qualche plasticità. 25 Pertanto, oltre a verificare bi-potenza geni con albumina e Krt19, ulteriori verifiche possono includere geni associati con le cellule stellate, come gliale fibrillare acida in proteine cellule stellate quiescenti e alfa-actina del muscolo liscio e desmina nelle cellule stellate attivate. 3,4,26 Inoltre, la popolazione CD133 + rappresenta una popolazione più ampia progenitore. L&#39;aggiunta di un secondo marcatore, come EpCAM, può aiutare a perfezionare ulteriormente la popolazione, ed eterogeneità limite.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> DMEM: F12 </td><td> Invitrogen </td><td> 10565-018 </td><td> Con il rosso fenolo </td></tr><tr><td> CD45 microsfere </td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td> Fattore di crescita degli epatociti </td><td> Sigma </td><td> H1404 </td><td></td></tr><tr><td> Epidermal Growth Factor </td><td> Sigma </td><td> E4127 </td><td></td></tr><tr><td> DNasi </td><td> Sigma </td><td> DN25 </td><td> 1 grammo </td></tr><tr><td> Collagenasi D </td><td> Roche </td><td> 1088874 </td><td></td></tr><tr><td> Pronasi </td><td> Roche </td><td> 0165921 </td><td></td></tr><tr><td> 70 micron mesh </td><td> Pescatore </td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50 reazioni </td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi LD colonna </td><td> Miltenyi </td><td> 130-042-901 </td><td></td></tr><tr><td> CD133-PE FACS Ab </td><td> eBioscience </td><td> 12-1331-82 </td><td></td></tr><tr><td> Piastre di laminina rivestiti </td><td> BD </td><td> 354410 </td><td> 96 pozzetti </td></tr><tr><td> Tripsina 0,05% EDTA </td><td> Invitrogen </td><td> 25300-354 </td><td> 100 ml </td></tr><tr><td> RNeasy Micro Kit </td><td> Quaigen </td><td> 74004 </td><td> 50 colonne di 5x10 5 celle o meno </td></tr><tr><td> Pharm Lyse </td><td> BD </td><td> 555899 </td><td> Concentrazione 10X </td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

Cellule staminali del fegato, o cellule ovali, proliferano durante la ferita cronica del fegato, e sono proposti a differenziarsi in epatociti e colangiociti sia. Inoltre, le cellule staminali del fegato sono ipotizzate essere i precursori di un sottoinsieme di cancro al fegato, il carcinoma epatocellulare. Una delle sfide principali per il lavoro delle cellule staminali in qualsiasi organo solido come il fegato è l&#39;isolamento di una rara popolazione di cellule per l&#39;analisi dettagliata. Per esempio, la stragrande maggioranza delle cellule nel fegato sono epatociti (frazione parenchimale), che sono significativamente più grandi rispetto ai non-cellule parenchimali. Arricchendo i compartimenti cellulari specifici del fegato (cioè parenchimale e non parenchimali frazioni), e la selezione di cellule CD45 negativi, siamo in grado di arricchire la popolazione di cellule staminali a partire da oltre 600-fold.The proceduresdetailed in questo rapporto consentono una popolazione relativamente rara di cellule di un organo solido da ordinare in modo efficiente. Questo processo può essere utilizzato per le cellule staminali isolateliver dal fegato normale topo e cronica modelli di danno epatico, che dimostrano un aumento arginare la proliferazione delle cellule del fegato. Questo metodo presenta chiari vantaggi rispetto immunoistochimica standard di surgelati o formalina fegato fissi studi funzionali utilizzando cellule vive può essere eseguita dopo i primi esperimenti di co-localizzazione. Per realizzare la procedura descritta in questo rapporto, un rapporto di lavoro con una base di ricerca citometria a flusso core è fortemente incoraggiato come i dettagli di isolamento FACS sono altamente dipendenti strumentazione specializzata e una conoscenza forte lavoro di base, le procedure di citometria a flusso. L&#39;obiettivo specifico di questo processo è quello di isolare una popolazione di cellule staminali del fegato che può essere clonale espanse in vitro.

Watch this Scientific Journal Video about Isolation of CD133+ Liver Stem Cells for Clonal Expansion at JoVE.com

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Qui si descrive l&#39;isolamento delle cellule epatiche che esprimono CD133 staminali e le cellule staminali del cancro al fegato da tutto murini, un processo che richiede la digestione dei tessuti, delle cellule di arricchimento, l&#39;isolamento e la citometria a flusso. Ci sono metodi avanzati per l&#39;isolamento singola cellula e l&#39;espansione clonale.

Isolamento di cellule CD133 + staminali del fegato per l&#39;espansione clonale

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

isolation-of-cd133-liver-stem-cells-for-clonal-expansion

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Biology

22.2K Views.  Pennsylvania State College of Medicine. Qui si descrive l'isolamento delle cellule epatiche che esprimono CD133 staminali e le cellule staminali del cancro al fegato da tutto murini, un processo che richiede la digestione dei tessuti, delle cellule di arricchimento, l'isolamento e la citometria a flusso. Ci sono metodi avanzati per l'isolamento singola cellula e l'espansione clonale.

Video: Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

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Biologia

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento di cellule staminali del mouse ghiandole salivari

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, Cultura e trapianto di cellule muscolari satellite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocollo per l&#39;isolamento di epatociti umani primari e corrispondenti principali popolazioni di cellule del fegato non-parenchimali

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...