Dr. Rountree erkennt aktuelle Unterstützung der Kinder-Miracle Network, National Institute of Health, K08DK080928 und R03DK088013, und die American Cancer Society Research Scholar Award, MGO-11651. Dr. Rountree erkennt an, dass dieses Verfahren ursprünglich entwickelt und verfeinert, während die Kinder Scientist Development Program (NICHD Grant Award K12-HD00850) finanziert.

<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 erhält Forschungsförderung von Bayer Pharma. Drs. Ding und Crooks und Frau Dang und Frau VanKirk haben keine Angaben zu melden.

Anders als das blutbildende System, in dem hämatopoetischen Stammzellen sind verantwortlich für die Aufrechterhaltung einer zellulären Differenzierung, das replacesnormal physiologischen turn-over von Leukozyten, rote Blutkörperchen und Blutplättchen, Leber Stammzellen oder erwachsenen Leber Vorläuferzellen, nicht in normalen Leber zu beteiligen Homöostase. 5,6 Nach einer akuten Schädigung der Leber oder teilweisen Hepatektomie, Hepatozyten, wie differenziert Leber Epithel, durchlaufen mehrere Runden der Proliferation, um die verlorenen Leber Masse zu ersetzen. 5,6 Nur bei chronischen Verletzungen sind Leber-Stammzellen beobachtet zu vermehren. 1,5 -11 Diese Erwachsenen, organspezifische Stammzellen werden vorgeschlagen, um sowohl in Hepatozyten und Cholangiozyten unterscheiden. 1,5-8 Interessanterweise ist die überwiegende Mehrheit von Leberkrebs auf dem Hintergrund der chronischen Verletzung entwickelt, und damit Leber-Stammzellen sind auch die Hypothese zu sein Vorläufer für eine Teilmenge von Leberkrebs. 2-4,12-17 Eine der wichtigsten Herausforderungen für die Zelle Arbeit in der Leber Stammzellen ist die Isolierung von seltenen Populationen von Zellen für die funktionelle Analyse. Zum Beispiel sind die überwiegende Mehrheit der Zellen in der Leber Hepatozyten, die deutlich größer als Cholangiozyten und andere kleinere nicht-Parenchymzellen sind. Durch das Brechen der ganzen Leber in Zellkompartimenten (große Hepatozyten - Parenchym-Fraktion und kleinere Zellen - nicht-parenchymatösen Fraktion) und weitere Auswahl für CD45 negativen Zellen (nicht-hämatopoetischen Zellen), können wir an den Start Population von Stammzellen durch bereichern über 600-fache. 1,18-21 Zu beachten ist, sind wir keineswegs darauf hinweist, dass die CD133 + Population ein 100% reines Stammzellen Bevölkerung ist, sondern ganz klar eine heterogene Population von Zellen mit unterschiedlichen Herkunft und Wiederbesiedlung Potenzial. Eine der wichtigsten Einschränkungen des Feldes Definitionen von Stammzellen und Vorläuferzellen. Wir verwenden den Begriff &quot;Stammzelle&quot; im weiteren Sinne in dieser Arbeit, aber in strengen Definition stellen CD133 + Nicht-Parenchymzellen ein bi-Linie Vorläufer-Population. Angesichts des Stands der aktuellen Stamm-und Vorläuferzellen Forschung in der Leber, gibt dieser Bericht einen Startplatz für die Ermittler, die in diesem Bereich interessiert sind. Als neuer Marker, entstehen wie EpCAM, 22,23 oder Transkriptionsfaktoren, wie Sox9, 24 sie eingebaut werden kann. Zum Beispiel haben wir eine ziemlich hohe Überlappung zwischen EpCAM + Zellen und CD133 + Zellen gefunden. In diesem Bericht detailliert ein Verfahren zur Stammzellen isoliert von normalen murinen Leber sowie chronische Leberschäden Modelle, die zeigen, erhöhte Leberwerte Stammzellproliferation. Diese Methode hat klare Vorteile gegenüber Standard-Immunhistochemie von gefrorenen oder Formalin fixiert Leber als funktionelle Studien mit lebenden Zellen nach der Isolierung durchgeführt werden kann. 1,3,4 Die spezifische Ziel dieses Prozesses ist es, eine relativ reine Population von Leber-Stammzellen, die isolieren können klonal in vitro erweitert werden. Die wichtigste Einschränkung dieses Verfahrens ist, dass die Mehrzahl der Zellen isoliert werden nicht nach Durchflusszytometrie lebensfähig (siehe Abschnitt Fehlersuche). Dies ist das Ergebnis der Stunden benötigt, um die Zellen herzustellen, und die zahlreichen Verfahren erforderlich, um die Bevölkerung vor der Isolierung zu verfeinern. Wenn die Leber in einzelne Zell-Suspension für die sofortige FACS-Analyse verdaut ist, wird der Größenunterschied zwischen Hepatozyten und anderen Nicht-Parenchymzellen effektiv FACS Tor Schöpfung unmöglich. Wenn die Leber nicht Parenchymzellen verwendet werden, ohne Abspaltung von hämatopoetischen Zellen, besteht die Gefahr, dass eine erhebliche Anzahl von CD133 + Zellen hämatopoetischen Ursprungs sein kann der Anteil verunreinigen. Weiterhin wird durch die Bearbeitung der Zellen durch die Miltenyi Filter sind nur einzelne Zellen und sehr kleine Gruppen von Zellen gesammelt. Dadurch wird sichergestellt, dass die Probe nicht verstopfen die FACS Einnahme Nadel. Alternativen für dieses Verfahren sind Änderungen für jede alternative Zelloberflächenmarker, wie CD49f, EpCAM, oder eine Kombination von Markern, wie CD133 + EpCAM + Ein zweiter Bericht veröffentlicht, nutzt einen Dichtegradienten zu Leber-Stammzellen, die aus einem Nicht-Parenchym-Fraktion zu isolieren. Dieses Verfahren erfordert eine Ultra-Zentrifuge (8000 xg), fügt viel Zeit, um das Verfahren, und in unserer Erfahrung, deutlich vor FACS zellulären Ertrag und post-FACS Lebensfähigkeit reduziert. 8 Zukünftige Experimente sind ein breiteres Spektrum von Genexpressionsanalyse, einschließlich fetale Leber Gene, wie HNF3, HNF4α und αfp.In Zusätzlich können Western-Blot-Analyse und Immunzytochemie von exprimierten Proteinen genutzt werden, um RT-PCR-Ergebnisse von Zellen in Kultur zu bestätigen. Ein Thema, zu beachten ist, neuere Arbeiten, die CD133-Expression auf Lebersternzellen identifiziert. 25 Wir haben jetzt routinemäßig Bildschirm unsere Proben für die Marker der Sternzellen (siehe Abschnitt Fehlersuche), und nicht identified signifikante Kontamination in unseren Fraktionen. Dies kann zu unterschiedlichen Techniken der Leber Verdauung und Zellisolation bezogen werden. 2,3,26 Basierend auf der Tatsache, dass die Mehrzahl der Zellen nicht sofort nach FACS isoliert lebensfähig, empfehlen wir, dass die Zellen ausplattiert werden, entweder als Bulk-CD133 + Zellen oder einzelne Zellen, vor Tieren zu verwenden. 5-7 Tage in vitro signifikant verbessert werden Ergebnisse der Tumor-Analyse. Darüber hinaus angesichts der Strapazen des Einzelzellisolierung, sollte diese nur durchgeführt, wenn Kolonien von Bulk-CD133 + isolierten Zellen erweitert werden kann. Eine genauere Diskussion der verschiedenen Kulturbedingungen und Gerüst Proteine, die zur Kultur Leber Stamm-und Vorläuferzellen verwendet werden kann, wurde auch von Lola Reid aus. Diese Arbeit bietet alternative Bedingungen und Modifikationen, die Ermittler in ihrem Forschungsprogramm integrieren kann einmal Basisisolierung Techniken beherrscht werden. 27,28 Dr. Reid Werk bietet auch eine detailliertere Analyse der Linie Biologie und Reifung von Leber-Stammzellen und Vorläuferzellen verpflichtet. In Bezug auf die in vivo Tumor-Analyse hatten wir Erfolg mit frisch isolierten Zellen und Zellen von klonal expandierten CD133 +-Zellen in vitro, in erster Linie mit 1x10 6 Zellen. / - - Und in der Leber bestimmte PTEN - / - Wir haben ontwo genetische Stämme von chronischen Leberschädigung, die MAT1A konzentriert Mäuse, und wir haben beide Nacktmäusen und Wildtyp-Mäusen als Gastgeber für das Tumorwachstum eingesetzt. Nach unserer Erfahrung wird CD133 + Leber-Stammzellen nur Tumoren bilden, wenn sie aus signifikante Schädigung der Leber Modelle, die prä-maligne sind isoliert. Beachten Sie, dass die Tumoren aus CD133 + Zellen gebildet haben sowohl hepatozellulären Karzinoms und Gallengangskarzinom Funktionen, was auf eine Stammzell-oder Progenitorzellen Entstehung der Tumoren. 2-4,29 Follow-up-Arbeit nach Tumoren dokumentiert umfasst Standard-pathologischen Analyse von Tumorgewebe (H &amp; E-Färbung) und immunhistochemische Färbung. Darüber hinaus können Tumoren zerkleinert und für die FACS-Analyse oder re-Kultur verdaut werden. 2-4,30 Zusammenfassend haben wir ein Verfahren zur Isolierung, Expansion und die grundlegende Charakterisierung von CD133 + Leber-Stammzellen und CD133 + Krebsstammzellen detailliert. Trouble Shooting: CD45 Kontamination: Für Schritt 3, wenn es Kontamination von CD45 +-Zellen, die durch Zugabe eines CD45-FITC Ab vor FACS-Analyse und isoliert betrachtet werden können, um sicherzustellen, dass der CD45 Mikrobead Antikörper nicht abgelaufen ist und dass das Filtrat nur während sammelten die Filter wurde in den Magnethalter. Jede Filtrat gesammelt, während der Filter nicht in der magnetischen Halterung wird CD45 +-Zellen enthalten. Niedrige Zellzahl: Für die unverletzten Leber, die Gesamtzahl der CD133 + Nicht-Parenchym isoliert werden können weniger als 10.000. Diese Zellen sind selten in den ruhenden Leber. Für eine chronische Verletzung Modell, wie die DDC 0,1% Ernährung, wird die Zahl deutlich erhöhen zu 100.000 Zellen. Wenn die gesamte Zellzahl liegt deutlich unter diesen Zahlen ist die eine Sache zu prüfen, FACS Ab Färbung. Stellen Sie sicher, dass die CD133 Ab nicht abgelaufen ist, wie arm Färbung in eine schlechte Ausbeute führt. Darüber hinaus empfehlen wir die Durchführung einer FACS-Analyse der Leber nicht Parenchymzellen, um die relative Bevölkerung vor dem Versuch FACS isoliert zu bestimmen. Low Lebensfähigkeit der Zellen nach FACS: Eine der Fragen der Lebensfähigkeit können, wie die Zellen verarbeitet werden und über welchen Zeitraum bezogen werden. Im Idealfall sollte die gesamte Zelle Isolierung, die Schritte 1-4, ohne Verzögerungen zwischen den einzelnen Schritten durchgeführt und abgeschlossen werden am selben Tag. Jede wesentliche Verzögerung zwischen den Schritten 1-4 wird erheblich reduziert die Lebensfähigkeit der Zellen. Ein zweites Problem im Zusammenhang mit Lebensfähigkeit der Zellen kann zum Mantel Druck für FACS Isolierung verwendet bezogen werden. Wir empfehlen die Verwendung einer geringeren Mantel Druck. Schließlich, nachdem die Zellen isoliert werden, sollten sie sofort vergoldet, als nennenswerte Lagerung auf Eis nach dem Sortieren reduzieren auch die Rentabilität. CD133 + Nicht-Parenchym-Heterogenität: Aktuelle Berichte zeigen, dass Lebersternzellen können auch CD133 Expression und haben einige Plastizität. 25 Darum, zusätzlich zur Überprüfung bi-Potenz Gene mit Albumin und Krt19, zusätzliche Überprüfung können Gene mit sternförmigen Zellen, wie saures Gliafaserprotein in assoziiert sind, zählen Ruhestrom Sternzellen und alpha-smooth muscle Aktin und Desmin in aktivierten Sternzellen. 3,4,26 Darüber hinaus stellt die CD133 + Population eine breitere Vorläufer-Population. Die Zugabe eines zweiten Marker, wie zB EpCAM, kann dazu beitragen, die Bevölkerung weiter zu verfeinern und zu begrenzen Heterogenität.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> DMEM: F12 </td><td> Invitrogen </td><td> 10565-018 </td><td> Mit Phenolrot </td></tr><tr><td> CD45 Microbeads </td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td> Hepatozyten-Wachstumsfaktor </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> DNase </td><td> Sigma </td><td> DN25 </td><td> 1 Gramm </td></tr><tr><td> Collagenase D </td><td> Roche </td><td> 1088874 </td><td></td></tr><tr><td> Pronase </td><td> Roche </td><td> 0165921 </td><td></td></tr><tr><td> 70 Mikron Mesh Sieb </td><td> Fischer </td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50 Reaktionen </td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi LD Spalte </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> Laminin beschichteten Platten </td><td> BD </td><td> 354410 </td><td> 96-Well- </td></tr><tr><td> Trypsin 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 Spalten für 5x10 5 Zellen oder weniger </td></tr><tr><td> Pharm Lyse </td><td> BD </td><td> 555899 </td><td> Konzentration 10X </td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

Leber-Stammzellen oder ovale Zellen, vermehren sich während der chronischen Leberschädigung, und vorgeschlagen, sowohl in Hepatozyten und Cholangiozyten unterscheiden. Darüber hinaus sind Leber-Stammzellen angenommen, dass sie die Vorläufer für eine Teilmenge von Leberkrebs, Leberzellkarzinom werden. Eines der wichtigsten Herausforderungen für die Zelle Arbeit in einem festen Organ wie die Leber Stammzellen ist die Isolierung von einer seltenen Population von Zellen, für eine detaillierte Analyse. Zum Beispiel sind die überwiegende Mehrheit der Zellen in der Leber Hepatozyten (Parenchym-Fraktion), die deutlich größer ist als Nicht-Parenchymzellen sind. Durch die Anreicherung der spezifischen zellulären Kompartimenten der Leber (zB Parenchym-und Nicht-Parenchym-Fraktionen), und die Auswahl für die CD45-negativen Zellen, können wir an den Start Population von Stammzellen durch bereichern über 600-fold.The proceduresdetailed in diesem Bericht ermöglichen eine relativ seltene Population von Zellen aus einem Organ-effizient sortiert werden. Dieser Prozess kann bis isolateliver Stammzellen aus normalen murinen Leber sowie chronische Leberschäden Modelle, die eine erhöhte Leber Stammzellproliferation demonstrieren genutzt werden. Diese Methode hat klare Vorteile gegenüber Standard-Immunhistochemie von gefrorenen oder Formalin fixiert Leber als funktionelle Studien mit lebenden Zellen nach der ersten Co-Lokalisation Experimente durchgeführt werden können. Um das zu erreichen das Verfahren in diesem Bericht dargelegt wurde, ist eine funktionierende Beziehung mit einem Forschungs-basierte Durchflusszytometrie Kern stark gefördert wie die Details der FACS isoliert in hohem Maße auf spezialisierte Mess-und ausgeprägte Kenntnisse der grundlegenden Durchflusszytometrie Verfahren. Das spezifische Ziel dieses Prozesses ist es, eine Bevölkerung von Leber-Stammzellen, die klonal in vitro erweitert werden kann isolieren.

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

Hier beschreiben wir die Isolierung von CD133 exprimierenden Leber-Stammzellen und Krebsstammzellen aus ganz murinen Leber, ein Prozess, der Gewebe Verdauung, Anreicherung von Zellen und Durchflusszytometrie Isolierung erfordert. Wir gehören Methoden für fortgeschrittene Einzelzellisolierung und klonalen Expansion.

Isolierung von CD133 + Leber-Stammzellen für die klonale Expansion

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

Research

JoVE Journal

Biology

22.3K Aufrufe.  Pennsylvania State College of Medicine. Hier beschreiben wir die Isolierung von CD133 exprimierenden Leber-Stammzellen und Krebsstammzellen aus ganz murinen Leber, ein Prozess, der Gewebe Verdauung, Anreicherung von Zellen und Durchflusszytometrie Isolierung erfordert. Wir gehören Methoden für fortgeschrittene Einzelzellisolierung und klonalen Expansion.

Serie: 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

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

Biologie

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.

Isolation und großräumige Ausdehnung der erwachsenen Menschen Endothelial Colony Forming Progenitorzellen

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.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (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.

Isolation und Ableitung von embryonalen Maus-Keimzellen

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.

Isolierung von Maus Speicheldrüsen 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 ...

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.

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

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.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

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.

Protokoll für die Isolierung von primären humanen Hepatozyten und entsprechende Hauptpopulationen von Nicht-parenchymale Leberzellen

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