Dr. Rountree reconhece o actual apoio da Rede das Crianças Miracle, National Institute of Health, K08DK080928 e R03DK088013, ea American Cancer Society Award Scholar Research, MGO-11651. Dr. Rountree reconhece que este processo foi inicialmente desenvolvido e refinado, enquanto financiado pelo Programa Pediátrica Scientist Desenvolvimento (NICHD Grant Award 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 recebe apoio de pesquisa da Bayer Farmacêutica. Drs. Ding e Crooks e Ms. Dang e Ms. Vankirk não têm divulgações para relatar.

Ao contrário do sistema hematopoético, em que as células-tronco hematopoiéticas são responsáveis ​​por manter um sistema de diferenciação celular que replacesnormal fisiológicas turn-over de leucócitos, glóbulos vermelhos e plaquetas, células-tronco do fígado, ou células progenitoras adultas fígado, não participam de fígado normal homeostase. 5,6 Após lesão hepática aguda ou hepatectomia parcial, hepatócitos, epitélio como fígado diferenciados, passam por várias fases de proliferação para substituir a massa hepática perdido. 5,6 Somente durante lesão crônica são células-tronco do fígado observada a proliferar. 1,5 -11 Estes adultos, órgão células-tronco específicas são propostas para diferenciar-se em ambos os hepatócitos e cholangiocytes. 1,5-8 Curiosamente, a grande maioria de câncer de fígado desenvolve no fundo da lesão crônica, e, assim, as células-tronco do fígado também são hipótese a ser os precursores de um subconjunto de câncer de fígado. 2-4,12-17 Um dos grandes desafios para conter o trabalho celular no fígado é o isolamento de populações raras de células para análise funcional. Por exemplo, a grande maioria das células do fígado são hepatócitos, que são significativamente maiores do que cholangiocytes e outros menores não-parenquimatosa células. Quebrando todo o fígado em compartimentos celulares (hepatócitos grande - fração de parênquima e células menores - não-parenquimatosa fração), e ainda a seleção de células CD45 negativo (não-hematopoiéticas células), somos capazes de enriquecer a população a partir de células-tronco por mais de 600 vezes. 1,18-21 De notar que estamos de maneira nenhuma o que indica que a população + CD133 é um 100% da população de células-tronco pura, mas representa claramente uma população heterogênea de células, com diferentes linhagens e potencial de repovoamento. Uma das principais limitações do campo é definições de células-tronco e células progenitoras. Nós usamos a &quot;célula-tronco&quot; termo mais amplamente neste trabalho, mas em definição estrita, CD133 + não-parenquimatosa células representam uma população progenitora bi-linhagem. Dado o estado atual da haste e pesquisa de células progenitoras no fígado, este relatório não fornece um ponto de partida para os investigadores que estão interessados ​​neste campo. Como marcadores surgem novos, como EpCAM, 22,23 ou fatores de transcrição, tais como Sox9, 24 podem ser incorporados. Por exemplo, nós encontramos uma taxa bastante alta de sobreposição entre células EpCAM + e células CD133 +. Neste relatório, detalhamos um processo para isolamento de células-tronco do fígado de camundongos normais, bem como modelos de lesão crônica no fígado, que demonstram um aumento da proliferação de células-tronco do fígado. Este método tem vantagens claras sobre imuno-histoquímica padrão de congelados ou formol fígado fixado como estudos funcionais utilizando células vivas podem ser realizadas após o isolamento. 1,3,4 O objetivo específico deste processo é isolar uma população relativamente pura de células tronco do fígado que pode clonalmente ser expandidas in vitro. A principal limitação para este procedimento é que a maioria das células isoladas não será viável após a citometria de fluxo (ver secção Resolução de Problemas). Este é o resultado de as horas necessárias para preparar as células, e os inúmeros procedimentos necessários para refinar a população antes de isolamento. Se o fígado é digerido em suspensão única célula para análise FACS imediato, a diferença de tamanho entre os hepatócitos e outras células não-parenquimatosas fará criação portão eficaz FACS impossível. Se o fígado não-parenquimatosa células são utilizadas, sem eliminação de células hematopoiéticas, há um risco de que um número significativo de células CD133 + pode ser de origem hematopoiéticas podem contaminar a fração. Além disso, através do processamento das células através do filtro Miltenyi, apenas uma única célula e clusters muito pequena de células são coletadas. Isso garante que a amostra não vai entupir a agulha ingestão FACS. Alternativas para este procedimento incluem a modificação de qualquer marcador alternativa da superfície celular, tais como CD49f, EpCAM, ou a combinação de marcadores, como CD133 + + EpCAM Um segundo relatório publicado utiliza um gradiente de densidade para isolar células-tronco do fígado de uma fração não-parenquimatosas. Este procedimento requer uma centrífuga Ultra-(8000 xg), acrescenta um tempo significativo para o procedimento, e em nossa experiência, reduziu significativamente o rendimento pré-FACS celular e pós-FACS viabilidade 8. Experimentos futuros incluem uma ampla gama de análise de expressão gênica, incluindo genes do fígado fetal, tais como HNF3, HNF4α, e além αfp.In, análise de Western blot e imunocitoquímica das proteínas expressas podem ser utilizados para confirmar a RT-PCR, os resultados de células em cultura. Uma questão a se notar é um trabalho recente que identificou CD133 expressão em células estreladas hepáticas. 25 Nós agora rotineiramente tela nossas amostras para os marcadores de células estreladas (ver secção Resolução de Problemas), e não identified contaminação significativa no nosso frações. Isso pode estar relacionado com as diferentes técnicas de digestão do fígado e do isolamento de células. 2,3,26 Baseado no fato de que a maioria das células não vai ser viável imediatamente após o isolamento FACS, recomendamos que as células sejam banhados, seja como massa CD133 + células ou células isoladas, antes da sua utilização em animais. 5-7 dias in vitro irá melhorar significativamente os resultados da análise do tumor. Além disso, dado os rigores de isolamento única célula, este deverá ser realizado apenas uma vez colônias pode ser expandida a partir de CD133 + granel células isoladas. Uma discussão mais aprofundada das condições de cultivo e várias proteínas de andaime que podem ser utilizadas para conter a cultura do fígado e células progenitoras foi bem caracterizada por Lola Reid. Este trabalho oferece condições alternativas e modificações, que os investigadores podem incorporar em seu programa de pesquisa uma vez que técnicas de isolamento básicos são dominados. 27,28 Dr. Trabalho de Reid também fornece uma análise mais detalhada da biologia linhagem e maturação entre as células-tronco e progenitores hepática comprometida. Em termos de análise de tumor in vivo, temos tido sucesso usando recém células isoladas e células de expansão clonal CD133 + células in vitro, usando principalmente 1x10 6 células. Nós nos concentramos cepas ontwo genética de lesão hepática crônica, a MAT1a - / - e PTEN específica do fígado - / - ratos, e nós utilizamos camundongos nude e camundongos selvagens como hospedeiros de crescimento do tumor. Em nossa experiência, CD133 + células-tronco do fígado só vai formar tumores se eles são isolados a partir de modelos hepática significativa lesão que são pré-malignas. Note-se que os tumores formados a partir de células CD133 + geral têm tanto carcinoma hepatocelular e características colangiocarcinoma, sugerindo uma célula-tronco ou células progenitoras origem aos tumores. 2-4,29 Trabalho de acompanhamento após tumores são documentados inclui a análise patológica padrão de tecido tumoral (coloração H &amp; E) e coloração imuno-histoquímica. Além disso, os tumores podem ser picadas e digeridas para FACS análise ou re-cultura. 2-4,30 Em conclusão, temos um procedimento detalhado para o isolamento, expansão e caracterização básica de CD133 + células tronco do fígado e CD133 + células-tronco cancerosas. Resolução de Problemas: CD45 contaminação: Para o Passo 3, se houver contaminação do CD45 células +, que pode ser avaliado pela adição de um Ab CD45-FITC antes FACS análise e isolamento, verifique se o anticorpo microbead CD45 não expirou e que o filtrado foi coletado somente enquanto o filtro foi no suporte magnético. Qualquer filtrado recolhido enquanto que o filtro não está no suporte magnético irá conter células CD45 +. Número de células de baixa: Para o fígado sem ferimentos, o número total de CD133 + não-parenquimatoso isolado pode ser inferior a 10.000. Estas células são raras no fígado quiescente. Para um modelo de lesão crônica, tais como a dieta DDC 0,1%, o número irá aumentar enormemente a 100.000 células. Se o número total de células é significativamente abaixo desses números, uma questão a considerar é o FACS coloração Ab. Verifique para garantir que o Ab CD133 não expirou, como coloração pobres irá resultar em um rendimento pobre. Além disso, recomendamos realizar uma análise FACS do fígado não-parenquimatosa células para determinar a população relativa antes de tentar o isolamento FACS. Viabilidade celular baixa após FACS: Uma das questões de viabilidade pode ser relacionado à forma como as células são processadas e sobre o que o tempo. Idealmente, todo o procedimento de isolamento de células, os passos 1-4, deve ser realizada sem atrasos entre as etapas e concluída no mesmo dia. Qualquer atraso significativo entre os passos 1-4 irá reduzir significativamente a viabilidade celular. Uma segunda questão relacionada com a viabilidade das células podem estar relacionados à pressão bainha utilizado para o isolamento FACS. Recomendamos a utilização de uma menor pressão bainha. Por último, uma vez que as células são isoladas, elas devem ser imediatamente banhado, como qualquer armazenamento significativo no gelo após a triagem também irá reduzir sua viabilidade. CD133 + não-parenquimatosa heterogeneidade: Relatórios recentes indicam que as células estreladas hepáticas também podem ter CD133 expressão e ter alguma plasticidade. 25 Por isso, além de verificar a potência bi-genes com albumina e Krt19, verificação adicional pode incluir genes associados com as células estreladas, como a proteína fibrilar glial ácida em células estreladas quiescentes e alfa-actina de músculo liso e desmina em células estreladas ativadas. 3,4,26 Além disso, a população CD133 + representa uma população mais ampla progenitor. A adição de um segundo marcador, como EpCAM, pode ajudar a refinar ainda mais a população, e heterogeneidade limite.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> DMEM: F12 </td><td> Invitrogen </td><td> 10565-018 </td><td> Com vermelho de fenol </td></tr><tr><td> CD45 microesferas </td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td> Fator de Crescimento do hepatócito </td><td> Sigma </td><td> H1404 </td><td></td></tr><tr><td> Fator de Crescimento Epidérmico </td><td> Sigma </td><td> E4127 </td><td></td></tr><tr><td> DNase </td><td> Sigma </td><td> DN25 </td><td> 1 grama </td></tr><tr><td> Colagenase 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 micron filtro de malha </td><td> Pescador </td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50 reações </td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi coluna LD </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> Placas revestidas laminina </td><td> BD </td><td> 354410 </td><td> 96 poços </td></tr><tr><td> Tripsina EDTA 0,05% </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 colunas de 5x10 5 células ou menos </td></tr><tr><td> Pharm Lyse </td><td> BD </td><td> 555899 </td><td> Concentração 10X </td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

Células-tronco do fígado, ou células ovais, proliferam durante a lesão hepática crônica, e são propostas para se diferenciar em ambos os hepatócitos e cholangiocytes. Além disso, as células-tronco do fígado são a hipótese de ser os precursores de um subconjunto de câncer de fígado, carcinoma hepatocelular. Um dos principais desafios para conter trabalho em células de qualquer órgão sólido como o fígado é o isolamento de uma população rara de células para análise detalhada. Por exemplo, a grande maioria das células do fígado são hepatócitos (fração parenquimatosa), que são significativamente maiores do que os não-parenquimatosa células. Através do enriquecimento das compartimentos específicos celulares do fígado (ou seja, parenquimatosas e não parenquimatosas frações), e selecionando para CD45 células negativas, somos capazes de enriquecer a população a partir de células-tronco por mais de 600 fold.The proceduresdetailed neste relatório permitem uma população relativamente raro de células de um órgão sólido a ser classificado de forma eficiente. Este processo pode ser utilizado para as células-tronco do fígado murino isolateliver normal, assim como modelos de lesão crônica no fígado, que demonstram um aumento da proliferação de células-tronco do fígado. Este método tem vantagens claras sobre imuno-histoquímica padrão de congelados ou formol fígado fixado como estudos funcionais utilizando células vivas podem ser realizadas após a primeira co-localização experimentos. Para realizar o procedimento descrito neste relatório, uma relação de trabalho com uma pesquisa do núcleo de citometria de fluxo baseado é fortemente encorajado, como os detalhes de isolamento FACS são altamente dependentes de instrumentos especializados e um forte conhecimento de básico citometria de fluxo dos procedimentos. O objetivo específico deste processo é isolar uma população de células-tronco do fígado que pode ser expandida clonal 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

Aqui nós descrevemos o isolamento de células do fígado CD133 expressando-tronco e células-tronco do câncer de fígado murino todo, um processo que requer a digestão dos tecidos, o enriquecimento de células eo isolamento de citometria de fluxo. Nós incluem métodos para o isolamento de células avançado única e expansão clonal.

Isolamento de CD133 + células tronco do fígado de Expansão Clonal

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

22.2K visualizações.  Pennsylvania State College of Medicine. Aqui nós descrevemos o isolamento de células do fígado CD133 expressando-tronco e células-tronco do câncer de fígado murino todo, um processo que requer a digestão dos tecidos, o enriquecimento de células eo isolamento de citometria de fluxo. Nós incluem métodos para o isolamento de células avançado única e expansão clonal.

Vídeo: 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 Transplante de Células-Tronco Hematopoéticas (HSCs)

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 expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

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 animais de Expansão de soro livre de cordão umbilical humano Derivado células mesenquimais estromais (MSCs) e endoteliais Células Progenitoras Colony Forming (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.

Isolamento e derivação de células embrionárias de rato 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 de Células-Tronco do rato das Glândulas Salivares

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 Cultura de miofibras individuais e suas células satélites do músculo esquelético

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 de culturas primárias de fibroblastos humanos

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.

Indução e Análise de epitelial para mesenquimal Transição

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 transplante de células musculares satélite

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.

Protocolo para o isolamento de hepatócitos humanos primários e Correspondente principais populações de células do fígado não-parenquimatosas

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