Ce projet a été financé par le NIH / NCI numéro de subvention R00 CA127357 et l&#39;Université du Kansas cancer dotation Center.

<ol>
	<li>Hayward, S., Haughney, P. C., Rosen, M. A., Greulich, K. M., Weier, H. U., Dahiya, R., Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+adult+human+prostatic+epithelium+and+rat+urogenital+sinus+mesenchyme+in+a+tissue+recombination+model.">Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model.</a> Differentiation. 63, 131-131 (1998).</li><li>Cunha, G., Hom, Y. K., Young, P., Brody, J., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Ethier, S. P., Ammerman, C. A., Dziubinski, M. L., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Medina, D., Kittrell, F., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat Rev Cancer. 6, 392-401 (2006).</li><li>DeNardo, D. G., Johansson, M., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+as+mediators+of+solid+tumor+metastasis.">Immune cells as mediators of solid tumor metastasis.</a> Cancer Metastasis. 27, 11-18 (2008).</li><li>Naito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+differentiation+and+function+in+health+and+disease.">Macrophage differentiation and function in health and disease.</a> Pathol Int. 58, 143-155 (2008).</li><li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+cometh:+interplay+between+adaptive+immunity+and+cytokine+networks+in+rheumatoid+arthritis.">The T cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis.</a> J Clin Invest. 114, 471-474 (2004).</li><li>Cheng, N., Chytil, A., Shyr, Y., Joly, A., Moses, 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=Enhanced+Hepatocyte+Growth+Factor+Signaling+by+Type+II+Transforming+Growth+Factor-{beta}+Receptor+Knockout+Fibroblasts+Promotes+Mammary+Tumorigenesis.">Enhanced Hepatocyte Growth Factor Signaling by Type II Transforming Growth Factor-{beta} Receptor Knockout Fibroblasts Promotes Mammary Tumorigenesis.</a> Cancer Res. 67, 4869-4877 (2007).</li><li>Qiu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+expression+profiling+identifies+signatures+of+tumor+virulence+in+MMTV-PyMT-transgenic+mice:+correlation+to+human+disease.">Global expression profiling identifies signatures of tumor virulence in MMTV-PyMT-transgenic mice: correlation to human disease.</a> Cancer Res. 64, 5973-5981 (2004).</li><li>Schaffhausen, B. S., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+polyoma+middle+T+antigen+on+signaling+and+transformation:+A+DNA+tumor+virus+contribution+to+the+war+on+cancer.">Lessons from polyoma middle T antigen on signaling and transformation: A DNA tumor virus contribution to the war on cancer.</a> Virology. 384, 304-316 (2009).</li><li>Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+neural+cells+via+retrovirus-mediated+oncogene+transduction.">Immortalization of neural cells via retrovirus-mediated oncogene transduction.</a> Annu Rev Neurosci. 12, 47-65 (1989).</li><li>O'Hare, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+freshly+isolated+human+mammary+fibroblasts+and+endothelial+cells.">Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.</a> Proc Natl Acad Sci U S A. 98, 646-651 (2001).</li><li>Shay, J. W., Wright, W. E., Werbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+molecular+mechanisms+of+human+cell+immortalization.">Defining the molecular mechanisms of human cell immortalization.</a> Biochim Biophys Acta. 1072, 1-7 (1991).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+macrophage+cell+lines+transformed+by+Abelson+leukemia+virus.">Functional macrophage cell lines transformed by Abelson leukemia virus.</a> Cell. 15, 261-267 (1978).</li><li>Shen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+endothelial+cells+differentiated+from+mouse+embryonic+stem+cells.">Immortalization of endothelial cells differentiated from mouse embryonic stem cells.</a> Shi Yan Sheng Wu Xue Bao. 35, 218-228 (2002).</li><li>Wang, S. J., Greer, P., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+propagation+of+yolk-sac-derived+endothelial+cells+from+a+hypervascular+transgenic+mouse+expressing+a+gain-of-function+fps/fes+proto-oncogene.">Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene.</a> In Vitro Cell Dev Biol Anim. 32, 292-299 (1996).</li><li>Tiede, B. J., Owens, L. A., Li, F., DeCoste, C., Kang, Y. <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+mouse+model+for+non-invasive+single+marker+tracking+of+mammary+stem+cells+in+vivo+reveals+stem+cell+dynamics+throughout+pregnancy.">A novel mouse model for non-invasive single marker tracking of mammary stem cells in vivo reveals stem cell dynamics throughout pregnancy.</a> PLoS One. 4, e8035-e8035 (2009).</li><li>Guzman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracarotid+injection+of+fluorescence+activated+cell-sorted+CD49d-positive+neural+stem+cells+improves+targeted+cell+delivery+and+behavior+after+stroke+in+a+mouse+stroke+model.">Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.</a> Stroke. 39, 1300-1306 (2008).</li><li>Duda, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+transplantability+of+tumor-associated+stromal+cells.">Differential transplantability of tumor-associated stromal cells.</a> Cancer Res. 64, 5920-5924 (2004).</li></ol>

Les expérimentations animales: Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le comité IACUC à l&#39;Université du Kansas Medical Center.

La contribution fonctionnelle des fibroblastes dans la progression tumorale a été démontrée par des modèles de transplantation, dans lequel le carcinome fibroblastes associés co-transplantés avec bénignes mammaires des résultats des cellules épithéliales dans la croissance tumorale et l&#39;invasivité accrue 5. Approches de transplantation conventionnelle ont impliqué l&#39;utilisation de souris SCID ou nude à la co-transplantation de cellules stromales et épithéliales de différentes origines...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Souris C57BL/6N </td><td> Harlan </td><td> N / A </td><td></td></tr><tr><td> MMTV-PyVmT souris transgéniques </td><td> Laboratoires Jackson </td><td> 002374 </td><td></td></tr><tr><td> Sérum de veau fœtal </td><td> Fisher </td><td> SH3039603PR </td><td></td></tr><tr><td> DMEM </td><td> VWR </td><td> 10000113873 </td><td></td></tr><tr><td> Pénicilline / streptomycine </td><td> Fisher </td><td> MT-30-001 </td><td></td></tr><tr><td> amphotéricine </td><td> Fisher </td><td> BP2645-20 </td><td></td></tr><tr><td> Amicon de filtration colonnes Ultracel 50k </td><td> Millipore </td><td> UFC905008 </td><td></td></tr><tr><td> Tubes pour Beckman TI du rotor </td><td> Beckman </td><td> 355618 </td><td></td></tr><tr><td> Collagène de queue de rat </td><td> Fisher </td><td> CB 40236 </td><td></td></tr><tr><td> EBSS 10x </td><td> Sigma-Aldrich </td><td> E7510-100ML </td><td></td></tr><tr><td> Trypsine 1X, 0,25% dans HBSS w / o de calcium et de magnésium </td><td> Fisher </td><td> MT-25-050-CI </td><td></td></tr><tr><td> L&#39;acide acétique glacial </td><td> Fisher </td><td> A491-212 </td><td></td></tr><tr><td> Coomasie bleue </td><td> Fisher </td><td> BP101 25 </td><td></td></tr><tr><td> Trypsine </td><td> Sigma-Aldrich </td><td> T3924-100ml </td><td></td></tr><tr><td> Une collagénase </td><td> Sigma AldrichC0130-50 </td><td></td><td></td></tr><tr><td> hyalronidase </td><td> Sigma-Aldrich </td><td> H3884 </td><td></td></tr><tr><td> DNase </td><td> Sigma-Aldrich </td><td> D5025 </td><td></td></tr><tr><td> Standard de protéines Kaléidoscope </td><td> Biorad </td><td> 1610375 </td><td></td></tr><tr><td> Lames de verre </td><td> Fisher </td><td> 12545-78 </td><td></td></tr><tr><td> Verre lamelles </td><td> VWR </td><td> 101400-042 </td><td></td></tr><tr><td> Vimentine anticorps S-20 </td><td> Santa Cruz Biotechnology </td><td> SC-7558 </td><td></td></tr><tr><td> α-actine musculaire lisse d&#39;anticorps </td><td> Abcam </td><td> ab5694 </td><td></td></tr><tr><td> CK14 d&#39;anticorps </td><td> Santa Cruz Biotechnology </td><td> sc-53253 </td><td></td></tr><tr><td> CK18 d&#39;anticorps </td><td> Abcam </td><td> ab668 </td><td></td></tr><tr><td> DAPI </td><td> Sigma-Aldrich </td><td> D9542 </td><td></td></tr><tr><td> Anti-souris biotinylé </td><td> Laboratoires Vecteur </td><td> BA9200 </td><td> Distribués au travers de Fisher </td></tr><tr><td> Anti-souris-alexa-568 </td><td> Invitrogen </td><td> A10037 </td><td></td></tr><tr><td> Anti-souris-alexa-488 </td><td> Invitrogen </td><td> A11001 </td><td></td></tr><tr><td> Streptavidine alexa-488 </td><td> Invitrogen </td><td> S11226 </td><td></td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td><td></td></tr><tr><td> Prolongez Antifade </td><td> Invitrogen </td><td> P-36930 </td><td></td></tr><tr><td> Ciseaux chirurgicaux </td><td> Outils Fine Science </td><td> 91400-12 </td><td></td></tr><tr><td> Ciseaux beau printemps </td><td> Outils Fine Science </td><td> 15000-02 </td><td></td></tr><tr><td> Blunt pince </td><td> Outils Fine Science </td><td> 11002-12 </td><td></td></tr><tr><td> # 5 pinces fines </td><td> Outils Fine Science </td><td> 11251-10 </td><td></td></tr><tr><td> De suture chromique Gut </td><td> Fisher </td><td> NC9326254 </td><td></td></tr><tr><td> Pasteur en verre pipette </td><td> Fisher </td><td> 22-042-815 </td><td></td></tr><tr><td> Éthanol </td><td> Fisher </td><td> A406P 4 </td><td></td></tr><tr><td> bétadine </td><td> Fisher </td><td> NC9386574 </td><td></td></tr><tr><td> Clips des plaies </td><td> Fisher </td><td> 12032-07 </td><td></td></tr><tr><td> Base des plaies </td><td> Fisher </td><td> 12031-07 </td><td></td></tr></tbody></table>

mammary transplantation of stromal cells and carcinoma cells in c57bl/6j mice

L&#39;influence de cellules stromales, y compris les fibroblastes sur la progression de la tumeur mammaire a été bien documenté par l&#39;utilisation de modèles de souris, en particulier grâce à la transplantation de cellules stromales et les cellules épithéliales de la glande mammaire de souris. Modèles de transplantation actuels impliquent souvent l&#39;utilisation de la souris immunodéprimées en raison de la génétique de différentes origines cellules stromales et les cellules épithéliales. Matrices extracellulaires sont souvent utilisés pour intégrer les deux types cellulaires différents pour cohérentes interactions cellule-cellule, mais impliquent l&#39;utilisation de Matrigel ou de collagène de queue de rat, qui sont des substrats immunogène. Le manque de cellules T fonctionnelles des souris immunodéprimées empêche une évaluation précise des cellules stromales sur la progression de la tumeur mammaire in vivo, avec des implications importantes sur le développement de médicaments et d&#39;efficacité. Par ailleurs, les souris immunodéprimées sont coûteux, difficiles à reproduire et exigent des conditions de soins spéciaux. Pour surmonter ces obstacles, nous avons développé une approche de la transplantation orthotopique de cellules stromales et les cellules épithéliales des souris de la même bagage génétique pour induire la formation de tumeurs cohérente. Ce système implique la récolte normale, le carcinome fibroblastes associés, PyVmT cellules de carcinome mammaire et du collagène de souris C57BL/6J donateurs. Les cellules sont ensuite intégrés dans le collagène et transplantées dans les glandes mammaires inguinales des souris C57BL/6J femelles. La transplantation de cellules PyVmT seuls forment des tumeurs palpables greffe 30-40 jours après. Analyse Endpoint moins 60 jours indique que la co-transplantation avec des fibroblastes améliore la croissance des tumeurs mammaires par rapport aux cellules transplantées PyVmT seul. Alors que les cellules et la matrice de souris C57BL/6J ont été utilisées dans ces études, l&#39;isolement de cellules et la matrice et l&#39;approche de transplantation peuvent être appliquées sur des souris de différentes origines génétiques démontrant la polyvalence. En résumé, ce système peut être utilisé pour étudier les interactions moléculaires entre les cellules stromales et les cellules épithéliales, et surmonte les limitations critiques dans les modèles murins immunodéprimés.

Watch this Scientific Journal Video about Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice at JoVE.com

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Dans ce rapport, nous démontrons un système pour isoler les cellules du donneur et de la culture de la glande mammaire de la souris, et la transplantation orthotopique ces cellules dans des souris bénéficiaires d&#39;analyser stromales: interactions épithéliales pendant le développement de tumeurs mammaires.

Transplantation mammaires de cellules stromales et les cellules de carcinome chez les souris C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

mammary-transplantation-stromal-cells-carcinoma-cells-c57bl6j

Research

JoVE Journal

Medicine

17.5K vues.  University of Kansas Medical Center. Dans ce rapport, nous démontrons un système pour isoler les cellules du donneur et de la culture de la glande mammaire de la souris, et la transplantation orthotopique ces cellules dans des souris bénéficiaires d'analyser stromales: interactions épithéliales pendant le développement de tumeurs mammaires.

Vidéo: Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Génération expérimentale de carcinome Associated fibroblastes (CAF) à partir des fibroblastes mammaires

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Recherche

Médecine

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Human artère mammaire interne (IMA) à la transplantation et stenting: un modèle humain pour étudier le développement de la resténose intra-stent

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Génération de sous-cutanée et intra-hépatique droits de carcinome hépatocellulaire xénogreffes chez la souris immunodéficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Système de co-culture avec un cerveau organotypique et 3D sphéroïde de cellules de carcinome

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injection Orthotopique des cellules du cancer du sein dans le Fat Pad mammaire de souris pour étudier la croissance de la tumeur.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Evaluation des propriétés de cellules souches en cellules humaines de l&#39;ovaire Carcinome utilisant MULTI et simples à base de cellules Sphères dosages

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotopic col de l&#39;utérus chez la souris Transplantation cardiaque

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...