Direct reprogramming Jana Bartoňková Lukáš Bálek Direct reprogramming = transdiferenciace Nobelova cena 2012 (S. Yamanaka) Sir John B. Gurdon Fibroblast Neuron ICM tisíce let Fibroblast Neuron hESC 1998 ICM tisíce let Fibroblast Neuron iPSC 2006 hESC 1998 ICM tisíce let Fibroblast Neuron iPSC 2006 hESC 1998 ICM tisíce let můžeme…?! náhrada/oprava buněk, tkání a orgánů model nemoci na buněčné úrovni Diferencovaná buňka Transdiferencovaná buňka in vitro Implantace Transdiferenciace in vivo iPSC Diferencovaná buňka Implantace Medicína IN VITRO IN SITU IN VITRO Jak na to? • transkripční faktory • epigenetické regulátory • miRNA • malé chemické molekuly Jak na to? transkripční faktory - hodiny po transfekci - stabilní i po odstranění stimulu Jak na to? epigenetické regulátory - Dnmt1 (Dhawan et al., 2011) - Baf60c (Takeuchi and Bruneau, 2009) - polycomb repressor complex 2 (PRC2) (Patel et al., 2012) Jak na to? miRNA - miR-9/9 and miR-124 (Yoo et al., 2011) - není tak efektivní jako TF Jak na to? malé chemické molekuly - především u iPSC - předmět výzkumu Huang P., Zhang L., Gao Y. et al. (2014). Cell Stem Cell 14, 370–384 HFF = human fetal limb fibroblast www.systembio.com Tbx5, Mef2c, Gata-4, Mesp1 Ieda, M., J. D. Fu, et al. (2010). "Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors." Cell 142(3): 375-86. Efe, J. A., S. Hilcove, et al. (2011). "Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy." Nat Cell Biol 13(3): 215-22. Oct4, Sox2, Klf4, C-Myc Zhou, Q., J. Brown, et al. (2008). "In vivo reprogramming of adult pancreatic exocrine cells to beta-cells." Nature 455(7213): 627-32. Ngn3, Pdx1, MafA, VP16 Szabo, E., S. Rampalli, et al. "Direct conversion of human fibroblasts to multilineage blood progenitors." Nature 468(7323): 521-6 Oct4, Gata1, Gata2, Gata3, Gata-4 Ivey, K. N. and D. Srivastava (2011). MicroRNAs as regulators of differentiation and cell fate decisions." Cell Stem Cell 7(1): 36-41. Mir-2861 IN VITRO Brn2, Ascl1, Mytl1, Zic1 Vierbuchen, T., et al. (2010). "Direct conversion of fibroblasts to functional neurons by defined factors." Nature 463(7284): 1035-41. Brn2, Ascl1, Mytl1, NeuroD1 Pang, Z. P., et al. (2011). "Induction of human neuronal cells by defined transcription factors." Nature 476(7359): 220-3. Mir-9, Mir-124, Ascl1, Mytl1 Yoo, A. S., et al. (2011). "MicroRNA-mediated conversion of human fibroblasts to neurons." Nature 476(7359): 228-31. Ascl1, Brn2, Mytl1, Lmx1a, FoxA2 Caiazzo, M.et al. (2011). "Direct generation of functional dopaminergic neurons from mouse and human fibroblasts." Nature 476(7359): 224-7. Mytl1, Brn2, Mir-124 Ambasudhan, R., M. Talantova, et al. (2011). "Direct Reprogramming of Adult Human Fibroblasts to Functional Neurons under Defined Conditions." Cell Stem Cell 9(2): 113-8. Kim, J., J. A. Efe, et al. (2011). "Direct reprogramming of mouse fibroblasts to neural progenitors." Proc Natl Acad Sci. 108(19): 7838-43. Oct4, Sox2, Klf4, C-Myc IN VITRO Pfisterer, U., A. Kirkeby, et al. (2011). "Direct conversion of human fibroblasts to dopaminergic neurons." Proc Natl Acad Sci.108(25):10343-8. Ascl1, Brn2, Mytl1, Foxa2, Lmx1a Son, E. Y., J. K. Ichida, et al. (2011). "Conversion of Mouse and Human Fibroblasts into Functional Spinal Motor Neurons." Cell Stem Cell. Lhx3, Ascl1, Brn2, Mytl1, Ngn2, Hb9, Isl1, NeuroD1 Huang, P., Z. He, et al. (2011). "Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors." Nature 475(7356): 386-9. HNF4-alpha, Foxa1, Foxa2, Foxa3 Sekiya, S. and A. Suzuki (2011). "Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors." Nature 475(7356): 390-3. Gata-4, HNF1-alpha, Foxa3 Foxa3, HNF1A, HNF4A Huang P., Zhang L., Gao Y. et al.(2014). Direct Reprogramming of Human Fibroblasts to Functional and Expandable Hepatocytes. Cell Stem Cell 14, 370–384 IN VITRO 2008 D. Melton: exokrinní buňky slinivky břišní > beta buňky (Ngn3, Pdx1 a Mafa) 2012 D. Srivastava: fibroblasty z jizvy srdce po infaktu > bijící kardiomyocyty (Gata4, Mef2c and Tbx5) 2014 Guo: gliové buňky > neurony (NeuroD1) 2015 Weider: satelitní gliové buňky > oligodendrocyte-like buňky (Sox10) IN SITU • udržení/expanze buněk • nízká efektivita • bezpečnost použití Překážky ve využítí http://www.eurostemcell.org/files/images/reprogramming_cells_for_dummies_small_0.img_assist_custom- 200x196.jpg