Meritxell Huch - Wellcome Trust/Cancer Research UK Gurdon Institute, Universidad de Cambridge, Cambridge, Reino Unido.
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El jueves 8 de febrero de 2018 se realizó en la Fundación Ramón Areces un Ciclo de Conferencias sobre células madre y organoides, en colaboración con Springer Nature.
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Meritxell Huch - Wellcome Trust/Cancer Research UK Gurdon Institute, Universidad de Cambridge, Cambridge, Reino Unido.
- 1. Meritxell Huch, Sir Henry Dale Fellow and Beit Prize Fellow Gurdon Institute, University of Cambridge Fundacion Ramon Areces/Springer Nature. Madrid 2018 Adult liver and pancreas organoids: present and future of their biomedical utility
- 2. Huch and Koo, Development, 2015 Generation of functional tissue ex-vivo: Pluripotent Stem Cell ESC, iPSC
- 3. Generation of functional tissue ex-vivo: Adult tissue cells/ stem / progenitors Barker & Huch et al., Cell Stem Cell 2010 Karthous et al., Cell 2014 Lee et al., Cell 2014 Hisha et al., Sci Rep 2013 Huch et al., Cell 2015 Sato et al., Nature 2009 Huch et al., Nature 2013 Huch et al., EMBOJ 2013 Boj et al. Cell 2015 Dontu et al., G&D 2003
- 4. 70-85% of the liver's mass Functions Detoxification, Digestion (bile production), Storage (glycogen, aa…), Blood glucose regulation… Hepatocytes Cholangiocytes Drain Bile Liver anatomy Endothelial Mesenchymal Resident Macrophages Cholangiocyte Hepatocyte
- 5. Mouse liver cells generate self-sustaining liver organoids in vitro HGF/FGF/Wnt/nic Huch et al.., Nature 2013
- 6. BME2 ERgFHNicTiFsK human liver culture Human liver organoids expand long term in culture
- 7. Human liver organoids express ductal and progenitor markers Krt19 Hoechst Lgr5 EphB2 EpCAM STEM CELL DUCTAL Huch et al., Cell, 2015
- 8. Liver organoids express HEPATOCYTE genes upon DM ZO1 Alb Hoecsht Huch et al.., Cell 2015
- 9. Human liver organoid cultures differentiate into functional hepatocyte-like cells in vitro EM DMDM B LDL-uptaken storage
- 10. Can we model human liver diseases using organoid culture technology? √ A1AT Deficiency (rare monogenic disease) √ Liver cancer (2nd most common worldwide)
- 11. Can we model Primary Liver Cancer using patient-derived organoids? Main subtypes of Primary Liver Cancer (PLC) Hepatocellularcarcinoma (HCC) Cholangiocarcinoma (CC) Mixed subtype (CHC) Tumour organoids that retain the patient and subtype specific features? ?
- 12. Organoid H&EBrightfield Tissue 50 um 50 um50 um 50 um 100 um 100 um100 um 100 um 50 um 50 um50 um 50 um * Type II: HCC/CCHealthy Type I: m/w HCC Type III: CC Human Liver Tumouroids EXPAND long term in vitro while retaining the histological architecture of the tumour sub-type Broutier et al., Nature Medicine 2017
- 13. Liver tumoroids expression profile correlates with the profile of the patient’s tumour-of-origin HCC CC CC-1_O CC-2_O CC-3_O CHC-1_O CHC-2_O HCC-1_O HCC-3_O -1 0 1 CLIC3 ALDOA S100A6 S100A11 KRT19 EPCAM FGA FGG CYP2D6 APOE APOA1 TTR ALB GPC3 AFP PROM1 CC-1_T CC-2_T CC-3_T CHC-1_T CHC-2_T HCC-1_T HCC-3_T FETAL HCC/hepatocyte PROGENITOR CC/ductal
- 14. H C C -1H C C -3C H C -1C H C -2C C -1C C -2C C -3 0 20 40 60 80 100 Concordant Tissue specific Organoid specific Cancer-relatedvariants(%) ~84% of the cancer-related somatic variants present in the patient’s original tissue were retained in the corresponding tumouroid cultures. Tumoroids recapitulate the genetic alterations present in the original tumour specimen
- 15. Do expanded tumor-organoids resemble the original tissue ? Patient organoid Donor organoid ?Tumor? (sc)
- 16. CC- derived organoids resemble the original tissue upon transplantation CC-derived tumoroids Patient organoid Donor organoid ?Tumor? (KidCap) CC-tissue
- 17. What can we use these for? √biomarker identification √drug sensitivities
- 18. PLC-organoids as platforms for personalized drug testing? Dr Mathew Garnett Dr Hayley Francies PLC patients PLC-Organoids Drug screening
- 19. Liver tumouroids are a valuable resource for drug screening and allow the identification of novel drug sensitivities HCC-1 HCC-3 CHC-1 CHC-2 CC-1 CC-2 Pathway Drug Name Target Cell cycle/ DNA damage and repair Cisplatin DNA crosslinker Olaparib PARP1, 2 KU-55933 ATM 5-Fluorouracil antimetabolite Doxorubicin DNA replication Gemcitibine DNA replication RTKi Axitinib PDGFR… PD-173074 FGFR1, 3 Sorafenib PDGFR.. AZD8931 ERBB1, 2, 3 Lapatinib ERBB2, EGFR CH5424802 ALK EMD-1214063 MET MAPK, PI3K, AKT, mTOR Trametinib MEK1/2 Dabrafenib BRAF SCH772984 ERK1/2 Deltarasin KRAS MK-2206 AKT1, 2 Taselisib PI3K OSI-027 mTORC1/2 Other Vorinostat HDAC BIRB 0796 p38, JNK2 Nutlin-3a MDM2 PD-0332991 CDK4, 6 LGK974 PORCN LY2109761 TGFB1 GSK126 EZH2 BIBR-1532 TERT Dasatanib ABL… sensitive resistant
- 20. SCH772984 / ERKi Liver tumouroids are a valuable resource for drug screening and allow the identification of novel drug sensitivities
- 21. Liver tumouroid lines are a valuable resource for drug screening and allow the identification of novel drug sensitivities
- 22. Pancreas ?
- 23. Why focus on the pancreas? The pancreas is an exocrine and endocrine tissue. Complications lead to debilitating diseases including Diabetes and Pancreatic cancer. Endocrine cells secrete hormones (e.g. insulin) that control blood sugar levels. Exocrine cells produce enzymes used to breakdown food.
- 24. Pancreas ducts grow into pancreas organoids with ductal phenotype Huch & Bonfanti & Boj et al., EMBO J 2013 DUCTAL
- 25. Pancreas organoids differentiate towards mono-hormonal endocrine cells in vivo INSULIN INSULIN +GFP
- 26. HUMAN Pancreas organoids derived from human pancreas biopsies expand long term in culture Passage 13 (130 days in culture) Prior et al., Unpublished ? Old medium V1 medium V2 medium
- 27. Summary Long term expanded in vitro Model Primary Liver Cancer PERSONALIZED MEDICINE?Healthy/ Tumor biopsy
- 28. Collaborators Acknowledgements • Mikel McKie • Luigi Aloia • Gianmarco Mastrogiovani • Olga Sidorova • E Cuppen and R. Van Boxtel (Hubrecht) • Kourosh Saeb Parsy, Nikitas G (Addenbrokes hospital, Cambridge) • Dr Susan Davies (Pathologist, Addenbrokes) • L. vd Laan, Monique Verstegen (Erasmus MC) • Steve Wigmore (Edinburgh) Huch lab • Laura Broutier • Lucia Cordero • Robert Arnes • Nicole Prior Funding
- 29. Pancreas Morphogenetic assay: Embryonic pancreas fully maturates into adult pancreas lineages Ductal cell INS + cell Acinar cell Paola Bonfanti / Harry Heimberg
- 30. Epcam HFN4aAlbunin CC-1_O CC-2_OHCC-3_OHCC-1_O CHC-1_O CHC-2_O DAPI Tumoroids retain the differentiation state of the original biopsy after long-term expansion in culture
- 31. Ductal cells undergo a partial de-differentiation in their transition to a proliferative/progenitor state DUCTAL MARKERS STEM CELL MARKERS PROLIFERATION MARKERS
- 32. Etiology: -acute or chronic liver damage -massive hepatic necrosis -hepatic fibrosis -hepatic cirrhosis Human liver disease is marked by a prominent ductular reaction Krt19
- 33. duct hepatocyte Adapted from Guldiken et al., 2016
- 34. Forbes lab (Lu et al Nat Cell Biol 2015) Mouse models with impaired hepatocyte regeneration show significant increase in ductular reaction
- 35. Methylcytosine is dynamically regulated during development
Hinweis der Redaktion
- As a final 3rd part of my I want to share with you the projects that are ongoing in the lab where we use this organoid technology to model human disease:
- Format: Abstract Send to Hepatology. 2013 Apr;57(4):1469-83. doi: 10.1002/hep.26159. Epub 2013 Mar 6. Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers. Oikawa T1, Kamiya A, Zeniya M, Chikada H, Hyuck AD, Yamazaki Y, Wauthier E, Tajiri H, Miller LD, Wang XW, Reid LM, Nakauchi H. Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4′s expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2). Conclusion: SALL4′s expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers. (HEPATOLOGY 2013)
- As a final 3rd part of my I want to share with you the projects that are ongoing in the lab where we use this organoid technology to model human disease:
- Exogenous insulin can induce hypoglycaemic crises and does not prevent the onset of acute and chronic complications of T1D. These complications impair the health and life expectancy of patients with T1D. Only advanced therapies, which restore a functional mass of β-cells will fundamentally change the way T1D is treated and will come close to the long-sought cure of T1D. The advanced therapy with hPOs has the potential to cure T1D and relieve patients from insulin injections. Pancreas islets transplantation restores a therapeutic pool of functional β-cells. The transplanted islets take over the precise physiological control of blood glucose levels in a manner that is not achievable with insulin injections. Thus, T1D patients transplanted with pancreas islets are relieved from injecting insulin. This fine-tuned glucose control allows T1D patients to develop a healthier life. In addition to improving their quality of life, the long-term complications of T1D are also expected to be diminished, thus significantly reducing morbidity and mortality. Pancreas islet transplantations have shown 1) that cellular therapy of T1D is feasible and 2) that cellular therapy of T1D is beneficial for the patient and the healthcare system. However, the transplantation of human islets suffers from the lack of suitable donors, surgical complications and the rejection of transplanted islets, which in turn requires continuous immunosuppression. Since it is currently not possible to culture and expand pancreas islets in vitro, only fresh islets are used for transplantation. Stem cells are an alternative source of functional islets. Several sources are available at present, from hESC/iPSC to adult endogenous pancreas cells. The use of human embryonic stem cells (hESCs) presents serious ethical and regulatory issues. Induced pluripotent stem cells (iPSCs) can be utilized. iPSCs do not raise the ethical concerns of hESCs, since they are reprogrammed from adult cells. However, iPSCs raise safety concerns, since iPSCs reprogramming induces genetic and epigenetic changes to the cells. Thus, iPSC-derived β-cells potentially lead to tumour (teratoma) formation once transplanted in the patient. Adult endogenous stem cells (progenitor cells), from which the pancreas organoids are generated, do not pose ethical dilemmas, as they are harvested from adult tissues. Endogenous stem cells are also safer
- Now if we isolate this ductal structures, normal cells and put them in culture we can generate similar organoids that are mainly single leyers epithelium of ductal like cells. And similarly to the liver, we can expand these in culture for many months splitiung 1:5-dilution.
- We generated organoids from single Lgr5 cells expandem in vitro and transplanted into the mouse, and then we aske dthe question whether if our organoid cells can differentiate into proper hepatocytes in vivo, we should be able to detect FAH + cells in the FAH neg background of the host. 3 months after transplantation we detected positive FAH clones in the liver parenquima of the FAH KO mouse, indicating that single lgr5 cells expanded in vitro can generate functional hepatocytes also in vivo after transplantation. To answer the question wether these were or not functional hepatocytes we lloked at the survial of themice and we saw that indeed, When we compare the survival of the mice that received cells and we could detec grafts (brown line) with the mice that received cells but we could not detect graft, blue line we observe a significant increase in the survivial of the mice. indicates, this cells can make functional hepatocytes in vivo upon transplantation,
- As a final 3rd part of my I want to share with you the projects that are ongoing in the lab where we use this organoid technology to model human disease:
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