Induction of functional hepatocyte like cells from mouse fibroblasts
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This study generated functional hepatocyte-like cells (iHep cells) from mouse fibroblasts by transducing them with three transcription factors - Gata4, Foxa3 and Hnf1a. The iHep cells exhibited epithelial morphology, expressed hepatic genes and markers, and displayed metabolic functions similar to hepatocytes. Transplanting iHep cells into a mouse model of liver failure partially rescued the mice and restored normal liver structure and function, without tumor formation. While not identical to hepatocytes, iHep cells represent a potential alternative cell source for modeling liver disease, transplantation and tissue engineering.
Induction of functional hepatocyte like cells from mouse fibroblasts
- 1. Inductionoffunctional hepatocyte-like cellsfrom mousefibroblasts bydefinedfactors Authors:PengyuHuang,ZhiyingHe,ShuyiJi,HuawangSun,DaoXiang,ChangchengLiu, YipingHu,XinWang&LijianHui1 Presented by Shubhda Roy 5/1/2014
- 2. Background • Generation of functional Hepatocyte great therapeutic interest to cure liver disease • Induced hepatic differentiation via - Embroynic Stem Cells - Induced Pluripotent stem cells - Patient’s induced Pluripotemt stems cells prevents immunological rejection • Very complicated process, which may be improved with technological advances • Overexpression of transcription factors induces terminally differentiated cells into other lineages
- 5. Hypothesis • Induced iHep Cells • Show epithelia morphology • Express Hepatic Gene • Acquire Hepatic functions • Repopulates livers of (Fah-/-) mice and restores liver functions, saves live of the mice • Does not form Tumors
- 7. Methods • 14 Mouse Transcription Factors • Important for Liver Development & Function • Transduced into Tail Tip Fibroblasts via Lentiviral Infection • iHep Cells expressed
- 8. Overcoming Constrains Wild Type TTF – in 7 days proliferation arrest, cell death p19arf hepatocytes grow without losing genetic stability P19arf -/-/ was used in culture to overcome growth arrest Cell showed epithelial morphology Cell expressed hepatic genes Albumin, Tdo2, Ttr Indicates Fibroblast ->converted into hepatocytes
- 9. DoesiHepcellsexpressHepaticGenes? AreFibroblasticGenesstillbeingexpressed? 11 epithelial Colonies After 21 Days, all expressed 14TF & Hepatic Genes Colony ET26 – RT PCR expressed Hepatic secretory genes – Alb, Ttr, Transferrin Epithelial cell adhesion genes- E-cadherin Cytokeratin genes – CK8, CK18 Endogenous hepatic transcription factors like Foxa2, foxa3, Hnf1s, Hnf4a, Gata4 Typical fibroblast genes not expressed Like col1a1, Pdgfrb, Postn and Fsp1
- 10. WhatkindofMorphologydoesET26show? DoesET26showGlycogendeposits? DoesET26showuptakeofDil–Labelledacetylatedlow-densityLipoprotein? ET26 shows Epithelial morphology ET26 shows glycogen storage with acid-Schiff (PAS) purple staining ET26 shows LDL presence with red staining
- 11. WhichgroupofTranscriptionfactors causedhepaticconversion? 6TF 8TF Hnf4a Hnf4a Hnf6 Hnf6 foxa3 foxa3 Hnf1a Hnf1a Gata4 Gata4 Foxa2 Foxa2 Foxa1 Hlf Either 6TF or 8TF causes the transformation? Removal of Hnf4a, Hnf4a from 6TF increased Epithelial colonies and Hepatic Gene expression Formed two groups of the remaining 4 in 6TF • 3TF had a stronger expression of hepatic genes than 3TF’ as in RT PCR • Removal of any of three factors decreased the colony growth as in graph
- 12. iHepcellsinducedbyoverexpressionof3TFfactorsshowed epithelialmorphology? iHep cells show typical Epithelial morphology Epithelial conversion of TTFs confirmed by immunofluorescence staining Tight Junctions in Tjp1 and E-cadherin - (red membrane staining) Nuclei are stained blue by DAPI
- 13. iHepcellsinducedbyoverexpressionof3TFfactors expressedhepaticgenes? Increased expression of hepatic genes With time shows progressively enhanced Reprogramming
- 14. iHepcellsinducedbyoverexpressionof3TFfactors showedmetabolicactivatescharacteristics? • iHep metabolic activities measured by liquid chromatography-tandem mass spectrometry according to standard curve • iHep cells metabolized • Acetaminophen • Testosterone • Diclofenac • iHep shows secretion of high amounts of Alb into medium measured by Eliza
- 15. DidTTFsgetconvertedtolineagesotherthanhepatocytes? Bile Duct Genes were not expressed in iHep Like Cftr, Aqp1, Ggt Pancreatic endocrine or exocrine cells not expressed in iHep Like Prss1, Cela2a and Amy2a5 Ins1, Ins2 and Glucagon Intestinal markers like CDX1, CDX2 were not expressed in iHep Induction of bile duct formation in a 3-dimention culture system Bile duct formed branches in LEPC iHep stopped growing TTFs are not converted to lineages other than hepatocytes
- 16. CaniHepcellspreventliverfailure? • Fah-/- mice defective in tyrosine metabolism require 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3- cyclohexanedione (NTBC) supply for survival • After NTBC withdrawal Fah-/- mice have liver failure and die • Mice can be rescued if transplanted with wild type primary hepatocytes • 10 Mice without iHep died in 6.5 weeks • 5 /12 Mice survived if transplanted with iHep, with increased body weight (not a 100%)
- 17. DoesiHeprestoredthedamanagedliver? • livers from NTBC-off Fah-/-Rag-/- control mice swelled with necrotic lesions • Hexagonal hepatic lobule were destructed due to massive cell death in NTBC-off • iHep-Fah-/-Rag-/- livers are normal and healthy • iHep cell repopulation restored liver architecture without cell death
- 18. Does iHep improve liver functions • iHep-Fah-/-Rag2-/- mice show reduced levels of • Tyrosine • Phenylalanine • Total bilirubin • Alanine aminotransferase (ALT) • Aspartate aminotransferase • iHep cell transplantation improves liver functions • By being able to breakdown these products
- 19. Does iHep cells cause Tumour ? • iHep and PLC/PRF/5 cells (human hepatoma cell line) transplanted into the left and right flanks of mice • 2months after transplantation • Ki67 staining revealed that iHep cells ceased proliferation 8 weeks after transplantation • iHep cells did not form tumors 8 weeks • 20 out of 25 analyzed iHep cells displayed 40 chromosomes after 17 passages • comparable with results from wild-type cells • iHep cells do not seem to be tumor prone.
- 20. Summary Cell lineages converted by regulating the transcriptional network Combination of Gata4, Foxa3 and Hnf1a as sufficient to induce hepatic conversion iHep cells shows epithelial morphology and express genes and functions very close to a hepatocyte but not a 100% iHep cell transplantation shows rescue of the liver is partial iHep cells are not identical to hepatocytes iHep cells do not seem to be prone to tumor formation iHep cells represent an alternative source of hepatocytes for disease modelling, transplantation and tissue engineering Future studies to address human fibroblasts and other cell types could be successfully converted to functional iHep cells
Editor's Notes
- #11: P19AF-/- TTFs were conveted into cells with significane low density lipporotein