Johns Hopkins Masters Project: Stem Cells

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

3. Analogy
Totipotent
Pluripotent
deprogramming
Freshman
Freshman

4. Hypothesis
http://stemcells.nih.gov/policy/statements/pages/SC_2008.aspx

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

6. Fibroblasts
http://stemcells.nih.gov/policy/statements/pages/SC_2008.aspx
http://www.youtube.com/watch?v=cPvidAvzmx0&app=desktop

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