ANHB3323 Lecture Notes - Lecture 14: Embryoid Body, Insertional Mutagenesis, Myc
3 Jun 2018
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LECTURE FOURTEEN: Human Pluripotent Stem Cells
Uses for Induced Pluripotent Stem Cells (iPSC):
• Disease modeling
• Drug screening/discovery
• Cardiac/liber/neural toxicity tests
• Preclinical trials in a tube
• Transplantation
Yamanaka Factors:
• 4 transcription factors from 24 predominantely ES cell specific genes
• Sufficient to reprogram embryonic and adult mouse fibroblasts when
expressed retrovirally
• Factors include
o Oct4
o Sox2
o Klf4
o c-Myc
• 4 factor expression needs to be transient for successful iPSC generation
• Steps
o Transfection of OSKM factors
o Induction of expression of endogenous OSKM genes
o Silencing of the transfected OSKM genes
• Methods of reprogramming → retroviruses
Testing iPSC Clones for Pluripotency:
• Formation of dense colonies
• Chimaera development and germline transmission
• Expression of pluripotency genes/proteins
• Teratoma formation (3 germ layers)
• Embryoid body formation and differentiation
Possible Adverse Effects:
• c-Myc is an oncogene and ectopic expression can cause tumor formation
• Insertional mutagenesis (viral DNA)
• Genomic instability
• May cause induction of tumor formation
Reprogramming:
• iPSC generation requires MET
• Morphological changes
• Cytoskeletal reorganization and cell polarization
• Eputhelial markers expressed (E-cadherin)
• Endogenous pluripotency factors are not yet expressed
• Oct4, Sox2 and c-Myc collaborate to inhibit the TGFb pathway → suppress
mesenchymal phenotype and initiate MET
• Klf4 → activator of epithelial programme
• miRNAs regulate programming
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