IPSCs Flashcards
Introduction about the IPSCs
What’s the procedure and the rational of this technology
Idea of Yamanaka, 2006 and Nobel Prize in 2012
Scientific scenario in which he was working: SNT, cell fusion of Ian Wilmut, treatment with extracts of pluripotent stem cells, stable expression of defined factors
Fundamental passages that helped Yamanaka: Gurdon experiments, identification of mESC and hESC and paper of Weintraub, 1987 (ability of MyoD to activate muscle genes in a variety of differentiated cell lines)
How did Yamanaka work?
Selection of 24 candidate genes, divided in three classes
Model of MEF cells from Fbx15-Bgeo transgenic mice: explain the strategy
Retroviral transfection of the 24 genes one by one: no drug-resistant colonies
Introduction of all the 24 genes together: 22 drug-resistant colonies, only 5 with stem cell-like morphology
introduction of group of genes: identification of 10 genes whose individual removal from the transduction pool resulted in no colony formation
Selection of OCT3/4, KLF4, SOX2 and C-MYC
Controls of the experiments
-real time PCR
-global gene expression profile analysis using DNA microarrays + Pearson correlation analysis
-formation of embryoid bodies in vitro and capacity to differentiate into the three germ layers
-formation of teratomas in vivo
-creation of living animals inserting the IPSCs into a mouse blastocyst
Complications in the production of IPSCs
Reactivation of the retroviral vector of c-MYC that is a proto-oncogene > 20% of the living animals obtained developed tumours
Remove c-MYC: the reprogramming happens but with a lower efficiency
New methods to insert the genes: adenoviruses or cell permeable recombinant proteins
IPSCs applications
Nowadays: drug screening, patient-specific or disease-specific modeling, toxicological tests of new drugs, regenerative medicine
At the beginning of: cell therapy (spinal cord injury) and disease modeling and drug screening (neurodegenerative diseases, fulminant type I diabetes, diseases of the embryo Zika virus)
