Induced Pluripotent Stem Cells (Lecture 4) Flashcards
Induced pluripotent stem cells (iPSCs)
- non-pluripotent cells engineered to become pluripotent
- a cell with a specialized function ‘reprogrammed’ to an unspecialized state
How is stemness maintained?
- expression of key stemness genes
- epigenetics (changes in gene function that occur independently of alterations to primary DNA sequence)
Epigenetics
heritable changes in gene function that occur independently of alterations to primary DNA sequence
Characterization of the pluripotent cell nucleus
- open/loose chromatin structure
- bivalent chromatin of developmental regulators (off, but ready to go)
- expression of Oct4, Nanog, and Sox2 to regulate expression of target genes
Why was reverting differentiated cells back to pluripotency thought to be unachievable?
- differentiated cells carry irreversible epigenetic modifications or genetic alterations that render induction of pluripotency impossible
- differentiated cells are remarkably stable and as a rule do not shift fate into other cell types
- the number of genes that it would require to induce pluripotency is likely too high to experimentally test
Three lines of research that led to discovery of iPSCs
- Antennapedia/MyoD master gene regulators
- – showed all cells have the intrinstic ability to become any other cell with the correct instruction
- SCNT
- – showed that adult cells have all the genetic material necessary to generate entire organisms and oocytes contain factors that can reprogram somatic cell nuclei
- Human ESCs
- – factors associated with ESC maintenance and cancer are candidates for reprogramming factors
Write down Yamanaka experimental approach and conclusions
Check with powerpoint
- 24 genes narrowed down to the 4
- Fbx15/B-geo/G418
- retroviruses
- hayflick limit
- bisulfate genomic sequencing
iPSCs and germ layers
- have ability to generate all three germ layers in teratomas and in vitro
- also in adult tail tip fibroblasts
iPSCs chimeras
- chimeric embryos were generated but not viable (three germ layer formation in embryos)
Significance of Yamanaka paper
- important step for creation of pluripotent stem cells directly from somatic cells of patients and possibly prevention of tissue rejection
- also overcoming ethical question of using embryos
Problems with Yamanaka paper
- very low yield
- – levels of 4 factors may have narrow ranges to generate iPSCs
- – generation of IPSCs may require additional chromosomal alternations, which take place spontaneously
- – many cells start process, few complete it
- assay based upon integration of DNA (avg. 20 per cell)
- – has been overcome with non-integrating factor
- viable chimeras were not generated from their approach –> is it truly like an ESC?
iPSCs in humans
Yamanaka and Thomson were both able to repeat experiment using human fibroblasts and were successful
Chimeras using nanog
- using nanog promoter instead of fbx15 generated adult chimeras and transmitted to the next generation
- nanog is better indicator of pluripotency that fbx15
Process of generating chimeras
- have transgenic mouse
- take transgenic mouse’s skin fibroblasts and insert retroviral transfection (oct3/4, sox2, klf4, c-myc)
- have antibiotic selection (cells that had drug resistance gene) and do growth in culture
- leads to iPSC line
- inject into a normal blastocyst
- place blastocyst in new host mother mouse
- leads to chimeric mouse
- chimeric mouse and normal mouse mate leading to a litter of mice where some will be iPS-derived
bisulfate sequencing using nanog
- reprogrammed oct3/4 (no longer methylated)
