Stem Cells - Induced Pluripotent Stem cells (IPSc)

Pluripotent stem cells in the adult body?

no! Adult stem cells are multipotent (Mature cells can be reprogrammed to be pluripotent)

Gurdon's Discovery

Awarded nobel prize for - reprogram a somatic cell

Gurdon's Discovery - process and result

PROCESS:
Nuclear transfer
The nucleus of a differentiated cell (i.e. intestinal cell) is transferred to an egg-cell without nucleus...

RESULT
A modified cell, that enters normal embryonic development...
...to ultimately form an organism (Tadpole)

Yamanaka's Discovery

Generation of iPS cells

Yamanaka's Discovery - EXPERIMENT

Took 4 genes for stem cell function
Transferred them into mouse fibroblasts...
This created induced pluripotent stem cells (iPS cells)
These were reprogrammed into pluripotent stem cells...
...which could then develop into all cell types of an adult mouse

proccess of generating iPS cells (simple)

Transduction of 4 transcription factors pluripotency (Yamanaka factors) into somatic cells (fibroblast)

proccess of generating iPS cells (detailed)

1. Priming of differentiated cell (making it amendable for reprogramming)
2. downregulation of "somatic programm" + epigentic events: Dnmt3a and Dnmt3b are activated - De-methylation of silenced endogenous Oct4 and Nanog
3. Oct4 and Nanog can now express -> expression of pluripotency factors -> Induced pluripotent cell

Yamanaka factors - what

- pluripotency factors

Yamanaka factors - example

- Oct4, Sox2, Klf4, c-Myc

Primed cell can also become a partially programmed cell. how can you further manipulated a partially programmed cell to become iPS?

DNMT inhibitor
Forces demethylation - hope it demethylated the silenced the endogenous factors

do you need all 4 Yamanaka Factors?

To reprogram a somatic cell (fibroblast) -> a neural stem cell
just need one of the Yamanaka factors (Oct4)

ways of generating iPS cells (viral methods)

1. Via viral transduction
2. Via Retroviral or lentiviral transduction
3. Via Adenoviral transduction
4. Via Plasmid transfection

ways of generating iPS cells (non-viral methods)

Intergrative methods (DNA-BASED)
Non-integrative methods (DNA-BASED, RNA, Proteins)

best method of generating iPS cells?

Non-integrative RNA methods - has been used the most

What are the genetic and epigenetic changes during reprogramming from fibroblast (differentiated cell) to iPS cell?

Pluripotency genes = activated
(lineage-sepcific genes) differentiation genes = repressed/down-regulated
global methylation = decreased
chromatin = more open / relaxed

Waddington Landscape Model

what are the different Pluripotency Tests?

In vitro differentiation
Teratoma formation
Chimera formation
Germ-line contribution
Tetraploid complementation

Pluripotency Test - Chimera formation?

Inject cells into host blastocyst
See if cells contribute to normal development of a new organism

Pluripotency Test - In vitro differentiation?

Observe - Put cells into culture with conditions that would induce differentiation
Test them for expression of lineage-specific markers

Pluripotency Test - Teratoma formation?

Inject immunodeficient (SCID) mice with cells
See if tumours (Teratoma) are induced - demonstrates potential to generate differentiated cell types of various lineages

Pluripotency Test - Germ-line contribution?

See if reprogrammed cells can generate functional germ cells

Pluripotency Test - Tetraploid complementation?

Reprogrammed cells (2n) are injected into 4n host blastocyst
4n host cells cannot contribute to somatic lineages
SO
Embryo is exclusively composed of the reprogrammed cells