IPSC

Induced Pluripotent Stem Cells … or what?

• embryonic stem cells – where would these be obtained? • naturally occurring stem cells – such as? • cord blood • nuclear transplantation • other? such as? IPSCs • origin: somatic cells – not stem cells • Treated with variety of agents 

STEM CELLS!

• first produced in 2006 from mouse cells • & first from cells in 2007 • Transfection of certain - HOW? associated genes • into non-pluripotent cells, e.g. fibroblasts • Using retrovirus • & reporter genes (~ selectable markers idea)

• Potential problems?

http://biol1020-2012-1.blogspot.com/2012/05/fountain-of-youth-induced-pluripotent.html Transfection

biol1020-2012-1.blogspot.com/2012/05/fountain-of-youth-induced-pluripotent.html What Genes Are Transfected? • Oct-3/4 • c- is an oncogene! • • ~ 20% chimeric embryos • c-myc developed cancer • • So now there is another way, • + reporter – 1st reporter didn’t work that takes longer and is less well efficient – but no c-myc gene is – Now use a gene that is added to the IPSCs required and easy to spot HOW? In ? How does it differ? • OCT4 (similar to mouse) • SOX2(similar to mouse) Pretty much • NANOG(similar to mouse) the same ! • LIN28 a different gene cells are cells • lentivirus vector A scheme of the generation of induced pluripotent stern (iPS) cells.

1. Isolate and culture donor cells. 2. Transfect stern cell-associated genes into the cells by viral vectors. Red cells indicate the cells expressing the exogenous genes. 3. Harvest and culture the cells according to ES cell culture, using mitotically inactivated feeder cells (lightgray). 4. A small subset of the transfected cells become iPS cells and generate ES-like colonies.

http://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell How do we get around c-myc?

• What is c-myc? A factor – That’s how it causes cancer – it is involved in regulating cell cycle starting and stopping

• Mimicking transcription factors with small compounds • Ultimately, the goal is to discover a cocktail of reprogramming factors and compounds that efficiently and reliably reprogram somatic cells to iPS cells. And find …. • Alternate vectors • Drug-Like chemicals, recombinant proteins • RNA molecules to regulate specific genes’ activity • Other regulators – based on learning the fundamentals of controlling – Cell cycle – differentiation Genes of induction

• SOX genes • Klf family • Myc family • protein NANOG • LIN28 Sox family of genes • associated with maintaining pluripotency – similar to Oct-3/4 • but associated with multipotent and unipotent stem cells – (in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells) • while Sox2 was the initial gene used for induction … • now other genes in the Sox family have been found to work as well in the induction process • Sox1 yields iPS cells with a similar efficiency as Sox2, • Sox3, Sox15, and Sox18 also generate iPS cells, although with decreased efficiency. OCT family of octamer transcription factors • Oct-3/4 (Pou5f1) plays a crucial role in maintaining pluripotency. – The absence of Oct-3/4 in Oct-3/4+ cells, such as blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation – The presence of Oct-3/4 gives rise to the pluripotency and differentiation potential of embryonic stem cells • Oct1 & Oct6 fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process Klf family

• Klf4: originally identified as necessary for the generation of mouse and human iPS • More recent reports: Klf4 may be unnecessary for generation of human iPS cells – in fact failed to generate human iPS cells in some labs • Klf2 and Klf4 were found to be factors capable of generating iPS cells, and related genes Klf1 and Klf5 did as well, although with reduced efficiency

• So – still up in the air? The Myc family of genes proto-oncogenes

• implicated in cancer • Originally used for generating mouse & human iPS cells • Other labs found work-around • Use of myc family of genes in induction of iPS cells is troubling for clinical therapies – 20-25% of mice transplanted with c-myc-induced iPS cells developed lethal teratomas. – N-myc and L-myc have been identified to induce iPS instead of c-myc with similar efficiency. Nanog

• Nanog along with Oct-3/4 and Sox2 in embryonic stem cells is necessary in promoting pluripotency • But Nanog was unnecessary for induction of mouse iPS (this was a surprise) • In other labs it is possible to generate iPS cells with Nanog as one of the factors LIN28: mRNA binding protein

• LIN28 is expressed in embryonic stem cells • and embryonic carcinoma cells • associated with differentiation and proliferation • Can be used to generate iSPCs, but is probably unnecessary Source of Cells

• Recipient's own somatic cells – Activating adult bone marrow stem cells • Donor bank? – Cord blood registry • Other future possibilities? – Building body parts – 3-D – Using a printer? links • Overview • Cellular reprogramming • Activating adult bone marrow stem cells • Cord blood registry • Building body parts – 3-D • Using a printer? • Reprogramming • Designing genes