Stem Cells from Skin Cells?!? The story of four little genes and a HUGE cellular change

Talk Outline Fibroblasts and Stem Cells Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

Fibroblasts

Fibroblasts Are fully differentiated cells Can not become any other cell type Can only divide to make more fibroblasts Contact inhibition

Stem cells

Stem Cells Can both make more of itself and create other, differentiated cells Totipotent Stem Cells can create everything needed to make a baby Pluripotent Stem cells can make only the cells of the baby Only Adult Stem Cells (multipotent) in your body Unipotent Cells can only make more of itself

Stem Cells

Talk Outline Fibroblasts and Stem Cells Before iPS Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

Before iPS Embryonic Stem Cells-good source of pluripotent cells, but unethical Somatic cell nuclear transfer-still requires oocytes

SCNT The basic concept is that the oocyte reprograms the DNA to be “embryonic stem cell-like” Very low efficiency No human stem cell lines have been made from SCNT Hwan Woo-Suk’s fake data Not fully reprogramed

Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

If the goal is to get stem cells from normal cells, what would you need to add?

Retroviruses Randomly inserts DNA into genome of cells Can make special retroviruses with whatever gene you want Can’t really control how many copies of genes

Different lines expressed different amounts of Klf4

Drug Selection Only turn on a drug resistance gene when stem cell state Do this by using a gene that is only expressed in stem cells Add drug resistance to promoter region of that gene Takes around 16 days for resistance gene to be expressed- some secondary change

Drug Selection

So They Picked 24 Genes

Four Magic Genes Sox2- Self Renewal Oct4- Differentiation switch Klf4- p53 pathway, Oncogene c-Myc- Global Histone Acetylation, Oncogene

Do you really need all 4? Without Oct 3/4 or Klf: no colonies Without Sox2: rough morphology Without c-Myc: flatter cells, now know actually can do without c-myc-just very low efficiency

No Sox2

Are they really stem cells?

Somewhere stuck in between

Teratoma formation

Pluripotent/Multipotent?

No baby mice! Tried to inject into blastocyst to make baby mice but failed Final and best test of pluripotency

The Next Step: 11 months later

Better iPS cells Still working with mouse model Used different drug selection marker Same 4 genes Much more closely resemble ES cells

Genes expressed in iPS group with ES cells not MEFs

Still Integration differences

Bisulfite Pyrosequencing Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected Introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues

ES cell-like Methylation

Gold Standard!

Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS Human iPS iPS used in treatment

4 months later

Technique Basically same technique as mouse Added the mouse retrovirus receptor to the human cells to increase transfection efficiency Used facial skin cells from a 36 year old female Takes 25 days for colonies to form

Gene expression profiles look like ES cells

And protein expression

DNA Methylation Profiles

Differentiates into all types of cells in culture

And in teratomas (injected into mice)

One month later Used Oct3/4, Sox2, Nanog and Lin28

Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment iPS used in treatment

Around the same time (Dec 2007)

Wow! Used the animal’s own cells- no immune rejection! Transfected with all four genes, but c-myc taken out after time- prevent tumors! Sickle Cell Anemia has known genetic basis-so target that gene and change it back to normal! Inject it back into the animal after radiation to reconstitute the whole blood system!

A Cure!

The Possibilities are Endless Any disease with a single genetic mutation could be easily cured! Tissue regeneration after accidents or diseases “Nanobots” Companies have already started testing iPS for therapy

But there are still obstacles No way FDA will approve a therapy with an oncogene Use of retroviruses can lead to mutations and cancers So many changes in the DNA can be harmful Probably hard to target to some areas