Induced Pluripotent Stem Cells (iPS) The story of four little genes and a HUGE cellular change

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

Stem cells

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 Not fully reprogramed

What is a stem cell? “A cell from which other types of cells can develop.” “An undifferentiated cell that divides to give rise to specialized cells.” “An undifferentiated active somatic cell that undergoes division and gives rise to other stem cells or to cells that differentiate to form specialized cells.” A cell that undergoes asymmetric cell division, giving rise to one stem cell and one partially differentiated cell

Types of stem 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

In case you missed it… Fertilized egg up to 8-cell stage Embryonic stem cells Adult stem cells (MULTIPOTENT)‏

2: Mouse iPS Techniques and theory Optimization

Cell 126, 663–676, August 25, 2006. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University.

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

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

Drug Selection Only turn on a drug resistance gene at 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

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 24

Do you really need all 4? Without Oct 3/4 or Klf: no colonies Without Sox2: a few colonies, rough surfaces 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 Whitehead Institute for Biomedical Research and Department of Biology,Massachusetts Institute of Technology,

Better iPS cells Still working with mouse model Used different drug selection marker Same 4 genes Much more closely resemble ES cells Oct4

Genes expressed in iPS group with ES cells, not MEFs

Bisulfite Pyrosequencing Sequencing-based analysis of DNA methylation that quantitatively measures multiple, consecutive CpG sites individually with high accuracy and reproducibility. use of bisulfite treatment of DNA to determine its pattern of methylation. Yields high-resolution information about the methylation status of a segment of DNA 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!

4. 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 Lin28 encodes a cytoplasmic mRNA-binding protein. Used Oct3/4, Sox2, Nanog and Lin28 Lin28 encodes a cytoplasmic mRNA-binding protein. Science 21 December 2007: Vol. 318. no. 5858, pp. 1917 - 1920

Around the same time (Dec 2007)

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