Download What is a stem cell?

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
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•
•
•
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
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
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•
•
•
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
• 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
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