Download Stem Cells from Skin Cells?!?

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