How do cells know what to become?

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How do cells know what to become?

• All cells in a person have the same DNA
• Yet eye cells differ from nose cells
• Central dogma of biology

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Signals to Stem Cells
Little, et al. Chemical Reviews (2008).
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Factors known to affect stem cells

• Low stress levels
• Regular exercise
• Enriching experiences
• Learning new information
• Healthy diets rich in antioxidants
• Avoid excessive drinking

Helping you help yourself
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Bone marrow transplantExample of adult stem
cell-based therapy
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Induced Pluripotent Stem (iPS) CellsGenetically
engineering new stem cells
Skin cells
iPS cells
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Fibroblasts
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Fibroblasts

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

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

• Randomly inserts DNA into genome of cells
• Can make special retroviruses with whatever gene
  you want
• Cant really control how
• many copies of genes

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Different lines expressed different amounts of
Klf4
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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

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Drug Selection
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So They Picked 24 Genes
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Four Magic Genes

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

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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

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No Sox2
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Are they really stem cells?
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Somewhere stuck in between
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Teratoma formation
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Pluripotent/Multipotent?
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No baby mice!

• Tried to inject into blastocyst to make baby mice
  but failed
• Final and best test of pluripotency

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The Next Step 11 months later
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Better iPS cells

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

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Genes expressed in iPS group with ES cells not
MEFs
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Still Integration differences
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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

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ES cell-like Methylation
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Gold Standard!
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4 months later
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Gene expression profiles look like ES cells
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And protein expression
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DNA Methylation Profiles
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Differentiates into all types of cells in culture
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And in teratomas (injected into mice)
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One month later
Used Oct3/4, Sox2, Nanog and Lin28
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Around the same time(Dec 2007)
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Wow!

• Used the animals 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!

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A Cure!
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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

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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