Cloning

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Lecture 8

• Cloning
• Stem cells

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problems with NT clones

• gt98 die for various random reasons
• 2 that survive often have immune or other
  dysfunctions frequently obese
• subtle/long term problems in reprogramming/lack
  of imprinting

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epigenetic reprogramming

• Major challenges to getting nuclei to readjust to
  new cytoplasmic environment
• heritable changes in chromatin structure
• heritable DNA modifications (methylation)
• programming occurs during development/differentiat
  ion and must be removed to get back to embryonic
  state

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Reprogramming of Oct4 gene

• Byrne et al 2003
• transfer mouse thymocyte nuclei into Xenopus
  oocytes, Oct4 gene activated after 4 days
• If Oct4 plasmid DNA injected, activation
  immediate
• is delay due to DNA modification or protein
  (chromatin)?
• if deproteinated thymocyte DNA injected,
  activated in 2 days
• conclusion?

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epigenetic reprogramming at Oct4

• Simonsson Gurdon 2004
• analyze methylation patterns at Oct4
• Show DNA demethylation after nuclear transfer, by
  as-yet unidentified DNA demethylase enzymes
• demethylation may be required to open up
  chromatin

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methylation in early development

• both maternal and paternal haploid DNAs are
  methylated
• early embryo has active demethylase
• only paternal DNA sensitive, gets demethylated
• in nuclear transfer from somatic cells, both
  genomes sensitive, so lose parental
  imprintscould be a big problem

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Summary nuclear programming

• Differentiated somatic cells have fixed gene
  expression patterns due to chromatin, methylation
  etc
• germline also has specific programming, but early
  embryo removes a lot of the marks
• cloning success depends on ability to reprogram
  nuclei--use embryonic stem cells.

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Interlude Stem cells

• What are stem cells?
• Why are they important?
• What is special about embryonic stem cells (ES
  cells, ESCs)

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What is a stem cell?

• Undifferentiated cell that is self renewing and
  can also give rise to differentiated cell types

Asymmetric Cell division
Aka transit amplifying Or TA populations
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Different ways of self renewal
Asymmetric Cell division
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Different ways of self renewal
Asymmetric Cell division
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Two very different kinds of stem cells

• somatic stem cells
• In adult tissues that undergo cell renewal
  (blood, skin, gut)
• May or may not be multipotent, pluripotent
• Embryonic stem (ES) cells
• Cultured cells derived from ICM of blastocyst
• totipotent
• Unclear if ICM normally has ES properties

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Hematopoietic stem cells

• All blood cells are derived from pluripotent
  hematopoietic stem cells
• Turnover of blood means stem cells active
  throughout life
• Accessible, mostly
• Section 9.6

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cell lineage of mammalian blood
erythroid
myeloid
lymphoid
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Bone marrow and the niche hypothesis

• Irradiation of bone marrow is lethal, can be
  rescued by transplant of bone marrow cells
• Bone marrow transplants populate spleen etc
• Spleen transplants do not regenerate all blood
  cell types
• Conclude something about bone marrow cellular
  environment is essential to maintain the
  hematopoietic stem cell population--the niche
  hypothesis (Schofield 1978)

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Where is the niche?
Stem cell niche regions easily found in model
organisms but harder in complex mammalian
tissues Location of HSC niche in bone marrow has
been especially elusive In 2003 located close to
spindle shaped N-cadherin positive osteoblasts
(SNOs) on inner surface of bone

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HSC niche as of 2005

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Other stem cells

• Found in any tissue with active proliferation
• Gut, skin, germline
• Pancreas, liver? unclear
• Brain? YES

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Intestinal stem cells
Close to base of Crypts of Langerhans 1 stem cell
per crypt? 300 new cells/day
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the niche in the crypt
Mesenchymal cells send signals BMPs inhibit ISC
self renewal BMP inhibitor noggin!
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neural stem cells
Subventricular zone Subgerminal zone Astrocytes
are the Stem cells (B)
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A stem cell needs a good niche

Fuchs et al Cell 2004
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Function of the niche

• Protects the stem cells
• Allows asymmetric cell division that releases
  differentiation (TA) daughter but retains stem
  cell
• Cell adhesion molecules glue the stem cells to
  their niche
• Cadherins
• etc

Drosophila spermatogonial stem Cells stick to a
hub cell via adherens junctions
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Adult stem cell plasticity?

• Can stem cells from one tissue make other
  tissues?
• Transdifferentiation again, except with stem
  cells?
• E.g. possible contribution of HSCs to muscle?
• Seems to be rare if it happens at all, which is
  controversial
• Wagers Weissmann review 2004

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Embryonic stem cells

• Nature. 1981 Jul 9292(5819) 154-6.
• Develop indefinitely in culture, normal karyotype

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How ES cells made

• Blastocyst stage embryos dissociated in culture
• ICM cells are Oct4, then expression declines
• After time some small fraction start to divide,
  turn Oct4 on (selection for proliferation)
• Now can divide indefinitely in culture, an ES
  cell line

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Human embryonic stem cells

• Federal funding only if using hESC lines derived
  pre August 2001.
• eligible cell lines
• http//stemcells.nih.gov/

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Cloning ES cells are totipotent

• Box 3C

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ES cells can make tumors if in wrong environment

• Fig 14.12
• Inner cell mass as a niche

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cloning via ES cells

• Recent successes in cloning from differentiated
  cells used a two-step procedure
• 1. NT from somatic cell into enucleated egg
• allow to develop to blastocyst
• then dissociate and culture cells in vitro to
  select for proliferating ES cells
• 2. inject these ES cells into host blastocyst,
  but allow to develop into injection chimera
• Development via ES cell stage seems to re-set
  the epigenetic marks of the somatic nucleus into
  an early embryonic state

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Stem cells summary

• Definition of stem cell
• Somatic vs Embryonic stem cells
• Niche hypothesis and role of niche
• Stem cell plasticity
• Use of ES cells in cloning

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Stem cells why the fuss?
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Are clones born old?

• Somatic cells senesce
• accumulation of mutations, despite repair
• shortening of telomeres
• Dollys telomeres started short and she died
  prematurely (2003, age 6)
• but other clones (cows, mice) show re-setting
  of telomeres in early embryo

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Other issues

• could mismatched nuclear and mitochondrial
  genomes cause problems? we dont know
• heterospecific transfers (Loi et al)
• mouflon (wild sheep) are endangered
• inject mouflon nuclei into domestic sheep egg
• same genus, different species
• a way to save endangered species?

The mouflon, Ovis orientalis musimon
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there are natural animal clones

• Parthenogenesis found in
• social insects (drone bees)
• whiptail lizards
• Bdelloid rotifers (60 million years without sex)
• monozygotic twins (share nuclei, mitochondria,
  cytoplasm--unlike NT clones)

Cnemidophorus uniparens the desert grassland
whiptail
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human reproductive cloning

• basic biology is likely to be similar --we are
  mammals
• but low success rates pose a problem
• USA no federally funded research
• UK, Canada restricted, but allowed
• Germany total ban

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Proposed outcomes (if it ever worked)

• Vanity cloning
• eternal life? but your clone would be even less
  similar than an identical twin
• Eugenics, Dysgenics
• clone Einstein, Hitler, armies
• Sentimental cloning
• clone dead child, parent, pet
• all the above rest on wrongheaded genetic
  determinism, i.e., that DNA determines identity.
• cloning is not photocopying
• Infertility treatment?
• there are easier ways...

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therapeutic cloning

• Goal is to make genetically matched ES cells
• Proof-of-concept in mice
• allows somatic (not germline) gene/cell therapy

Rideout WM et al. 2002. Correction of a genetic
defect by nuclear transplantation and combined
cell and gene therapy. Cell 109 17-27.
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therapeutic cloning

• goal is to make ES cells
• ethical worries are with use of blastocysts
• isnt there any other way to convert regular
  cells into ES cells?
• not yet
• adult stem cells very controversial, and not as
  pluripotent as ES

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therapeutic cloning questions

• what makes blastocysts special
• biologically?
• ethically? why are other cells not so
  privileged?
• they are potential individuals
• but any cell is

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The ethical debate

• Kant humans are an end in themselves, not a
  means
• Bentham the greatest good for the greatest
  number
• is a zygote an individual? totipotency means any
  cell is a potential individual!

Immanuel Kant
Jeremy Bentham