Samenvatting Introduction to Stemcells

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Samenvatting - Introduction to Stemcells

  • 1.1 From embryo to embryonic stem cells (Roelen)

  • Zygote
    A fertilized diploid cell of an embryo. 
    It is a totipotent stem cell.
  • Totipotent
    The ability of a single cell to produce all of the differentiated cells in an organism.
  • Oocyte (egg cell)
    Large immotile cell.
  • Zona pellucida
    The layer of cumulus cells that contact the oocyte.
  • Sperm cell
    Very motile, small cell.
  • Cleavage
    First divisions of the fertilized egg.
  • Cleavage 
    The cells remain totipotent at first – all cells can still give rise to a complete, new embryo. Here, a cell can be removed for genetic testing (removal causes no damage).
  • Morula
    First differentiation -> formation of inside and outside cells.
  • Morula
    Hippo signaling is involved in trophectoderm formation.
    • Inner cells: Yap protein is phosphorylated and destroyed. Oct4 leads to formation of the inner cell mass (ICM)
    • Outer cells: Yap is a transcription factor for Cdx2, which leads to formation of trophectoderm (TE) 
  • Blastocyst
    A structure formed in the early development of mammals. It possesses an inner cell mass (ICM) which subsequently forms the embryo. The outer layer consists of cells called the trophoblast. 
  • ICM
    The ICM is pluripotent – it can form the complete fetus (ectoderm, mesoderm, endoderm, germ cells), but not extra tissues like the placenta and yolk sack.
  • Pluripotent
    Ability to form all the cells of an individual, so without the extra-embryonic tissue such as placenta.
  • Formation of blastocyst
    ICM forms into:
    1. Epiblast (under influence of Nanog) 
    2. Primitive endoderm/hypoblast (under influence of Gata6) 
      • In humans the hypoblast forms the yolk sack.
  • Embryonic stem cells (ESC)
    Pluripotent cells derived from the ICM of the blastocyst.
  • Testing pluripotency of ESCs
    By chimaera formation. Inject cells in the blast stage of the embryo. Check if cells form all germ layers to eventually call them pluripotent.
  • Two mechanisms of epigenetics:
    • DNA methylation
    • Histone modification  
  • Implantation of an embryo: 
    In humans, the embryo migrates in five days to the uterus, where it “hatches” (zona pellucida is lost) and implants (very invasively) into the womb. In other mammalians, the implantation is much less invasive.
  • Placenta
    Disc-shaped organ in mammalians, the connection between mother and fetus. Forms between the 2nd and 4th week in humans.
  • Important note:
    There is a huge structural difference between embryos from mammalian species. Info cannot be translated 1:1 to humans. Depending on what you study, you choose an appropriate animal model.
  • Nuclear reprogramming
    Changes in gene activity that are induced experimentally by introducing nuclei into a new cytoplasmic environment.
  • Nuclear reprogramming
    Epigenetic info of the two mature cells is restored when the oocyte and spermatozoid come together.
    1. The nucleus of a fully differentiated somatic cell is extracted from one individual
    2. This nucleus is injected into an oocyte (of which the DNA is removed) from another individual. 
    3. Activation with an electrical pulse leads to a genetically identical clone from the somatic cell.

    So, a differentiated cell is reprogrammed to become a totipotent stem cell
  • Induced pluripotent stem cells (iPSC)
    Oct4, Sox9, Klf4, cMyc are used to reprogram a differentiated cell into a pluripotent stem cell. The iPSC can form cells from the ectoderm, endoderm, and mesoderm.
  • Which tissues can a pluripotent stem cells differentiate towards?
    Endoderm, mesoderm, germ cells (NO yolk sack and placenta).
  • Trophectoderm
    The first epithelium to appear during mammalian embryogenesis. A polarized transporting single-cell layer that comprises the wall of the blastocyst.
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Laatst toegevoegde flashcards

Tetraploid complementation
A technique in which cells of two mammalian embryos are combined to form a new embryo. It is used to construct genetically modified organisms, to study the consequences of certain mutations on embryonal development, and in the study of pluripotent stem cells.
Gene editing of EPS cells
High gene targeting efficiency, high chimeric ability from edited cells and high success rate of tetraploid complementation (very fast process).
Why can EPS cells make extra-embryonic tissues?
There is an upregulation of a collection of genes that upregulate in embryonic cells early during preimplantation.
Can we generate single-mEPS-cell-derived postnatal chimeric mice (showing both embryonic and extraembryonic chimerism)?
Yes. Higher chimeric efficiency compared to 2i-ESCs. High germline transmission efficiency.
EPS cells
Extended pluripotent stem cells. Cells that can integrate both embryonic and extra-embryonic tissues (placenta and yolk sac).
Naive pluripotent state
Major advantage is that there is no differentiation bias, can be used for gene targeting, high blastocyst chimeric potency, high developmental potential
Primed pluripotent state
The most commonly used pluripotent SC -> genetic instability is a major safety issue here
Differentiation bias
There is a difference in tendency due to originating from different patients. Long-term culturing can change the genetics (instability).
Heritable chemical modifications of the genome that affect gene expression without alteration of the DNA sequence.
Association with non-coding RNAs
  • Binding of non-coding RNAs to the DNA blocks transcription
  • E.g. X-inactive specific transcript (XIST) and TSIX
    •  XIST and TSIX can neutralize each other