lecture 3: pluripotency and iPS cells Flashcards Preview

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Flashcards in lecture 3: pluripotency and iPS cells Deck (18):
1

How do we define pluripotency?

  • functionally and molecularly

2

What is potency?

  • stem cells are categorised by potency, which denotes the potential of the cell to derive other cell types – how many and what cell types
  • potency: the range of developmental options available to a cell
  • totipotent: ability to form the entire organism (includes placenta/extra embryonic tissues). In a mammal only the zygote and the blastomeres are totipotent. Not demonstrated for any other mammalian stem cell type. 
  • pluripotent: ability to form all the lineages of the body. example: embryonic stem cells and Embryonic Germ (EG) cells 
    • cannot form placenta 
  • multipotent: ability to form multiple cell types from onelineage. e.g. haematopoietic stem cells which form all the blood type cells 
  • unipotent: ability to form one cell type. e.g. spermatogonia which can only form sperm 

3

What is the first test for pluripotent stem cells?

  • in vitro differentiation
  • take away culture conditions and see random differentiation 
  • differentiate spontaneoulsy in vitro into derivatives of the three germ layers: ectoderm, mesoderm, endoderm 
  • least stringent test
  • the expression for differentiation markers is not a test for functionality:
    • any changes in culture conditions can stress the cells or induce differentiation  

4

What is the second test for pluripotency?

  • teratoma formation 
  • take 200-400 cells, inject under skin, kidney, abdomen of SCID mouse (no functional immune system)
  • formation of teratomas when injected into immune-deficient mice
  • differentiate spontaneously in vivo into derivatives of the three germ layers; ectoderm, mesoderm, endoderm due to loss of pluripotency and exposure to signals in the new environment that induce differentiation 
  • does not test for the ability to promote normal development 
  • should we be using eggs instead of mice since it is a big imposition on the mouse?

5

What is the third test for pluripotency?

  • germline chimerism 
  • white mouse donates blastocyst
  • ES cells from black mouse 
  • inject ES cells from black mouse into blastocyst from white mouse 
  • inject into pseudopregnant mouse
  • generate chimera 
  • when injected into donor blastocyst, ES cells contribute to all tissues of the resulting offspring 
  • can test for germline competency 
  • does not test for complete pluripotency i.e. problems caused by epigenetic defects affecting development 
  • one of the most important tests for pluripotency 

6

What is the fourth test for pluripotent stem cells?

  • tetraploid complementation 
  • produced by injecting ES cells into a tetraploid (4n) blastocyst
  • the tetraploid embryo can only produce the placental tissues 
  • epiblast are from the cells that you injected and only from the cells that you injected 
  • most stringent test for pluripotency 
  • because 4n host cells cannot contribute to somatic lineages, embryo is exclusively composed of the cells formed from the injected cells 
  • can test for germline competenct
  • does not test for the ability for form trophoblast (placental) lineage

7

What are characteristics of mouse embryonic stem cell colonies?

  • maintain normal karyotype 
  • pluripotent ES cells express these stem cell markers: e.g. AP (alkaline phosphatase, highly expressed), SSEA (Stage Specific Embryonic Antigen 1, cell surface marker on ES cells), Oct4, Nanog and Sox 2
  • conventional ES culture requires: (mitotically inactivated) Fibroblast feeder layer and serum 
  • serum and feeder layer-free ES culture 
    • Feeder layer replaced by LIF (leukaemia inhibitory factors) - suppresses mesoderm and endoderm 
    • serum can be replaced by BMP (bone morphogenic protein) - suppresses neuroectoderm 

8

What are characteristics of human ES cells?

  • do not depend on LIF
  • grow as flat, epithelial colonies (mouse ES cells grow as non-epithelial, domed colonies) 
  • unlike mouse ES cells, cannot be passaged as single cells  
  • colonies must be mechanically broken into smaller pieces for passaging 
  • serum- and feeder-free culture requires Activin (activates Nodal signalling pathway) and FGF growth factors 
  • why are mouse and (currently used) human ES cells so different?

9

How do mouse and human ES cell characteristics compare?

  • both express Oct 4, Sox 2, nanog
  • human:
    • different surface markers (e.g. SSEA-4, hTRA-1 proteins) 
    • do not express LIF receptor, and gp130 
    • express some trophoblast markers (e.g. esomesodermin)
    • express some differentiation markers (Brachyury, Fgf5, AFP< keratin 14) 
    • difference in cell cycle, cell death pathways, cytokine gene expression
    • differences in signalling pathways
    • express vimentin
    • can form teratomas, but germline chimera not tested
    • require FGF and IGF both in vivo and in vitro
    • conclusions:
      • human culture conditions not optimal? or
      • human cells isolated at later stage of development?

10

Compare pluripotent ES cells and EpiSC

  • ES cells derived from embryonic day 3.5, 4.5 
  • EpiSC, 4.5, 5.5 
  • preimplantation epiblast derived ES cells - best stage for deriving pluripotent stem cells
  • post-implantation EpiSC are different - features
  • ES are rounded 
  • EpiSC from both post implantation and preimplantation - easier post

11

What is the difference between pluripotent pre-implantation and post-implantation epiblast stem cells? 

Pre vs Post

  • embryonic tissue: late blastocyst (epiblast) vs egg cylinder (epiblast)
  • cultured stem cell: ES cells vs Epi Stem Cells (EpiSC) 
  • chimeras: yes vs no
  • teratomas: yes vs yes 
  • pluripotency factors: oct4, nanog, sox 2, Klf2, vs oct4, nanog, sox2 
  • differentiation markers: absent vs Fgf5, Brachyury
  • response to LIF: self-renewal vs none
  • response to FgF/Erk: differentation vs self-renewal
  • response to 2i: self-renewal vs differentiation/apoptosis  

 

  • both cell lines are pluripotent: able to form cells of the three primary germ layers, express pluripotency factors but are clearly different 
  • 'primed epiblast' represents a more advanced stage of development than the 'Naive epiblast' 
    • thus naive pluripotency versus primed pluripotency 

12

What are the mouse post-implantation epiblast-derived stem cells (epiSC)?

  • derived from the epithelial, post-implantation epiblast and maintained under similar conditions to human ES cells (Activin & FGF2; not LIF-dependent) 
  • pluripotent, but not-germline-competent 
  • very similar to human ES cells in morphology and gene expression profile: hypothesised to be equivalent
  • thus current hES cells are more like mouse EpiSC than ES cells 

13

Why is studying mammalian development and understanding the molecular basis of pluripotency so important?

For transplantation therapies:

  • how cells form in the embryo
  • how to maintain SC and form differentiated cells
  • when and what type/stage of cells to transplant into patients 
  • because we don't understand there are always some stem cells that defy attempts to turn into a differentiated cell

for intellectual interest

14

How do stem cells divide to produce daughters with different fates?

  • stem cell divides to produce two daughter cells: one goes on to become terminally differentiated cell and one a stem cell (self renewal)
  • million dollar question: why is this cell unaffected?

15

What is the transcriptional regulatory circuitry in ES cells?

  • thought they by knowing the 353 genes controlled by nanog, sox 2 and oct 4 it would all work out and we would understand pluripotency 
  • if only it were this simple
  • this data suggests that Oct4, Sox2, and Nanog function together to regulate a significant proportion of their target genes in ES cells

16

What is a model of transcriptional regulatory circuitry in ES cells? 

  • all three factors regulate the others (and themselves) 
  • these factors are now known as: core pluripotency regulators 
  • Oct4, Sox2, and Nanog collaborate to form a regulatory circuitry in ES cells

17

What is the expanded transcriptional regulatory cicuitry in ES cells?

  • Activated:
    • factors that induce:
      • chromatin remodelling
      • histone acetylation
      • histone methylation
      • TGF-beta signalling
    • ES cell transcription factors
      • including oct4, Sox2, nanog
  • Repressed:
    • factors that induce:
      • neurogenesis
      • mesoderm
      • endoderm
      • extra-embryonic endoderm

18

What is the Bivalent chromatin state?

  • In ES cells, regulatory regions of differentiation genes, contain both activating and repressive histone modifications 
  • thus differentiation genes are both silenced and 'poised' for activation – allowing cell to respond to signals for differentiation or renewal easily 
  • so repression of differentiation genes is not permanent 
  • ES cells are in a constant state of flux 
  • In mouse (conventional) ES cell cultures:
    • all cells express Oct4 but only some express Nanog
    • Nanog levels fluctuate and these are correlated to ES cell state