Pluripotent stem cells

• What are stem cells?

  Unspecialised cells that can turn into many other cell types
• How are stem cells functionally classified?

  Abiltity to...

  1. Self-renew - generate at least one daughter cell identical to mother cell
  2. Be clonal - have a single cell generate more stem cells and differentiated progeny
  3. Produce progeny with more restricted potential - able to differentiate
• Where can stem cells be found?

  • Fertilised egg
  • Embryos
  • Umbilical cord
  • Adult tissues and organs
  • Cancers
  • Bone marrow
  • Intestine
• What is the concern with iPSC-based therapies?

  Potential for teratoma formation (as iPSCs undergo an induced pluripotent stage) after being injected into tissues

  
  

  Transdifferentiation does not carry this risk - instead relies on ectopic expression of transcription factors and/or other stimuli
• What is the difference between unipotent, totipotent, pluripotent, and multipotent?

  • Unipotent - forms one type of cell, e.g. committed progenitor cells
  • Totipotent - forms all tissues required for reproduction, e.g. fertilised egg
  • Pluripotent - forms all the cells of an embryo and adult, e.g. ESCs
  • Multipotent - forms a limited number of cells types, e.g. HSCs, MSCs

  
• What are embryonic stem cells?

  Pluripotent cells which give rise to all somatic cell types in the body

  • Derived from the inner cell mass of a blastocyst (pre-implantation embryo)
  • Requires a balance of self-renewal and differentiation to maintain pluripotency
  • Generates the 3 major germ layers: ectoderm (external), mesoderm (middle), endoderm (internal)
• How can we replicate the phenomenon of pluripotent stem cells?

  Understanding of how pluripotency is maintained during ESC self-renewal, i.e. by

  1. Promotion of proliferation
  2. Suppression of differentiation - extrinsic signals (GFs and cytokines), signalling pathways, intrinsic factors (TFs)
• How are mouse embryonic stem cells (mESCs) cultured?

  • Original derivation required culture on fibroblast feeder layers, e.g. mouse embryonic fibroblasts (mEFs) - isolated ICM cells are layered on top
  • Feeders increase plating efficiency, help maintain pluripotency, and facilitate survival and growth of SCs - but also may introduce animal viruses and unwanted immunogens
  • Leukaemia inhibitory factor (LIF) is the main factor produced by feeders
  • Later found that LIF (with serum) can maintain mESCs in a pluripotent state without needing the feeders themselves
• What can LIF tell us about self-renewal maintenance?

  • Understanding the signalling pathways activated by LIF can reveal how self-renewal is maintained - no longer need LIF
  • Serum is used alongside LIF but is not reproducible batch-batch - so better not to use
• What other factors maintain mESC pluripotency?

  • Bone morphogenetic proteins 2 and 4 (BMP2 and 4) are members of TGFB family and normally in serum
  • BMP2 and 4 act via. TGFBR2 and activate Smad 4 and Id (inhibitor of differentiation) - promotes self-renewal
  • Thus, in the absence of serum (where LIF cannot soley maintain pluripotency in mESCs), BMP2 and 4 can replace serum - work with LIF to maintain pluripotency of mESCs

  
• Describe LIF signalling in mESCs

  • LIF binds to LIFR and GP130 heterodimer receptors
  • LIF phosphorylated
  • Through the Jak-STAT3 pathway, the c-myc oncogene/TF is activated
  • c-myc drives self-renewal
  • Can also activate MAPK pathway

  Looking downstream, activated forms of STAT3 can maintain pluripotency without LIF while dominant negative forms of STAT3 reduce self-renewal

  
• Describe Wnt signalling in mESCs

  • Wnt binds to Frizzled (receptor) and inhibits glycogen synthase kinase 3 (GSK3)
  • This stabilises B-catenin (unphosphorylated)
  • B-catenin accumulates in the nucleus
  • B-catenin relieves TCF-3 repressive effect on transcription/pluripotency and reinforces self-renewal

  
• What agent can be used that targets the Wnt signalling pathway to maintain pluripotency?

  Small molecule inhibitors of GSK-3 - don't need to add Wnt
• What are the states of pluripotency?

  Naive, primed, and ground states of pluripotency - derived from different stages of the early developing embryo

  1. Naive, e.g. mESCs - undifferentiated, resembling the blastocyst, ICM-like (pre-implantation embryo)
  2. Primed, e.g. hESCs - resemble a transitional between naive state and differentiation, primed to differentiate (post-implantation epiblast)
  3. Ground - stable naive state, maintained under conditions promoting 1) inhibition of signalling pathways promoting differentiation and 2) activation of signalling pathways promoting self-renewal/pluripotency
• Using knowledge of pluripotency to culture ground state ESCs

  Norml extrinsic factors which maintain pluripotency

  • LIF activates STAT3 and either serum or BMP induces Id - promotes self-renewal
  • Mitogen-activated protein kinase (ERK1/2) pathway by fibroblast growth factor-4 (FGF4) - promotes differentiation

  In specialised conditions...

  • 2i = dual inhibition of MEK and GSK-3
  • 3i = 2i + additional third inhibitor - of FGFR
  • LIF + FGFRi and MEKi - replaces requirement for BMP/serum and promotes pluripotency
  • GSK-3i further promotes growth and viability of cells
• What is the history of induced pluripotent stem cells (iPSCs)?

  • Dervied by Yamanaka et al. (2006)
  • Generated from mouse embryonic fibroblasts
• What are the Yamanaka factors?

  • Oct-4
  • Sox-2
  • c-myc
  • KIf-4

  These factors are necessary for the reprogramming to pluripotency. Other starter cell types may require different TFs
• What are the considerations for deriving iPSCs?

  1. Choice of transcription factor - dependent on choice of starter cell type
  2. Method of TF delivery - retrovirus, adenovirus, etc.
  3. Choice of starter cell type
  4. Parameters of factor expression - timings and levels
  5. Derivation conditons - e.g. how to select the colonies
  6. Identification of iPSC colonies - i.e. morphological, evidence of pluripotency (pluripotency markers, test differentiation potential, identification of teratomas etc.)
  7. Expansion and characterisation
• What are the applications of iPSCs?

  • Cell replacement therapies (regenerative medicine)
  • Patient and disease specific iPSCs
  • Use in drug discovery
  • Cellular agriculture

  Must ensure a stable phenotype of the iPSCs - otherwise cell reverts back (anaplastic)
• What are induced pluripotent stem cells?

  • Adult somatic cells that are reprogrammed via. inducing genes and transcripton factors to generate cell lines that have the capacity for self-renewal and pluripotency
  • Similar to ESCs
• How are human embryonic stem cells (hESCs) cultured?


  • Require feeder cell layer for best growth
  • Unlike mESCs, dissociate the cells into small clumps rather than completely single cells - prevent them from growing slowly
  • Not maintained in LIF - instead use FGF-2 and activin A
  • hESCs express Oct-4, Sox-2, and Nanog
• What happens when mESCs are cultured under hESC conditions?

  • Flat compact colonies
  • Cannot colonise the early mouse embryo
  • Undergo multi-lineage differentiation
  • More similar to hESC than conventional or naive mESCs
  • Considered "primed" epiblast cells - rather than naive ICM cells