Concepts and Techniques in Cellular Reprogramming

DEVB3002/7002 Lecture Notes - Concepts and Techniques in Cellular Reprogramming

1. Introduction to Reprogramming

  • Reprogramming Process:

    • Fibroblast -> iPSC -> Lineage Stem Cell -> Terminal fate
    • Blood cell -> iPSC -> Lineage Stem Cell -> Terminal fate

  • Induced Pluripotent Stem Cells (iPSC):

    • ~35,000 citations since the original study published in 2006.
2. Key Components of Reprogramming
2.1. Understanding iPSC Generation

  • Fbx15 Gene:

    • Specifically expressed in ES cells and early embryos.
    • A β-gal-neomycin reporter construct was inserted into the Fbx15 locus to track expression.
    • Mouse embryonic fibroblasts (MEFs) derived from these mice exhibit neomycin resistance and β-gal expression.

  • Transcription Factors (TFs):

    • 24 transcription factors have been tested for their ability to activate Fbx15-βgeo and enable MEFs to grow under G418 selection, indicating pluripotency.
    • Tests using recombinant retroviruses (Moloney murine leukemia virus) showed that introducing all 24 factors allows MEFs to proliferate in G418 selection.
3. Key Transcription Factors Involved in Reprogramming
3.1. Yamanaka Factors
  • Key TFs:
    • KLF4: Zn-finger factor involved in controlling ES cell properties.
    • SOX2: HMG-factor family member influencing neural stem cell maintenance.
    • OCT3/4 (POU5F1): Crucial for maintaining pluripotency in ES cells.
    • c-MYC (c-Myc): bHLH factor associated with cell proliferation.
4. Stem Cell Differentiation
  • Differentiation Capability:
    • iPSCs can differentiate in vitro to all three germ layers and can form teratomas containing multiple cell types.
    • iPSCs are also capable of participating in embryogenesis, contributing to the formation of chimeric animals.
5. Technology Development in Reprogramming
5.1. TF Cocktail Optimization

  • Skipping Myc in Reprogramming:

    • Allows for reduced tumorigenesis during transplantation.
    • Myc was necessary for early robust growth in original study protocols, but could be omitted by changing the selection timeline.

  • Skipping Oct4:

    • Results in iPSCs that integrate into embryos more efficiently compared to traditional methods (OSKM)
    • Diminished expression of the retrovirus was observed when Oct4 was omitted from the reprogramming cocktail.
5.2. Growth Factor Treatments
  • Patterning Factors:
    • Complexity of signaling pathways can be navigated with the use of growth factors and small molecules which mimic embryonic development.
    • Development utilizes various factors like TGF-β, Wnt, and BMP to influence cell fate decisions.
5.3. DNA-Free Technologies for Introducing Reprogramming Factors
  • Usage of Sendai RNA virus for the delivery of transcription factors is emerging as a practical method for iPSC generation without the downsides of DNA integration.
6. Direct Reprogramming
  • Directing ESCs to Specific Lineage:
    • Lineage-specific cocktails can convert ESCs directly into terminally differentiated cells such as motor neurons.
    • Demonstrated effective integration into embryonic structures and function.
7. Overriding Waddington’s Landscape
  • Conceptual Framework:
    • Traditional developmental trajectories are challenged by the ability to reprogram or differentiate cells along various pathways (de- and trans-differentiation).
8. Summary of Key Learning Points
  1. Reprogramming Pathways: Fibroblast and blood cell routes to iPSC.
  2. Technologies: Emphasis on TF cocktails and patterns of growth factor applications.
  3. Integration-Free Methods: Use of Sendai RNA viruses highlighted for safer reprogramming.
  4. Direct Differentiation Strategies: Utilization of lineage-specific cocktails for targeted cell types.