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Stem Cell Biology and Reprogramming

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Examenjaar 2021-2022

1 examen
2de Semester1 item

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Stem Cell Biology and Reprogramming

Examen 2021-2022

Deel Björn Heindryckx:

  1. How are iPSCs and SCNT-ESCs generated? What are the key differences in their reprogramming mechanisms?
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  • iPSCs: Somatic cells transduced with defined transcription factors (Oct4, Sox2, Klf4, c-Myc); gradual, stochastic transcription factor-driven reprogramming over weeks.
  • SCNT-ESCs: Somatic cell nucleus transferred into an enucleated MII oocyte; rapid, maternal cytoplasmic factor-driven epigenetic reprogramming within hours.
  1. A 33-year-old woman has defective oocytes. How can you generate de novo oocytes?

Deel Jolanda van Hengel:

  1. Explain the CRISPRi (CRISPR interference) experiment presented in the lectures.
  2. A schematic figure is provided displaying iPSCs with arrows towards CRISPR, another culture dish, diseased cells, drug screening, and a mouse model:
    • a. Fill in the empty boxes and explain the workflow.
    • b. Which other pluripotent stem cells exist, and could they be used in this assay?
    • c. What are the disadvantages and limitations of using iPSCs?
  3. Spinal cord disease in a patient leads to the degeneration of nerve cells. What can be a clinical approach to help this patient? (Figure provided with numbers 1–6 to identify).

Deel Susana Chuva de Sousa Lopes:

  1. Explain genomic imprinting and the role of DNA methylation.
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Mondelinge vragen:

  • What is the name of the long non-coding RNA (lncRNA) responsible for X-chromosome inactivation?
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  • What is the difference between random and imprinted X-chromosome inactivation?
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  • Which histone modifications are involved in genomic imprinting? Give an example of a silencing and an activating histone modification.
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  • Silencing modification: $\text{H3K9me3}$ or $\text{H3K27me3}$.
  • Activating modification: $\text{H3K4me3}$ or $\text{H3/H4}$ acetylation.

Examenjaar 2020-2021

1 examen
2de Semester1 item

Examen Vragen

Geen datum

Stem Cell Biology and Reprogramming

Examen 2020-2021

Deel Jolanda van Hengel:

  1. Spinal cord disease in a patient leads to the degeneration of nerve cells. What can be a clinical approach to help this patient? (A figure was provided with numbers 1–6 indicating steps/cell types to be identified).
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  1. What is the biological function of transmembrane proteins?
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  1. What do you know about mechanobiology? How is this related to stem cell differentiation?
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  • Definition: An interdisciplinary field investigating how physical forces and changes in mechanical properties influence cell behavior, fate, and development.
  • Cellular effects: Mechanical cues regulate cell morphology, lineage specification, migration, organelle and nuclear remodeling, and stretch-activated ion channels.
  • Substrate stiffness:
    • In hESCs:
      • $< 0.1\text{ MPa}$ (soft) $\rightarrow$ ectoderm differentiation
      • $0.1 - 1\text{ MPa}$ (intermediate) $\rightarrow$ endoderm differentiation
      • $1.5 - 6\text{ MPa}$ (stiff) $\rightarrow$ mesoderm differentiation
      • $> 6\text{ MPa}$ $\rightarrow$ maintenance of undifferentiated state
    • In MSCs:
      • Soft matrix ($\sim 0.1 - 1\text{ kPa}$) $\rightarrow$ neurogenesis / adipogenesis
      • Stiff matrix ($\sim 30 - 40\text{ kPa}$) $\rightarrow$ osteogenesis
  • Micropatterned topography & cell shape:
    • High cytoskeletal tension (star-shaped, spread out) $\rightarrow$ osteogenesis
    • Low cytoskeletal tension (round, confined area) $\rightarrow$ adipogenesis
  • Dynamic mechanical forces:
    • Fluid shear stress: Endothelial differentiation.
    • Cyclic mechanical strain: Osteogenic differentiation.
    • Hydrostatic pressure: Chondrocyte differentiation.
    • Uniaxial stretch: Myogenic or tenogenic differentiation.

Deel Björn Heindryckx:

  1. How can you reset mESCs to the ground state? Compare the properties of mESCs and mouse epiblast stem cells (mEpiSCs).
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  • Resetting to the ground state is achieved by culture in 2i medium (supplemented with LIF):
    • PD0325901: MEK/ERK inhibitor (blocks differentiation signals).
    • CHIR99021: GSK3$\beta$ inhibitor (activates Wnt signaling to promote self-renewal).
  • Comparison:
    • mESCs: Naïve state, dome-shaped colonies, survive single-cell dissociation, high chimera-forming capacity.
    • mEpiSCs: Primed state, flat colonies, sensitive to single-cell passaging, do not integrate efficiently into blastocysts.
  1. A 33-year-old woman presents at the fertility clinic without functional oocytes. How can you clinically/experimentally generate de novo oocytes from stem cells?
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Deel Susana Chuva de Sousa Lopes:

  1. What is X-chromosome inactivation (XCI)? Discuss the differences between males and females.
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  1. What are bivalent chromatin domains? Give two examples of histone modifications involved.
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  • Bivalent domains are promoter regions of developmentally important genes that simultaneously carry activating and repressing epigenetic histone marks, keeping the gene silenced but primed for rapid activation upon differentiation.
  • Activating mark: $\text{H3K4me3}$ (histone H3 lysine 4 trimethylation).
  • Repressive mark: $\text{H3K27me3}$ (histone H3 lysine 27 trimethylation).

Mondeling Examen:

  • Jolanda van Hengel: Look at the figure provided and identify which mechanobiological mechanisms are displayed.
  • Björn Heindryckx:
    • What are interspecies chimeras? What are they used for and are they efficient?
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  • How can SCNT efficiency be improved?
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  • What is an artificial embryo?
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  • History of cloning: which cells were used first for nuclear transfer?
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