Lecture 7 – Induced Pluripotency Flashcards
Reprogramming Somatic Cells:
Concept: DNA in somatic adult cells is reprogrammable.
Applications: Cloning technology, regenerative biology, and genetic manipulation.
Example: Tracy the sheep (1990) showcased genetic manipulation for biotherapeutic purposes.
Ethical Considerations in Cloning:
Concern: Ethical issues arise with blastocyst disruption and human ES cell creation.
Milestone: In 2006, Takahashi and Yamanaka introduced an alternative approach with induced pluripotent stem cells (iPS cells).
Discovery of iPS Cells:
Method: Adult somatic cells reverted to ES-like state without cloning, using forced expression of 4 genes (c-Myc, Oct 4, Sox2, Klf4).
Alternative Source: iPS cells provided an ethical alternative for regenerative technologies.
Genes Involved in Reprogramming:
Key Genes: Oct4, Sox2, Nanog maintain pluripotency in ES cells.
Additional Genes: Genes expressed in tumors (Stat3, E-Ras, c-Myc) are essential for ES cell maintenance.
Hypothesis: Forced expression of these genes could induce pluripotency in somatic cells.
iPS Cell Generation Process:
Retroviral Lines: 24 retroviruses, each expressing candidate genes.
Selection Method: Neomycin resistance promoter used to select pluripotent cells.
Success: Forced expression of 10 genes initially, later narrowed down to 4 genes (Oct4, Sox2, Klf4, c-Myc).
Assessment of Pluripotency:
Teratoma Formation: iPS-MEF10/4 cells formed teratomas in nude mice, indicating stem cell tumor formation with differentiated cells.
Embryoid Bodies: iPS-MEF10/4 cells formed embryoid bodies in culture, similar to ES cells.
Chimeric Mice: Injection into blastocysts resulted in chimeric mice with a mixture of host and iPS-derived cells.
Significance of iPS Cells:
Application: Potential use in regenerative medicine, therapeutic applications without ethical concerns.
Challenges: Ensuring safety, understanding long-term effects, and improving efficiency for broader use.
Reprogramming with iPS Cells:
Source of Cells: iPS cells behaved like ES cells in experiments with skin cells from embryonic mice and adult mice.
Human iPS Cells: Created in 2007, opening new avenues for research and therapy.
Pluripotency Induction:
Gene Combinations: Several gene combinations have been identified as sufficient for inducing pluripotency.
Downstream Targets: C-myc has numerous downstream targets with widespread effects in the mammalian genome.
Key Transcription Factors:
Oct4 and Sox2: Core transcription factors maintaining pluripotency.
C-myc: Oncogene enhancing proliferation and associated with histone acetyltransferase complexes, possibly facilitating Oct4 and Sox2 binding.
Klf4: Represses p53, which represses Nanog, potentially contributing to pluripotency induction.
Tetraploid Cells and Pluripotency:
Placental Contribution: Tetraploid cells contribute to the placenta but not the embryo proper.
Milestone: Adult mice entirely derived from iPS cells demonstrated full ES-like pluripotency.
Human iPS Cells in Research and Therapy:
New Fields: Human iPS cells opened up research and therapeutic possibilities.
Ethical Approach: Ethically appropriate method—reprogramming patient skin cells to an ES-like state for various applications.
iPS Cells in Disease Treatment:
Sickle Cell Anemia Model: Treatment using iPS cells generated from autologous skin in a mouse model.
Procedure: Repairing IPS cells by knocking in a wild-type β-globin gene to replace the mutated allele.
Outcome: Successful correction of sickle cell anemia in treated mice.
Safety Concerns and Limitations:
Retrovirus Risks: Retroviral infection used in creating iPS cells raises mutation risks.
Oncogenic Activity: Use of c-Myc (oncogene) poses risks; knocking out c-Myc is recommended before therapeutic use in humans.
Challenges in Clinical Applications:
Risk Assessment: Consideration of potential mutations and oncogenic risks in retroviral-infected cells.
Alternative Strategies: Exploration of safer reprogramming methods for clinical applications.
Neural Differentiation of iPS Cells (2010):
Source of iPS Cells: Derived from human blood and skin fibroblasts.
Application: Transformed into neural stem cells and further differentiated into dopaminergic neurons.
Animal Model: Transplanted into 6-hydroxydopamine-lesioned rats (Parkinson’s disease model).
Outcome: Improved behavioral deficits in rats, suggesting potential therapeutic application in humans.
Comparison with Embryonic Stem (ES) Cells:
Advantages of iPS Cells: Potential similar to ES cells without ethical concerns of embryo destruction.
Safety Concerns: Tumor Formation—A major concern associated with iPS cells.
Study by Feng et al. (2010):
Cell Type Formed: iPS cells induced to form haemangioblasts.
Comparison: Compared with haemangioblasts derived from human ES cells.
Findings:
iPS cells less efficient at forming haemangioblasts.
Higher apoptosis rates in iPS cells.
Severely limited growth and expansion capacity compared to ES cells.
Less efficient in forming haematopoietic lineages.
Epigenetic Memory and Differentiation:
Induction of Pluripotency: Requires somatic cells to lose epigenetic memory.
Methylation Patterns: iPS cells retain some methylation patterns of the somatic cells they originated from.
Differentiation Bias: More prone to differentiate into the original somatic cell types than into new cell types.
Efficiency in Cell Type Formation:
Cell-Type Specificity: iPS cells show varying efficiency in forming different cell types.
Example: iPS cells from blood cells better at making blood cells; iPS cells from fibroblasts better at generating new fibroblasts.
Potential for Biomedical Research:
Nucleus Reprogramming: Nucleus of somatic cells can be reprogrammed to pluripotency through cloning or iPS technology.
Biomedical Potential: Offers immense potential for biomedical research and human therapy.
Ethical Considerations:
Ethical Issues with ES Cells: Considerable ethical problems associated with human ES cells.
Safety Verification: Need for extensive proof of safety associated with iPS cells for ethical acceptance and clinical applications.
