Lecture 3: iPS Cells
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Questions and Answers

What is a primary characteristic of iPS cells?

  • Their supply is difficult to scale.
  • They can differentiate into any cell type of the adult body. (correct)
  • They have a limited ability to self-renew.
  • They require donor human embryos for generation.

Which of the following factors is NOT one of the original four Yamanaka factors used for reprogramming cells?

  • c-MYC
  • KLF4
  • OCT3/4
  • GTF2I (correct)

What is a potential application of iPS cells in disease modeling?

  • Creating genetically modified animals
  • Generating embryonic stem cells
  • Screening for therapeutic compounds using differentiated cells (correct)
  • Repairing genetic mutations in embryos

What is the significance of patient-donor specific iPS cells?

<p>They alleviate concerns of immune rejection in therapies. (A)</p> Signup and view all the answers

Which statement correctly describes the transition between different cell states during development?

<p>The genetic information within cells is maintained during differentiation. (B)</p> Signup and view all the answers

What advantage do iPS cells have over traditional stem cell sources?

<p>They can be generated without ethical concerns regarding embryos. (D)</p> Signup and view all the answers

What is a common reprogramming method for converting differentiated cells into iPS cells?

<p>Viral transduction (B)</p> Signup and view all the answers

What primarily influences the differentiation ability of different iPSC lines?

<p>The genomic, epigenomic, and transcriptome differences between lines (A)</p> Signup and view all the answers

What is a defining characteristic of embryonic stem cells (ESCs)?

<p>They contribute to all tissues of the chimeric embryo and have naive pluripotency. (A)</p> Signup and view all the answers

How do epiblast-derived stem cells (EpiSCs) differ from embryonic stem cells (ESCs) in terms of pluripotency?

<p>EpiSCs exhibit a primed pluripotent state and have begun transitioning towards specific developmental pathways. (D)</p> Signup and view all the answers

Which statement about the differentiation responsiveness of EpiSCs is true?

<p>EpiSCs tend to differentiate spontaneously in response to a broad range of cues. (A)</p> Signup and view all the answers

What effect do epigenetic memories have on iPSCs?

<p>They bias the differentiation pathways based on the original cell type. (A)</p> Signup and view all the answers

What is the primary role of Oct-3/4 in the context of pluripotent stem cells?

<p>It promotes the expression of genes that maintain an undifferentiated state. (C)</p> Signup and view all the answers

Which histone modification is typically associated with active transcription?

<p>Me-H3(K4) (D)</p> Signup and view all the answers

During the early phase of reprogramming into iPS cells, which of the following events occurs?

<p>Transition from mesenchymal to epithelial states. (D)</p> Signup and view all the answers

What is the effect of DNA methylation in the context of gene expression?

<p>It is associated with long-term gene silencing. (B)</p> Signup and view all the answers

Which of the following factors is NOT associated with the maintenance of pluripotency?

<p>Me-H3(K9) (A)</p> Signup and view all the answers

What is a key change occurring during the late phase of reprogramming?

<p>Suppression of developmental genes. (D)</p> Signup and view all the answers

Which histone modification is associated with gene repression?

<p>Me-H3(K9) (C)</p> Signup and view all the answers

Which process must occur to ensure successful reprogramming of a somatic cell into an iPS cell?

<p>Completion of specific transitions and suppressions. (D)</p> Signup and view all the answers

In which types of organisms has reprogramming to iPS cells been shown to be universal?

<p>In mammals including mice, monkeys, and humans. (B)</p> Signup and view all the answers

What happens to Oct4 and Nanog during the transition from differentiated cells to ES or iPS cells?

<p>They are demethylated. (D)</p> Signup and view all the answers

What is the primary role of epigenetic modifications in gene expression?

<p>They regulate transcription factor binding and chromatin structure. (C)</p> Signup and view all the answers

How does DNA methylation affect gene expression?

<p>It decreases accessibility of DNA to transcription factors. (C)</p> Signup and view all the answers

Which factor is critical for maintaining pluripotency in stem cells?

<p>Oct3/4 transcription factor (D)</p> Signup and view all the answers

What is the process by which chromatin becomes more accessible for transcription called?

<p>Chromatin remodelling (D)</p> Signup and view all the answers

What is one of the primary functions of transcription factors in relation to the genome?

<p>To regulate the expression of specific genes. (B)</p> Signup and view all the answers

What characterizes the stochastic phase of reprogramming?

<p>Random and variable changes with uncertain outcomes (A)</p> Signup and view all the answers

Which of the following statements about histone modifications is true?

<p>Post-translational modifications can influence transcriptional access to genes. (C)</p> Signup and view all the answers

What is a defining feature of the deterministic phase in iPSC reprogramming?

<p>Predictable and orderly progression to a stable pluripotent state (D)</p> Signup and view all the answers

What characterizes epigenetic regulation of gene expression?

<p>It involves heritable changes in gene expression without altering the DNA sequence. (C)</p> Signup and view all the answers

Which method is considered the 'gold standard' for assessing iPSC pluripotency?

<p>Teratoma generation (A)</p> Signup and view all the answers

What role do transcription factors play in chromatin structural regulation?

<p>They facilitate chromatin remodelling to enhance transcription accessibility. (A)</p> Signup and view all the answers

What does the bivalent methylation of H3K4m33/H3K27me3 indicate during reprogramming?

<p>Regulatory control over pluripotency genes (B)</p> Signup and view all the answers

What does the phrase 'Genetics proposes, epigenetics disposes' imply?

<p>Epigenetic modifications can influence how genetic information is expressed. (B)</p> Signup and view all the answers

Which of the following is NOT typically assessed when determining iPSC pluripotency?

<p>Expression of lineage specific genes (D)</p> Signup and view all the answers

During reprogramming, what shift occurs alongside cellular proliferation?

<p>Changes in metabolic pathways (C)</p> Signup and view all the answers

What is a primary characteristic of the mesenchymal-epithelial transition in iPSCs?

<p>Acquisition of epithelial characteristics (A)</p> Signup and view all the answers

Which conclusion can be drawn from the generation of teratomas by iPSCs?

<p>iPSCs can generate tissues from all three germ layers (B)</p> Signup and view all the answers

In which phase of reprogramming do lineage specific genes undergo down-regulation?

<p>Stochastic phase (C)</p> Signup and view all the answers

What morphological change is associated with the progression to a stable pluripotent state?

<p>Flat, round morphology (A)</p> Signup and view all the answers

Flashcards

What are iPS cells?

Induced pluripotent stem cells are generated from adult cells and are capable of giving rise to various cell types in the body. This process of reprogramming adult cells back into a pluripotent state was pioneered by Shinya Yamanaka.

How are iPS cells utilized?

With iPS cells, researchers can create an unlimited supply of specific cell types without the need for embryonic tissues. This allows for the development of disease models, drug screening, and potential therapeutic applications.

What are the Yamanaka factors?

The four Yamanaka factors (OCT3/4, SOX2, KLF4, and c-MYC) are essential transcription factors that can reprogram differentiated cells back into a pluripotent state. These factors act like switches that turn on specific genes responsible for pluripotency.

How are iPS cells generated?

Reprogramming methods involve introducing the Yamanaka factors into adult cells using various techniques. These methods aim to efficiently and safely reprogram cells back to a pluripotent state.

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What is the role of epigenetics in cell reprogramming?

During development, cells transition from a totipotent state to a pluripotent state and then to more specialized states. This differentiation process involves epigenetic changes, which are alterations in gene expression without changing the underlying DNA sequence.

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How can iPS cells be used for drug discovery?

iPS cells can be used to create in vitro disease models, allowing researchers to study the disease process in detail and develop targeted drug therapies.

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How can iPS cells be used for gene therapy?

iPS cells can be genetically modified to repair faulty genes, potentially leading to the development of new therapies for genetic disorders.

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Cellular Differentiation

The process by which cells become specialized with distinct functions, involving selective activation or inactivation of specific genes.

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Epigenetics

Changes in gene expression without alterations in DNA sequence, including DNA methylation and histone modifications.

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DNA Methylation

A chemical modification of DNA, mainly by adding a methyl group (CH3), which can affect gene expression.

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Transcription Factors

Proteins that bind to DNA and regulate gene expression by promoting or inhibiting transcription.

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Chromatin Remodeling

A process that modifies the structure of chromatin, the complex of DNA and associated proteins, influencing gene accessibility.

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Histones

The fundamental protein units of chromatin, around which DNA wraps.

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Oct3/4

A transcription factor crucial for maintaining pluripotency, the ability of a cell to develop into any cell type.

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Pluripotent Cells

Cells with the ability to differentiate into any cell type in the body.

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Pluripotency

The ability of a cell to develop into any cell type in the body.

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What is Oct-3/4?

A transcription factor that promotes gene expression to maintain an undifferentiated, pluripotent state.

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What is Me-H3(K4)?

A histone modification associated with actively transcribed genes, indicating an open chromatin state.

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What is Me-H3(K9)?

A histone modification often associated with repressed genes and tightly packed chromatin.

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What is DNA methylation?

A key epigenetic modification associated with long-term gene silencing. It prevents transcription factor binding and compacts chromatin.

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What is epigenetic reprogramming?

The process of reversing epigenetic modifications to restore a cell to a pluripotent state, like an embryonic stem cell.

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How do the Yamanaka factors work?

During reprogramming, the four Yamanaka factors work together to remodel chromatin, leading to changes in gene expression.

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What happens in the early phase of reprogramming?

The early phase of reprogramming involves changes in gene expression, cell shape, and metabolism.

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What happens in the late phase of reprogramming?

The late phase of reprogramming involves activation of pluripotency genes, repression of tissue-specific genes, and further chromatin remodeling.

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What is the universality of iPS cell reprogramming?

Reprogramming into iPS cells is possible in a variety of species, suggesting a universal mechanism.

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Why are not all iPSCs equal?

iPSCs are generated from adult cells and can differentiate into different cell types, but they retain epigenetic memories of their origin which can bias their differentiation.

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What are ESCs?

ESCs are derived from the pre-implantation blastocyst and exhibit naive pluripotency, capable of forming both embryonic and extraembryonic tissues.

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What are EpiSCs?

EpiSCs are derived from the post-implantation epiblast and are primed for specific developmental pathways, with limited extraembryonic potential.

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What are the differences in lineage cue response between ESCs and EpiSCs?

ESCs require specific signals for differentiation, while EpiSCs respond to a broader range of cues and are more prone to spontaneous differentiation.

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How do differences in PSCs impact differentiation?

Differences in PSCs may explain why differentiation to certain cell types remains elusive.

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Stochastic Phase

A phase during iPSC reprogramming characterized by random and variable changes with uncertain outcomes. It involves down-regulation of lineage-specific genes and commencement of mesenchymal-epithelial transition. This phase is marked by uncertainty and a dynamic shift towards pluripotency.

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Deterministic Phase

A phase during iPSC reprogramming characterized by predictable and orderly progression towards a stable pluripotent state. This phase involves bivalent methylation of H3K4m33/H3K27me3 on promoter regions, sequential activation of pluripotency genes, and a flat, round cell morphology.

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Teratoma Formation

A method of assessing iPSC pluripotency that involves transplanting iPSCs into a host organism and observing their ability to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm). The resulting tumor is called a teratoma.

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Chimera Formation

A method of assessing iPSC pluripotency that involves injecting iPSCs into blastocysts (early-stage embryos). The iPSCs contribute to the development of the embryo and eventually give rise to different tissues in the chimeric animal.

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Reprogramming

The process of converting a differentiated cell (e.g., skin cell) back into a pluripotent state. This is achieved by introducing specific transcription factors, such as the Yamanaka factors, into the cell.

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Pluripotent State

The state of a cell that has not yet differentiated into a specialized cell. These cells are characterized by the ability to self-renew and differentiate into any cell type in the body.

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Study Notes

Lecture 3: iPS Cells

  • Induced pluripotent stem cells (iPS cells) are adult cells reprogrammed back into a pluripotent state.
  • iPS cells are exciting because they are self-renewing and can differentiate into any cell type in the adult body.
  • A scalable supply of iPS cells is possible, eliminating the need for donor human embryos.
  • iPS cells are relatively easy to generate from donor tissue in a lab.
  • Patient-specific iPS cells can be used for in vitro disease models.
  • iPS cells have potential in drug, anti-cancer discoveries, and clinical cell-based therapies.
  • iPS cells hold significant promise for the future.

Key Factors for Reprogramming

  • Four key factors, OCT3/4, SOX2, KLF4, and c-MYC (OSKM), can be used to reprogram adult fibroblasts into pluripotent cells.
  • These factors were identified in the experiments conducted by Yamanaka and colleagues.
  • Many other reprogramming methods have been attempted/tested.

Different Reprogramming Methods

  • Various methods exist for delivering reprogramming factors, each with unique efficiency and safety profiles.
  • These methods include retro/lentiviruses, lentiviruses, excisable lentiviruses, adenoviruses, RNA, transposons, episomal vectors, small molecules, and proteins.

Molecular Events Underlying Reprogramming

  • The process of cellular differentiation involves epigenetic modifications, such as DNA methylation and histone modifications, regulating gene expression.
  • These modifications control the access of transcription factors to DNA and rearrange chromatin structure which determines cell type characteristics and functions.
  • Oct3/4 is a regulatory transcription factor that is crucial to sustaining pluripotent cells.
  • Modifications like histone acetylation (Ac-H3) and DNA methylation are involved in regulating gene expression during reprogramming.

Assessing iPS Cell Pluripotency

  • iPSCs are assessed for pluripotency through various methods, including teratoma formation, transcriptome analysis, and chimera formation.
  • These methods assess a cell's potential to differentiate into various cell types.

Types of Pluripotent Stem Cells (PSCs)

  • Embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs) are two types of PSCs.
  • ESCs are derived from pre-implantation blastocysts.
  • EpiSCs are derived from the epiblast of the post-implantation embryo.
  • Differences in their behaviours relate to their different pluripotent states.

Methods for differentiating iPSCs

  • iPSCs can be differentiated into various cell types using different culture approaches, including 2-Dimensional and embryoid bodies.
  • Removal of pluripotency support (like LIF) allows for directed differentiation.
  • Cell-cell aggregation or random association of cells supports differentiation.
  • Culture approaches mimic conditions in the embryo.

Safety Considerations for Clinical Use

  • Careful assessment of the reprogramming process, identification of incompletely differentiated cells, and characterization of genomic and epigenetic changes during culture are essential in clinical translation.
  • Development of standard operating procedures (SOPs) for clinical-grade iPSCs is crucial for safety.

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Lecture 3: iPS Cells PDF

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This quiz covers the concept of induced pluripotent stem cells (iPS cells), their generation, and key factors involved in their reprogramming. It also explores various methods for generating iPS cells and their potential applications in medicine and research. Test your knowledge on iPS cells and their transformative role in biotechnology.

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