Epigenetics & Reprogramming

Introduction to Epigenetics and Regenerative Medicine

• Definition of Epigenetics:

  • Literally translates to "above" genetics.

  • It involves the development and maintenance of an organism orchestrated by chemical reactions that switch specific parts of the genome off and on at strategic times and locations.

  • Cold Spring Harbor (2008) Definition: An epigenetic genetic trait is a stably inheritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.

• Central Role in Biotechnology: Epigenetic modification is fundamental to the process of genome reprogramming, which is a cornerstone of regenerative medicine.

Mechanisms of Epigenetic Modulation

• DNA Methylation:

  • Mechanism: Involves the transfer of a methyl group ($CH_3$) to DNA.

  • Location: Usually occurs at the C5 position of cytosine to form $5\text{-methylcytosine}$.

  • Characteristics: Typically inhibits gene expression; these changes are largely erased and re-established between generations (transient nature).

• Histone Modification:

  • Definition: Refers to chemical modifications of core histones, specifically $H2A$, $H2B$, $H3$, and $H4$.

  • Residues Affected: Modifications target lysine, arginine, and other residues.

  • Types of Modification: Includes methylation, acetylation, phosphorylation, and ubiquitination.

  • Functional Impact: These modifications are catalyzed by specific enzymes and can alter the intensity of interaction between histones and DNA, leading to chromatin remodeling.

• Chromatin Remodeling:

  • Process: The rearrangement of chromatin from a condensed (silent) state to a transcriptionally accessible (open) state.

  • Causality: Often considered an output or result of other modifications, primarily histone modifications, which eventually switches transcription ON or OFF.

• Non-coding RNA:

  • Description: A cluster of RNAs that do not encode functional proteins.

  • Function: Originally considered post-transcriptional regulators; they act as scaffolds, guides, or decoys that interact with other epigenetic machineries to target specific sites.

External Influences on Epigenetic Expression

• Environmental and Lifestyle Factors:

  • Factors such as environmental exposures, stresses, diet, and lifestyle can induce epigenetic changes that determine gene activity.

• Multigenerational Impact Example:

  • A woman who smokes while pregnant induces epigenetic changes in three generations simultaneously: herself (Generation I), her unborn daughter (Generation II), and the reproductive cells of the unborn daughter (Generation III).

• Epigenetics and Obesity:

  • Exposure Sources: Maternal and paternal exposures lead to epigenetic changes in gametes.

  • In-utero Exposures: May cause changes in the fetus, though timing remains unclear.

  • Affected Pathways: Adipogenesis, the gut-brain axis, and fetal growth.

  • Markers: Changes are often demonstrated in umbilical cord blood, correlating with gene expression levels.

  • Persistence: Epigenetic changes may or may not persist into childhood; childhood exposures (diet, exercise, socioeconomic status) can introduce new changes or affect obesity risk.

Genetic Regulation via Epigenetic States

• Gene "Switched ON":

  • Characterized by active (open) chromatin.

  • Cytosines are unmethylated.

  • Histones are acetylated.

  • High access for RNA Polymerase II ($RNA\,pol\,II$).

• Gene "Switched OFF":

  • Characterized by silent (condensed) chromatin.

  • Cytosines are methylated ($5\text{-methylcytosine}$).

  • Histones are deacetylated.

  • Access for RNA Polymerase II is blocked.

The Epigenetic Landscape and Cellular Reprogramming

• Waddington’s Model of Epigenetics (1957):

  • Visualized as a ball rolling down a valley (landscape).

  • A pluripotent cell (top of the hill) differentiates into specialized cells (muscle, liver, leg, eye) as it moves down specific paths.

  • Transdifferentiation: The direct conversion of one differentiated cell type into another.

  • Reprogramming (Dedifferentiation): Forcing a differentiated cell back up the "landscape" to a pluripotent or progenitor state.

• Evolution of the Concept:

  • C.H. Waddington (1939-1942): Defined the "Epigenotype" as a network of organizing relations between genes and the environment.

  • D.L. Nanney (1958): Focused on heredity across cell division not based on template-replicating mechanisms (dual origin theory).

  • Contemporary Usage: Distinguishes between "Molecular Epigenetics" (mechanisms like DNA methylation and histone modification) and "Epigenetic State" (dynamical systems theory involving multi-stability and transition probabilities).

Induced Pluripotent Stem Cells (iPSCs)

• Discovery (Shinya Yamanaka, 2006/2009):

  • Proposed that most or all cells have the potential to become pluripotent.

  • The Stochastic Model: Reprogramming is viewed as an inefficient, stepwise process where cells must overcome an "epigenetic bump."

• Yamanaka’s Cocktail (Reprogramming Factors):

  • Core factors: $KLF4$, $SOX2$, $OCT\text{-}3/4$, and $c\text{-Myc}$.

  • Additional factors: $Nanog$ and $LIN\text{-}28$.

• Derivation and Delivery:

  • Source: Adult fibroblast cells or Peripheral Blood Mononuclear Cells (PBMCs).

  • Vectors: Retrovirus, Lentivirus, Adenovirus, Sendai virus, Episomal DNA, and mRNA/Protein-based methods.

  • Delivery Methods: Magnetofection, Nanoparticles, Liposomes, Electroporation, or Human Artificial Chromosomes.

• Potential for Differentiation:

  • iPSCs are pluripotent and can be directed to become: Cardiomyocytes, Adipocytes, Dopaminergic Neurons, Hematopoietic Progenitor Cells, Neural Cells, Pancreatic $\beta\text{-Cells}$, and Motoneurons.

Barriers and Challenges in Reprogramming

• Technical and Biological Hurdles:

  • Efficiency: The process is often incomplete or inefficient.

  • Stress and Senescence: Overexpression of factors can stimulate the $p53$ pathway, leading to apoptosis and reduced cell viability.

  • Partially Reprogrammed States: Many cells become trapped in a state where they self-renew but are not yet pluripotent; these cells may form tumors rather than teratomas.

  • Non-permissive Chromatin: Regulatory regions may remain closed, preventing the activation of endogenous pluripotency genes.

  • Epigenetic Instability: Human iPSCs have shown instances of X-chromosome instability.

• Stoichiometry of Factors: The levels of factors like $Oct4$, $Sox2$, and $Myc$ determine the success and pathway of reprogramming.

Clinical and Research Applications

• Disease Modeling (Young-onset Parkinson’s Disease - YOPD):

  • iPSCs generated from patients with onset at < 50 years.

  • Phenotype: Increased accumulation of soluble $\alpha\text{-synuclein}$ protein and reduced lysosomal membrane proteins (e.g., $LAMP1$).

  • Therapeutic Candidate: $PEP005$ (a phorbol ester) was found to reduce $\alpha\text{-synuclein}$ through proteasomal degradation and increase $LAMP1$ abundance.

• Regenerative Medicine (Bone Regeneration):

  • Study involving human iPSC-derived Mesenchymal Stem Cells (iMSCs) in mini-pigs.

  • Method: Combined HFF-iMSCs with Calcium Phosphate Granules (CPG) in critical-size defects.

  • Result: Significantly better osseous consolidation after 6 weeks compared to CPG alone, performing similarly to autologous bone marrow concentrate (BMC).

• Other Applications:

  • Basic research (human development, chromosomal abnormalities).

  • Drug toxicity screening and candidate testing.

  • Generation of cultivated meat.

  • Therapeutic delivery systems.

Human Embryonic Stem Cells (hESCs) vs. iPSCs

• Derivation of hESCs:

  • Derived from the Inner Cell Mass (ICM) of a blastocyst (Day 5) following In Vitro Fertilization (IVF).

• Genetic Engineering Goals:

  • Manipulation of Major Histocompatibility Complex (MHC) genes to prevent rejection (e.g., MHC-deficient tissue or isogenic tissues via nuclear reprogramming).

  • Hematopoietic chimera creation to establish immunologic tolerance.

• Advantages of iPSCs over hESCs:

  • iPSCs provide de-differentiated cells without the ethical concerns associated with embryonic destruction.

  • Ability to create patient-specific (isogenic) cell lines for transplantation.