Lecture 13 - Repair and Regeneration

Question 1

What are non-cell-based therapies focused on?

Answer 1

Activating the body’s own cells and repair mechanisms or blocking disease-causing pathways

This approach can utilize minimally invasive acellular scaffolds or pharmacological methods.

Question 2

What are some examples of pharmacological approaches in non-cell-based therapies?

Answer 2

• Small molecules
• Proteins (including growth factors & monoclonal antibodies)
• Nucleic acids (including siRNA and miRNA)
• Gene therapy
• Gene editing

Question 3

What are some limitations of traditional gene therapy?

Answer 3

• Size of cargo
• Transient effects
• Oncogenicity

Question 4

What is the most well-known method for gene editing?

Answer 4

CRISPR/Cas9

Question 5

How can CRISPR/Cas9 be modified for gene expression control?

Answer 5

Using dCas9, which has a mutation that deactivates nuclease activity and can be used to control gene expression.

Also known as ‘dead’ Cas as it doesn’t cleave DNA.

Question 6

What are recent approaches that offer more precise control than traditional CRISPR/Cas9?

Answer 6

• Base editing
• Prime editing

Question 7

What is the role of miRNA-34a in cancer regulation?

Answer 7

Regulates signaling pathways controlling cell proliferation, migration, invasion, resistance to apoptosis, and immune evasion

Question 8

What challenges are associated with miRNA therapy?

Answer 8

• Stability
• Immunogenicity
• Non-specific delivery

Question 9

What is the significance of AMD3100 (Plerixafor) in stem cell recruitment?

Answer 9

Mobilizes BM stem cells into the bloodstream

Question 10

What effect does FK506 (Tacrolimus) have on cell regeneration?

Answer 10

Augments cell regeneration and repair at sub-immunosuppressive doses

Immunosuppressant as inhibits IL-2 expression which typically stimulates T-cells.

Question 11

What is the impact of combining AMD3100 and FK506 on wound healing?

Answer 11

Reduces healing time by 25% with a synergistic effect

Question 12

What markers are associated with stem cells and macrophages in regeneration?

Answer 12

• CD133: stem cell marker
• F4/80: macrophage marker

Question 13

What is the role of HGF in stem cell proliferation?

Answer 13

Increases proliferation & differentiation of HSPCs

Question 14

What is rejuvenation in the context of cell biology?

Answer 14

Reversing the causes of cell senescence to rejuvenate cells

Senescence is characterized by features such as flattened morphology, increased cell size, loss of physiological function, and release of inflammatory cytokines.

Question 15

What are the characteristic features of senescent cells?

Answer 15

• Flattened morphology
• Increased cell size
• Loss of physiological function
• Release of inflammatory cytokines

Question 16

What role does the protein AP2A1 play in cellular signaling?

Answer 16

Involved in transport of integrin β1 receptors along stress fibres

Question 17

What is the function of integrin β1 in cells?

Answer 17

Enables stable focal adhesions to form, maintaining cell-ECM interactions and increased cell volume

Question 18

What happens when AP2A1 is knocked down using siRNAs?

Answer 18

Reverses hallmarks of senescence, including cell size, proliferation, and expression of SA-β-gal, p53, and p21

Question 19

What is the focus of current research in de-aging cell populations?

Answer 19

Improving healing and reducing the impact of aging, especially in stem cells

Question 20

What is dedifferentiation and how is it relevant to regeneration?

Answer 20

A process that doesn’t happen in mammals but is involved in regeneration in species like axolotls

Question 21

What is the Tet-On system used for?

Answer 21

To control the expression of OSKM (Oct4, Sox2, Klf4, c-Myc) for regeneration purposes

Question 22

What are the effects of long-term partial reprogramming in treated mice?

Answer 22

• Prevented or reversed epigenetic aging in skin and kidney
• Downregulation of senescence and inflammatory genes
• Restoration of metabolomic and lipidomic profiles to youthful levels (systemic)

Question 23

What was the outcome of using an adeno-associated virus to deliver the OSK system to elderly mice?

OSK system = Oct4, Sox2, Klf4

Answer 23

Median remaining lifespan extended by 109% and reduction in frailty scores

Question 24

What was the challenge faced with human somatic cells in regenerative studies?

Answer 24

Human epigenome is stable and needs to be reset for successful reprogramming

Question 25

What potential do chemical reprogramming processes hold?

Answer 25

May partially or fully dedifferentiate human somatic cells

Question 26

What role does bioelectricity play in true regeneration?

Answer 26

Influences cell patterning and healing through electrical fields and ion concentration gradients

Question 27

What is the 'bioelectric code'?

Answer 27

Combines with the genetic code to determine the final shape and function of tissues and organisms

Question 28

What are the implications of gene editing in regenerative medicine?

Answer 28

Can repair defects in cells, potentially curing disease states

Question 29

What is the current best method for replacing lost tissues?

Answer 29

Tissue engineering

Question 30

Fill in the blank: The reversal of the aging process is possible in isolated cells and _______.

Answer 30

whole animals

Question 31

True or False: Pharmacological reprogramming of cells is not currently possible.

Answer 31

False

Question 32

What combination of methods may be required for effective regeneration?

Answer 32

* Gene editing * Stimulation or inhibition of endogenous pathways * Bioelectrical approaches

Question 33

What is the basic mechanism of miRNA-based therapies?

Answer 33

miRNA therapies regulate gene expression by inhibiting or restoring the function of specific miRNAs involved in disease, either by using miRNA mimics or antagomirs (miRNA inhibitors).

Question 34

What are the two main strategies in miRNA therapy?

Answer 34

1. miRNA mimics: Synthetic miRNAs that restore the function of downregulated tumor suppressor miRNAs. 1. Antagomirs: Chemically modified oligonucleotides that inhibit overactive, disease-causing miRNAs.

Question 35

Give an example of a disease targeted by miRNA therapy.

Answer 35

miR-34a is a tumor-suppressor miRNA that regulates genes involved in cell cycle and apoptosis; MRX34, a synthetic miR-34a mimic, was developed to treat cancers by restoring its function, though clinical trials were halted due to immune-related side effects.

Question 36

How is miRNA-34a disrupted in cancer?

Answer 36

>50% of cancers have reduced activity of miRNA-34a often through decreased expression by genomic deletion or epigenetic silencing.

Question 37

How is Myoseverin used in true regeneration?

Answer 37

Myoseverin is a small molecule that promotes muscle regeneration by inhibiting dynein motor proteins, which enhances myogenic progenitor cell activity and muscle fiber repair.

Question 38

How is the OSKM system used for regeneration and rejuvenation?

Answer 38

The OSKM system refers to the four transcription factors Oct4, Sox2, Klf4, and c-Myc, which can reprogram somatic cells into induced pluripotent stem cells (iPSCs).

Question 39

How is the OSKM system used in the Tet-On system?

Answer 39

In the Tet-On system, the OSKM factors (Oct4, Sox2, Klf4, Myc) are controlled by a tetracycline-responsive promoter. When tetracycline or doxycycline is added, the OSKM factors are activated, allowing induced pluripotent stem cell (iPSC) formation or partial reprogramming in a controlled, reversible manner.

Question 40

How have murine somatic cells been converted to pluripotent stem cells (PSCs) using small molecules?

Answer 40

Murine somatic cells have been chemically reprogrammed into PSCs by using small molecules that activate pluripotency-related pathways, such as MEK/ERK inhibitors, GSK3β inhibitors, and TGF-β pathway inhibitors. These molecules replace the need for transcription factors like OSKM, enabling the generation of iPSCs without genetic modifications.

Question 41

What are some small molcules that have been used for chemical reprogramming?

Answer 41

* i (MEK inhibitor + GSK3β inhibitor): inhibits differentiation. * Y-27632: A ROCK inhibitor that enhances reprogramming efficiency. * Valproic acid: A histone deacetylase inhibitor

Question 42

Why has chemical reprogramming of human somatic cells been unsuccessful?

Answer 42

Greater complexity in humans: * Differences in signaling pathways * Stronger epigenetic barriers - humans have more modifications * Incomplete reprogramming - often leads to partial reprogramming where cells do not fully acquire pluripotency * Cellular differences - adult human cells often more differentiated

Question 43

What are hCiPSCs?

Answer 43

hCiPSCs (human chemically induced pluripotent stem cells) are generated from somatic cells using only small molecules, without introducing genetic material.

Question 44

How have fibroblasts been used to create human chemically induced pluripotent stem cells (hCiPSCs)?

Answer 44

Human fibroblasts have been reprogrammed into hCiPSCs by exposure to cocktail of small molecules that modulate pathways involved in pluripotency and epigenetic remodeling. These molecules typically target the Wnt, TGF-β, MAPK, and epigenetic pathways to gradually erase somatic identity and activate endogenous pluripotency genes, leading to the formation of fully reprogrammed pluripotent stem cells.

Question 45

How has bioelectricity been used to regenerate tissue?

Answer 45

Bioelectricity has been used to regenerate tissue by modulating membrane voltage and ion channel activity, which control cell behavior and patterning.

Question 46

How do bioelectric circuits influence cell patterning?

Answer 46

Bioelectric circuits, formed by ion channels and gap junctions, create voltage gradients across tissues that act as instructive signals for cells. These gradients guide cell proliferation, migration, and differentiation, helping establish spatial patterns during development and regeneration by encoding positional information.