Stem Cells & Regenerative Medicine

Question 1

What is a stem cell?

Answer 1

Cells that:

1. Are capable of self-renewal via cell division
2. Can differentiate into many different cell types

Question 2

What is a stem cell’s biological function?

Answer 2

Provide new cells as an organism grows and can replace cells that are damaged or lost

Question 3

What are the three main sources of stem cells?

Answer 3

1. Adult SCs
2. Embryonic SCs
3. Induced pluripotent SCs

Question 4

Discuss examples of therapeutic roles for stem cells in disease and injury

Answer 4

Blindness, wound healing, myocardial infarction, spinal cord injury and cancers

Question 5

Describe adult stem cells

Answer 5

• Quite rare; found in specific places e.g. brain, retina, pancreas etc.
• Supply new cells as organism grows and replace damaged cells
• • Ability to do this varies with different organs e.g. skin has greater ability to replace damaged tissue than heart muscle.
• Tissue-specific and multipotent i.e. they can differentiate into a subset of cell types, usually linked to their location and not every type of cell. E.g. epithelial stem cells provide the different types of cells making up the layers of skin
• Some ASCs have some plasticity (e.g. haematopoietic stem cells) and can differentiate into other types of cells
• Scientists can amplify and manipulate these cells in vitro and use them for a variety of purposes.

Question 6

Describe embryonic stem cells

Answer 6

• Supply all the cells of the developing embryo
• Pluripotent (can differentiate into every type of cell)
• Derived from embryos at blastocyst stage. Before implantation and only a few days old. Found in inner cell mass which eventually give rise to the embryo
• In vivo and in culture, these cells can proliferate for multiple rounds before differentiating.
• All three germ layers can be used. Each layer gives rise to specific tissues e.g. ectoderm gives rise to nervous, epithelial and sensory tissues.

Question 7

Describe induced pluripotent stem cells

Answer 7

• Made in lab
• Scientists take normal differentiated tissue and reprogram these cells by exposure to a specific set of pluripotency factors (e.g. SOX2). This produces pluripotent stem cells with similar characteristics to ESCs.
• Can be used for cell therapy by repairing gene mutations and then differentiating healthy cells in vitro and transferring back to patient. This reduces graft rejection by the host since its the patient’s own cells.
• Can be used as models for basic and translational research. Cells can be grown in a dish as a layer or a 3D organoid model. They can then be used in developmental biology studies, cell differentiation studies, disease modelling, drug screening and cell replacement therapy.

Question 8

Describe the process of stem cell production

Answer 8

1. Undifferentiated totipotent stem cells give rise to both placenta and embryo. They self renew and give rise to
2. Pluripotent embryonic stem cells. These can differentiate into 3 germ layers. These further divide into
3. Multipotent stem cells. These are adult, tissue-specific stem cells which will eventually lead to specialised cell types.

Question 9

What are stem cell niches?

Answer 9

Special supportive microenvironments that maintain tissue-specific stem cells.
They interact with stem cells to regulate cell fate. They protect cells from depletion and the host from excessive proliferation.

Question 10

Describe some specific features of stem cell niches

Answer 10

• supportive extracellular matrix molecules e.g. fibronectin or collagen
• neighbouring niche cells
• secreted soluble signalling factors e.g. growth factors and cytokines
• physical parameters; shear stress, tissue stiffness and topography
• environmental signals e.g. metabolites, hypoxia, inflammation etc.

Question 11

Compare and contrast the different properties of the differentiation potential of all three stem cells. This will help choose the most suitable cell type for a lab study or therapy

Answer 11

ESCs:

1. pluripotent - almost unlimited growth potential - may differentiate into any kind of cell
2. higher risk of tumour creation
3. risk of being genetically different from the recipient’s cells - higher risk of rejection
4. unlimited numbers of cells due to high cell potency
5. very low probability of mutation-induced damage in the DNA - [low spontaneous mutation rate & high genetic stability]
6. ethical issues as these are derived from surplus in-vitro fertilised embryos. Requires parental consent and adherence to strict legal guidelines. Some believe it is unethical.

ASCs:

1. oligopotent - unipotent - limited cell potency (the least out of the 3)
2. less risk of tumour creation
3. compatible with recipient’s cells - low risk of rejection
4. limited numbers may be obtained
5. higher probability of mutation-induced damage in the DNA - risk of diseases
6. no ethical issues - direct patient consent

iPSCs:

1. less growth potential than ESCs
2. less risk of tumour formation
3. compatible with recipient’s cells - low risk of rejection
4. rather limited numbers may be obtained
5. higher probability of mutation-induced damage in the DNA - risk of diseases
6. no ethical issues - direct patient consent

Question 12

Describe how iPSCs are generated

Answer 12

1. Adult somatic cells exposed to pluripotency factors e.g. Sox2, Oct 3/4, Klf4 and c-Myc. These work together to reprogram the cell.
2. c-Myc relaxes chromatin structure and promotes DNA replication thus allowing Oct3/4 to access target gene promoters.
3. Sox2 and Klf4 also co-operate with Oct3/4 to activate target genes. These encode transcription factors which establish the pluripotent transcription factor network.
4. Result in the activation of the epigenetic processes (more open chromatin) that establish the pluripotent epigenome.
5. iPSCs have a similar global gene expression profile to that of ESCs.

Question 13

Describe stem cell tracking

Answer 13

A reporter gene can be inserted into stem cells in vitro to see how they behave in vivo e.g. fluorescent genes. In vivo imaging can identify where the stem cell goes and how they behave once they are back in the body of the model. This helps the development and clinical translation of cell-based therapies. It is also non-invasive and allows long-term cell tracking in preclinical and clinical settings.

Question 14

What is neovascularisation?

Answer 14

• the natural formation of new blood vessels, usually in the form of functional microvascular networks, capable of perfusion by red blood cells, that form to serve as collateral circulation in response to local poor perfusion or ischaemia.

Question 15

How is neovascularisation used in CM regeneration?

Answer 15

• Promote neovascularisation either via stem cells which differentiate into new coronary vessels or by cell-free methods
• Causes improved circulation at injury site
• In turn promotes paracrine effects improving CM replacement

Question 16

Explain how models regenerate cardiac tissue

Answer 16

• We see re-expression of developmental gene programs early after injury e.g. Raldh2 and Wt1, particularly in epicardium - surface layer of heart
• this is known as reactivation of epicardium
• epicardium is important source of cells for coronary vessels and for signals that promote CM proliferation
• this is followed by similar activation of endocardium and then CM dedifferentiation and then formation of fibrin clot
• this degrades as CM regenerates

Question 17

What happens in models that do not regenerate cardiac tissue?

Answer 17

• after fibrin clot forms, it does not resolve and instead remodels to form a fibrotic scar which affects cardiac function

Question 18

Research has shown the lymphatics and immune response play a role in CM regeneration.
What is the difference in immune response in adult vs neonatal mice (in terms of CM regeneration)?

Answer 18

• in neonatal mice hearts, there is infiltration of the injury by embryonic macrophages, revascularisation and global cardiomyocyte proliferation
• whereas, in adults, there is infiltration by monocyte-derived macrophages, limited revascularisation and no cardiomyocyte proliferation

Question 19

With regards to lymphatic response to cardiac injury, what therapeutic strategy has been found to better cardiac repair?

Answer 19

Using VEGFC administration because it increases clearance of excessive tissue fluid and inflammatory cells, and decreases oedeme and inflammation. This in turn improves cardiac repair and function.

Question 20

How do pre-clinical and initial clinical studies suggest cell therapy maybe therapeutic?

Answer 20

• because of possible direct integration of grafted cells to the myocardium/coronary vessels
• or by re-expression of developmental gene programmed and paracrine signals which help the host tissue to regenerate

Question 21

Describe some ways stem cell therapy can be used in the treatment of cancer

Answer 21

1. Chemo/radiotherapy kills cancerous cells. Transplantation of stem cells reconstitutes healthy cells. e.g HSC transplantation for blood cells and leukocytes (lymphoma, leukemia) (ASC, iPSC)
2. Effectorr immune cells from iPSC/ESCs e.g. engineered T and NK cells targeted for immunotherapy.
3. Production of anti-cancer vaccines
   More on slides

Question 22

Describe different types of stem cells used in therapy for burns

Answer 22

• Fetal fibroblasts (from ESCs); improve skin repair due to the high expansion ability, low immunogenicity (lesser immune reaction), and intense secretion of bioactive substances such as growth factors.
• Epidermal stem cells; high proliferation rate and easy access and keep their potency and differentiation potential for long periods. Generate most skin cell types for repair and regeneration
• also mesenchymal stem cells - high differentiation potential and a degree of plasticity and iPSCs - differentiated into dermal fibroblasts, keratinocytes and melanocytes.

Question 23

How does stem cell-based therapy for burns take place?

Answer 23

• isolation and/or production of stem cells
• then selection and amplification of the required phenotypes in culture
• cells are chosen for lots of things but include: secretion of growth factors and secretion of extracellular matrix molecules (acts as scaffold for cell regeneration)
• cells delivered to pt. via cell spray, dressing or 3D bioprinting

Question 24

How can stem cell therapy be used to treat injury to the cornea?

Answer 24

• Limbal SCs are at edge of cornea. They make new corneal cells to replace damages ones.
• If SCs are lost due to disease or injury, pt. can have significant loss of vision.
• LSCs collected from healthy donor eye, expnaded in lab to sufficient numbers and transplanted into damaged eye.
• repairs cornea and permanently restores visions.
• avoids immune rejection if pt. has healthy section of limbus.
• iPSCs can be induced to make corneal epithelial cells for transplant and exposure to the right signals can transform fibroblast cells into LSCs.

Question 25

How can stem cell therapy be used to treat injury to the retina?

Answer 25

• Injury to retinal pigment epithelium can cause loss of vision
• RPE cells have been made from both ESC and iPSCs.
• Stem cells are cultured with growth and differentiation factors to form RPEs. These are transplanted back into eye and replace damaged/dead ones.
• vision is restored

Question 26

How can stem cell therapy be used to treat spinal injury?

Answer 26

• Take somatic cell biopsy from pts
• reprogram cells to form iPSCs
• these differentiate into neural stem cells
• then implanted at site of spinal cord injury
• can then differentiate into different cells e.g. neurons, astrocytes oligodendrocytes etc