Stem cells and regenerative medicine

Question 1

Are stem cells capable of self-renewal?

Answer 1

Yes by asymmetric or symmetric cell division - but I believe that as stem cells lose potency, some may lose the ability to self-renew

Question 2

Name the 3 main sources of stem cells

Answer 2

Embryonic stem cells (ESCs) (pluripotent)

Adult stem cells (ASCs) - rare (multipotent)

Induced pluripotent stem cells (iPSCs)

Question 3

What is the function of stem cells?

Answer 3

• Can differentiate into many different cell types
• capable of self-renewal via cell
  division
• provide new cells as an organism grows
• can replace cells that are
  damaged/lost

Question 4

Describe what we get ESCs from and their potency

Answer 4

Pluripotent (all types of cells)

Derived from embryos at the
blastocyst stage before implantation when the embryo is just a few days old, the stem cells reside in the inner cell mass, which will eventually give rise to the whole embryo in vivo culture, these cells can proliferate via multiple round of cell division before differentiating,
-Can give rise to all embryonic germline layers: ectoderm, mesoderm and endoderm

Question 5

What does the ectoderm vs mesoderm proliferate into eventually?

Answer 5

ectoderm - neural and epithelial and sensory

mesoderm - skeletal muscle, blood vessels, cardiac muscle

endoderm - organs

Question 6

Name the factors that can allow un-differentiation and so formation of iPSCs

Answer 6

OCT4

SOX2

KLF4

Myc

Question 7

What can we use CRISPR for in the process of iPSC formation?

Answer 7

Repairing any pathological DNA mutations before allowing iPSCs to differentiate and then giving them to the patient

Question 8

Describe the composition of a stem cell niche - so the signals involved

Answer 8

Supporting extracellular matrix in a supportive microenvironment.
-Has secreted cell signals (growth factors, cytokines etc.),
-physical parameters (shear stress, tissue stiffness and topography)
-environmental signals (metabolites, hypoxia, inflammation etc.)

Question 9

Name the functions of stem cell niches

Answer 9

Regulate (dictate) stem cell fate (differentiation)

Question 10

Why is there a lower risk of tumour formation in adult stem cells and iPSCs than in embryonic stem cells?

Answer 10

As ESCs have the highest growth potential, then iPSCs, then ASCs

Question 11

Explain why ESCs have a higher risk of rejection in the patient

Answer 11

ASCs and iPSCs are usually taken from the host and so have a low risk of rejection, but ESCs are not taken from the host and so are genetically different

Question 12

Describe the function of c-Myc

Answer 12

C-Myc is part of the yamananka cocktail and it relaxes chromatin structure - this allows OCT3/4 to access its genes

• from a later lecture they tell us that Myc is an oncogene (I think)

Question 12

Compare the mutation rate/stability in the 3 different stem cell types

Answer 12

Embryonic stem cells have a low genetic mutation rate and high genetic stability - ASCs and iPSCS are less genetically stable

Question 13

Describe the function of SOX2 and KLF4

Answer 13

Also co-operate with OCT3/4 to activate target genes

Question 13

So explain how these TFs will undifferentiate the cell to make an iPSC

Answer 13

They will cause activation of genes that in turn code for TFs - this will ‘establish the pluripotent transcription factor network’. Results in the activation of the epigenetic processes (more open chromatin) that is in a pluripotent cell epigenome

Question 14

What is a reporter gene and its use?

Answer 14

This is a gene that can be inserted into a stem cell to then track it in vivo (to study where the stem cells go and how they behave once they are back within the body of the model)- for example, this could be a fluorescent gene used in imaging

is non-invasive

Question 14

Explain what it is that we try to do with stem cell therapies for CV disease

Answer 14

If you survive a heart attack (70% of people do) then you are likely to suffer from heart failure due to loss of cardiac myocytes as well as fibrosis and scarring - so we aim to replace lost cardiac muscle as well as improve blood supply with stem cells

stem cell therapies may also be able to treat cardiac myopathies or conduction defects

Question 15

Describe how cardiac regeneration differs in different animal models

Answer 15

Zebrafish, some reptiles and neonatal mice can regenerate very large parts of the heart.

Question 15

Why is improving neovascularisation after cardiac damage particularly important?

Answer 15

As improved circulation to injured area allows for paracrine effects which promotes endogenous cardiac stem cells

Question 16

Explain how cardiac regeneration occurs in these animals

Answer 16

This is done by re-expression of developmental genes early after the injury such as WT1 and RILDH2 - this is particularly seen in the epicardium of the heart and is known as reactivation of the epicardium

As well as activation of the epicardium, there is activation of the endocardium, cardiomyocyte undifferentiation and formation of a fibrin clot that then degrades as cardiomyocytes proliferate and regenerate the heart

Question 16

Describe the function of the epicardium in the process of cardiac regeneration

Answer 16

Provides cells and signals that promote cardiomyocyte proliferation

Question 17

How is the fibrin clot different in cardiac regeneration in larger animals such as mammals?

Answer 17

The fibrin clot does NOT resolve, but instead remodels to form a fibrin scar which effects cardiac function

Question 18

Describe the differences in cardiac regeneration in neonatal mice vs adult mice: immune components, revascularisation and cardiomyocyte proliferation

Answer 18

Infiltration of the injury by embryonic macrophages. There is also an increase in vascularisation and increased cardiomyocyte proliferation. In adult mice, there is infiltration by monocyte derived macrophages, limited revascularisation and NO cardiomyocyte proliferation

Question 19

Describe the role of the lymphatic system in cardiac injury normally and what we can do to improve this

Answer 19

In a normal response, the lymphatic system would not clear excess tissue fluid and inflammatory immune cells enough and so there would be oedema and inflammation which causes poor cardiac repair and function but if we stimulate it with a modified IGF C there is increased lymphatic response and there is less oedema and inflammation

Question 20

What does inhibition of glycogen synthase kinase 3B allow us to do?

Answer 20

This is a downstream switch for a number of signalling pathways including Wnt - so we inhibit Wnt signalling. Further inhibition of Wnt signalling leads to the differentiation of cardiac progenitor cells from the iPSCs

Question 21

What allows us for further differentiation of these cells?

Answer 21

‘provision of specific signalling molecules’

Question 22

Are stem cells allowed to differentiate before injection into the heart or after?

Answer 22

After - so in the Shiba et al paper, differentiated cardiomyocytes were injected directly into the myocardium after infarction

Question 23

From the paper about injecting cardiomyocytes from iPSCs into the heart after infarction, discuss a downside to these iPSC-derived cardiomyocytes

Answer 23

There was shown to be more ventricular tachycardia in the test patient hearts as opposed to the control hearts - this is as iPSC-derived cardiomyocytes contain nodal-like cardiomyocytes that spontaneously contract FASTER than normal cardiomyocytes

Question 24

So how can thymosin beta 4 be used clinically? - from the smart et al paper

Answer 24

Can stimulate epicardial outgrowth and neovascularisation that NORMALLY DOES NOT OCCUR

Question 25

Explain what thymosin beta 4 is

Answer 25

A small molecule produced by cardiomyocytes necessary for coronary vasculature survival as well as cardiomyocyte survival and the migration of the epicardium to form vasculature in the myocardium

Question 25

How does thymosin beta 4 work?

Answer 25

Re-expression of a key developmental gene Wt1 - activated Wt1 with epicardial cells gave rise to cardiac progenitors in the MI-injured heart

Question 26

What is FSTL1 and what does it do?

Answer 26

Is normally expressed in the epicardium and promotes cardiomyocyte proliferation but this expression is lost after MI - the study below restored this expression

Question 27

With the grafting of large sheets of iPSC-derived cardiomyocytes, why is it thought that there is an increase in cardiac function despite not very good incorporation of the cells?

Answer 27

Is thought that it may be due to paracrine signalling from the iPSCs

Question 28

List some ways that stem cells can be used in cancer treatment (6)

Answer 28

• can be used to replace cells damaged by radio/chemotherapy, such as HPSCs in the bone marrow in leukaemia
• effector immune cells such as T and NK cells can be made from iPSCs/ESCs for targeted immunotherapy
• production of anti-cancer vaccines
• delivery of genes, viruses and nanoparticles to tumour niche with stem cells as they migrate towards tumours
• exosomes extracted from the culture of drug-priming stem cells can target drugs to tumour sites
• mutation correction in vitro and then replacement in vivo

Question 29

Name some of the complications of severe burns

Answer 29

sepsis, fatal shock and dehydration leading to renal failure

Question 30

In what ways can stem cell therapy help with burns

Answer 30

replace lost skin cells, speeding up endogenous healing, generate ECM and produce paracrine signals which aid healing

Question 31

Name the 4 stem cells used in stem-cell based therapy for burns in clinical trials

Answer 31

Fetal fibroblasts (from ESCs)
Epidermal stem cells
Mesenchymal stem cells
iPSCs

Question 32

Name some benefits of each stem cell type

Answer 32

Fetal fibroblasts improve skin repair due to high expansion ability and intense secretion of bioactive substances

Epidermal stem cells are highly proliferative and keep their potency for long periods

Mesenchymal stem cells have high differentiation potential and some plasticity - and migrate to injured tissues and regulate tissue regeneration by production of signalling molecules

iPSCs can be differentiated into dermal fibroblasts, keratinocytes and melanocytes

Question 33

Which stem cells can be used to repair damaged cornea?

Answer 33

limbal stem cells

Question 34

Describe the process of how we use L_____ stem cells to treat a damaged eye

Answer 34

Limbal stem cells are collected from a healthy donor eye, expanded in the lab and then transplanted into the damaged eye

limbal stem cells are at the edge of the cornea
otherwise, corneal cells can be made from fibroblasts → iPSC → corneal epithelial cells OR fibroblast → limbal stem cell directly by exposure to the right signals

Question 35

What is the retinal pigment epithelium?
- its structure
- its function
- diseases that damage it
- how stem cell treatments can heal it

Answer 35

-

Question 36

Describe the general process of stem cell therapy for spinal injury

Answer 36

You take somatic cell biopsy as normal (like from fibroblasts from the skin of the elbow) and then you reprogram to iPSC then iPSC → neural stem cells

neural stem cells will then be implanted at the site of the injury and will differentiate (so unlike cardiac stem cell treatment, it is not mature cells being implanted to the site of treatment?)