Stem Cells and regenerative medicine

Question 1

What are stem cells?

Answer 1

• They are cells which can differentiate into many different cell types
• They can undergo self-renewal through cell division

Question 2

What are the different types of stem cells?

Answer 2

Induced Pluripotent ,Embryonic and Adult

Question 3

Describe Adult stem cells

ASCs

Answer 3

• These are tissue specific and multipotent which means they can differentiate into set number of cells within a specific location
• There are exceptions such as adult bone marrow stem cells which have shown variation

Question 4

Describe embryonic stem cells

ESCs

Answer 4

• These are pluripotent which means they can differentiate into every type of cells and these originate from the blastocyst.
• This is before implantation when the embryos are just a dew days old.
• They can form all cells from all three germ layers.

Question 5

What are the different germ layers?

Answer 5

The endoderm (Internal)-Lungs,pancreas,stomach,liver ,germ cells

The ectoderm (External)-Nervous, epithelial and sensory tissues

The Mesoderm(Middle)-Skeletal and cardiac muscle, blood and connective tissues

Question 6

Describe Induced Pluripotent stem cells (iPSCs)

How are they created and how are they used ?

Answer 6

These are induced in the lab by taking normal differentiated tissue and reprogram through exposure to pluripotency factors:
OCT4,Sox2,cMyc,Klf4-Transcription factors
(Yamanka factors)

This produces pluripotent stem cells with similar characteristics to embryonic stem cells -Cell therapy

Crispr Cas -gene editing techniques differentiate to healthy cells in vitro and transplanting back to patient
Reduces graft rejection

Question 7

How can iPSCs be grown and what can this be used for ?

Answer 7

They can be used as models for research
Grown in layer in dish or 3D organoid model
-Cell differentiation study
-Developmental biology
-Cell replacement therapy

Question 8

What are organoids?

Answer 8

These are tiny self organised three dimensional tissue cultures that can be derived from stem cells .
They can replicate complexity of an organ

Question 9

What are three categories of stem cells in terms of potency ?

Answer 9

Totipotent -Undifferentiated , self renew and produce pluripotent stem cells

Pluripotent +(Induced pluripotent ) -These can differentiate into the three germ layers

Multipotent-These will differentiate into tissue specific stem cells which become specialised cell types.

Question 10

What do tissue specific stem cells require?

Answer 10

Microenvironments called stem cell niches (specific anatomical locations )

Required for :

• Regulation of cell fate (What the cell will become )
• Protect from depletion and host from excessive proliferation

Question 11

What are the features of a stem cell niche?

Answer 11

-A supportive extracellular matrix
(Collagen ,Fibronectin)

-Secreted soluble signalling factors
(Growth factors, Cytokines

-Physical parameters-
(Shear stress, tissue stiffness and topography)

-Environmental signals -
(Metabolites, hypoxia ,inflammation)

Question 12

What is Hedgehog(Hh)and WNT signalling ?

Answer 12

These are pathways which direct growth and pattern ins during embryonic development. Regulate in epithelia of skin and intestine which undergoes constant renewal

Question 13

What can unregulated stem cell proliferation cause ?

Which stem cells is this most likely to occur in?

Answer 13

It may cause cancers/tumours

Embryonic stem cells as they have an unlimited growth potential
There is a large number of embryonic stem cells which can be used

Question 14

Outline some pros and cons of embryonic stem cells

Answer 14

Pros:

• Almost unlimited growth potential meaning they can differentiate into any kind of cell
• Unlimited number of cells due to high cell potency
• Very low probability of mutation induced damage in DNA (Low spontaneous mutation +high genetic stability)

Cons:
-Higher risk of tumour creation due to uncontrolled stem cell proliferation

Question 15

Outline some pros and cons of adult stem cells

Answer 15

Pros:

• Compatible with recipient’s cells -low risk of rejection
• Less risk of tumour creation

Cons-

• A limited number can be obtained
• Higher probability of mutation-induced damage in the DNA -risk of disease
• Limited cell potency

Question 16

Outline some pros and cons of Induced Pluripotent stem cells

Answer 16

Pros:

• Compatible with recipients cells-low risk of rejection
• Limited number can be obtained
• Less risk of tumour formation

Cons:

• Less growth potential than embryonic stem cells
• Higher probability of mutation-induced damage in DNA -risk of disease

Question 17

Why are Adult stem cells more susceptible to mutations /diseases?

Answer 17

Adult stem cells are thought to be especially vulnerable to cell cycle mutations since these cells already have the capacity to self-renew and can pass mutations to their daughter cells.

Question 18

What are the ethical concerns surrounding embryonic stem cells ?

Answer 18

These are derived from surplus in vitro fertilised embryos

• Parental consent +legal guidelines
• Unethical to destroy embryos

Question 19

Outline how the Yamanaka factors and how they induce iPSCs

Answer 19

The four Yamanaka factors :
-Sox 2, Oct3/4,Klf4,c-Myc

•c-Myc promotes DNA replication and relaxes chromatin structure,
•This allows Oct3/4 to access its target genes.
•Sox2 and Klf4 also co-operate with Oct3/4 to activate target genes
-These genes encode transcription factors which establish the pluripotent transcription factor network •This results in the activation of the epigenetic processes (more open chromatin) that establish the pluripotent epigenome.
The iPCS cells have a similar global gene expression profile to that of ES cells.

Question 20

Outline how stem cells can be tracked

Answer 20

• Stem cells can be manipulated in vitro which can make them easier to track in vivo.
• A reporter gene is inserted (fluorescent )
• The cells are then transplanted back into pre-clinical animal models + clinical.
• Non invasive cell tracking can track where stem cells go and how they behave.
• Development of cell based therapies

Question 21

What takes place in a heart attack

Answer 21

The blood supply to a heart muscle is lost. Cardiac muscle will die and is not replaced.
This is because adult cardiomyocyte turnover is low.
There will instead be fibrosis and scarring
=decreased cardiac function and heart failure

Question 22

What can research help to solve in terms of the heart?

Answer 22

It can potentially replace lost cardiac muscle ,increase blood supply.

Useful for
cardiac myopathies -
heart muscle to become enlarged, thick or rigid.

Conduction defects-
Electrical regulation not properly conducted

Question 23

What are two regulation strategies used to treat the heart?

Answer 23

Cell based therapies

Cell free therapies

Question 24

Expand on cell based therapies (heart)

Answer 24

Cell transplantation -
Promotes cardiac regeneration and repair
Replenishes the lost cardiac myocytes

Challenges:
Immune rejection
Manufacture /isolation of sufficient cells
Mode of delievery
Clinical regulation

Question 25

Expand of cell free therapies | Heart

Answer 25

Therapies which involve direct stimulation of endogenous cardiomyocyte production. Includes reactivation of developmental pathways- epicardium based on models where there is no scarring and full cardiac regeneration

Question 26

What is neovascularisation?

Answer 26

This is improved circulation to an injured area Paracrine effects improve Cardiac myocyte replacement -Inhibiting apoptosis+anti-inflammation

Question 27

Explain the cardiac regeneration in adult zebrafish

Answer 27

Following injury: - Zebra fish can regenerate their heart following injury - Apex is removed (Injury) - 1 day = there is a large blood filled clot which seals the site - 9 days = the apex wound is sealed by fibrin clot - 14 days , this is reduced /resolved and starts to be replaced by heart muscle - 1 month later =new cardiac wall - 2 Months= the entire site is regenerated with cardiac wall - We also see re-expression of developmental gene programmes (WT1,RHald H2??) - These are found on epicardium (surface level)-causes reactivation

Question 28

Explain what's different in larger mammals in the heart following injury.

Answer 28

The fibrin clot will not resolve,remodels to form a fibrotic scar

Question 29

What is the epicardium and what is it important for ?

Answer 29

It is the surface level of the heart and it contains cells for coronary vessels and signals for cardiac myocyte proliferation.

Question 30

Expand on zebrafish regeneration (cardiac)-Adult

Answer 30

There is an injury Embryonic gene expression Endocardium activation Fibrosis degradation

Question 31

Explain the immune response during regeneration of a neonatal mouse heart

Answer 31

Infiltration of injury by embryonic macrophages | Revascularisation and global cardiac myocyte proliferation

Question 32

Explain the immune response during regeneration of adult mouse heart

Answer 32

Infiltration by monocyte derived macrophages | There is limited revascularisation and no cardiac myocyte proliferation

Question 33

Outline the lymphatic response to cardiac injury. | Explain the two different response types

Answer 33

It will modulate the immune response during cardiac regeneration. In normal controlled injury response: (Endogenous) -Does not clear excess tissue fluid and inflammatory immune cells efficiently and this will cause oedema + inflammation (poor cardiac repair /function) If stimulated by form of VEGFC, this will increase the response and reduce oedema and improve cardiac repair and function

Question 34

How are iPSCs specified towards different cardiac lineages ?

Answer 34

- The iPSCS will specify into pre-cardiac mesoderm through inhibition of GSK-3b. - Through inhibiting glycogen synthase kinase 3b - This kinase acts as a downstream switch for signalling pathways such as WNT signalling. - Inhibition of WNT signalling causes differentiation of cardiac progenitor cells - These further differentiate into cardiac lineages

Question 35

What are the cardiac lineages?

Answer 35

``` Epicardial Cardiomyocytes Smooth muscle cells Endothelial cells Endocardial cells ```

Question 36

Describe iPSC evidence found in monkey model

Answer 36

- iPSCS used to create patient-specific cardiomyocytes to aid with cardiac repair . - Tested on monkey model with identical MHC (Major histocompatibility complex)as humans. - They used fibroblast-derived iPSCS then direct intra-myocardial injection to monkeys after myocardial infarction. Result - The graft cardiomyocytes survived for 12 weeks without rejection - electrical coupling between host and graft - Improved cardiac contractile function -Ventricular arrhythmias were found to be present

Question 37

Describe the paracrine signalling based studies | Developmental gene reactivation

Answer 37

Myocardial thymosin beta 4 is necessary for epicardial migration, coronary vasculature, cardiomyocyte survival - If added to adult heart = * Stimulate epicardial outgrowth + neovascularisation (normally don't take place ) It reactivated embryonic epicardial gene Wt1 -These activated genes give rise to cardiac progenitors in Myocardial infarction injured adult heart and these could become (new )de novo cardiomyocytes

Question 38

Describe how secreted factors from epicardium are important for cardiac regeneration?

Answer 38

- The secreted factor FSTL1 is expressed in epicardium and can promote cardiac myocyte proliferation - Epicardial expression is lots after a myocardial infarction - If it becomes restored it can promote the regeneration of pre-existing cardiomyocytes in mouse /pig models

Question 39

Outline different ways in which stem cell therapy is used in treating different cancers

Answer 39

chemotherapy/radiotherapy kills cancerous cells: Transplantation of stem cells can reform healthy cells. -Haematopoietic cell transplantation for blood cells and leukocytes -iPSCS engineered by treating them using specific factors: IL7, FLT3L ,SCF = produce T and N killer cells targeted for immunotherapy ( tumour cells). - Stem cells can be used to produce anti cancer vaccines - MSC/NSCS (Mesenchymal /neural stem cells) have been used to deliver genes, nanoparticles and oncolytic viruses delivering anti cancer therapy to the tumours - Exosomes can be extracted from the culture of drug-priming MSCS/NSCS and can target the drugs to tumour sites Mutation correction in-vitro, drug testing before replacement with in vivo

Question 40

Describe how stem cell-based therapy can be used to treat burns.

Answer 40

The skin = largest organ in the body, protects against heat ,light injury and infection Wound healing of the skin includes: - Coagulation - Inflammation - Proliferation of skin cells - Angiogenesis (new blood vessels) - Maturation Foetal fibroblasts(ESCs)- high expansion ability, low immunogenicity (types of immune responses are activated and their magnitude over time.) + secretes bioactive substances such as asFGFS,VEGFs,KGFs (skin cell proliferation + maintenance) Epidermal stem cells-High proliferation rate, easy access High potency and differentiation potential for long time. Mesenchymal stem cells -High differentiation potential and plasticity .Migrate to injured tissue ,differentiate and regulate tissue regeneration by producing growth factors, cytokines and chemokines iPSCS can be differentiated into all cells of skin layers : Dermal fibroblasts, keratinocytes and melanocytes Stem cell therapy involves isolating stem cells , selecting and amplifying required phenotype. Delivered through different methods such as dressings

Question 41

What are some complications which can result due to burns ?

Answer 41

Sepsis Fatal shock + dehydration Scarring

Question 42

Describe how stem cell-based therapy can be used to treat eye injuries

Answer 42

Cornea- Protective barrier against injury + outer lens(focuses light ) -Stem cells at edge of cornea= Limbal stem cells and these make new corneal cells instead of damaged ones. - If lost =cornea can't be repaired. - Collected from healthy donor, expanded and transplanted into damaged eye. - To avoid rejection ,healthy section of limbus from same patient used - Using iPSCs to make corneal epithelial cells for transplant and exposure to right signals = fibroblast cells into limbal stem cells

Question 43

What is the retina and how can it be damaged

Answer 43

The retinas receives light and converts into neural signals sent to brain Contains rods and cones photoreceptor cells Retinal pigment epithelium (RPE ) single layer of post mitotic cells which act as selective barrier and regulate the photoreceptor layer. If not functional -parts of retina can die =loss of vision Diseases that can damage = -age related macular degeneration (AMD) (eye disease that can blur the sharp, central vision) Retinis pigmentosa -breakdown and loss of cells in the retina-loss of peripheral vision Leber's congenital aneurosis (severe vision loss)

Question 44

Describe how stem cell-based therapy can be used to treat retina injury

Answer 44

Retinal pigment epithelium have been made from ESC and iPSCS (They are cultured using various growth factors ) These are then replaced in the diseased eye leading to restored vision

Question 45

Describe how stem cell-based therapy can be used to treat spinal injuries

Answer 45

These can be life threatening injuries Somatic cells are taken from patients , reprogrammed and differentiated Transplanted to the local where they are required. Cells created - Neural progenitor cells oligodendrocytes (glial cell found in the central nervous system) Neurons Astrocytes (glial cells in the central nervous system) Mesenchymal stromal cells (multipotent stem cells found in bone marrow)

Question 46

Describe engraftment and evidence of it

Answer 46

This is when neural stem /progenitor cell grafts can integrate into sites of spinal cord injury. (piece of living tissue that is transplanted surgically.) Calcium imaging of grafts in spinal cord injury sites in vivo and in adult spinal cord slices showed NSPC grafts organise into localised active synaptic networks Optogenetic (biological technique that involves the use of light to control neurons) stimulation of host axons produced a neuronal response in the graft. In vivo imaging showed that behavioural stimulation elicited focal synaptic responses within grafts - Therefore neural progenitor grafts can form functional synaptic subnetworks and the activity pattenrs resemble the intact spinal cord.