Stem cells

Question 1

What are stem cells?

Answer 1

Immature, undifferentiated, non-specified cells with the capacity for prolonged or unlimited self-renewal.

Question 2

What is differentiation?

Answer 2

A process whereby a cell acquires distinctive morphological and functional features determined by gene expression and micro-environment allowing it to perform specialised functions but have limited ability to proliferate. The differentiation process usually involves intermediate formation of committed progenitors (transit amplifying cells).

Question 3

What are the different types of stem cells?

Answer 3

Totipotent – can form all tissues of an organism including extraembryonic membranes and tissues (like a fertilised egg).

Pluripotent – can give rise to most tissues of an organism. Examples of pluripotent stem cells include:
o Embryonic stem cells (ES cells) – a lab created stem cell type isolated from the early embryo (blastocyst). These include embryonic germ (EG) cells (present in the gonads and early fetus) and embryonal carcinoma (EC) cells (cancers that affect the gonads and germ cells and produce large teratomas – monstrous tumours containing hair, teeth, skin, bone).
o Induced pluripotency stem cells (iPS cells) – a lab made stem cell type created by reprogramming differentiated, somatic cells from skin biopsy with transcription factors.

Multipotent – “adult” stem cells capable of forming a restricted number of cell types. Examples of multipotent stem cells include:
o Haematopoietic stem cells (HSC) - form all our blood cells
o Mesenchymal stem cells (MSC) - form many musculoskeletal tissues e.g., bone, muscle, fat.
o Cord blood stem cells (includes foetal/neonatal HSC and MSC)
o Many other tissues in our body have a repository of stem cells e.g., bone marrow.

Unipotent, bipotent, and tripotent – give rise to one/two/three cell type(s).

Question 4

How do stem cells divide?

Answer 4

By asymmetric division, meaning they divide into 2 non-identical daughter cells: 1 of which is identical to the parent (self-renewal) and the other which is slightly changed (differentiated progenitor cell).

Question 5

What are progenitor cells?

Answer 5

Descendents of stem cells which can further differentiate into different cell types. However, they have a finite number of divisions so each cell division results in increased differentiation but decreased proliferation potential.

Question 6

What factors can affect the differentiation process?

Answer 6

• Cytokines
• Transcription factors
• Cell-cell interactions
• Cell-matrix interactions
• Nutrient/waste exchange
• Oxygen concentration

Question 7

What does the self-renewal cell do?

Answer 7

Acts as semi-conservative division as the stem cell compartment is maintained.

Question 8

What are the pros and cons of using maturing/non-stem cells in practice?

Answer 8

Pros:
Easily obtained from bone/cartilidge/skin/liver of patient.
No need for immunosuppression if used for re-implantation.

Cons:
Often have poor growth potential in the lab.
May change phenotypic characteristics when cultured in lab. Can take a lot of time to get enough cells so the earlier cells are sat waiting in the petri dish and can start to de-differentiate.
Tens of millions of cells required which may take several weeks to grow/
Prolonged patient morbidity and possible death mortality.
Creates 2 wound sites - site of injury and site of tissue biopsy.

Question 9

Where can adult stem cells be obtained from?

Answer 9

• Epidermal
• Follicular
• Intestinal
• Neural
• Haematopoietic

Question 10

What are the advantages of adult stem cells?

Answer 10

• Rapid growth potential – can generate lots of cells
• Plasticity, can form various cell types
• Possible to exert control over stem cells in vitro (& in vivo) and derive specific cell and tissue types. However, we need to know enough about the biology of the stem cells and the target cells to be able to supply the appropriate stimuli / environments.

Question 11

What are the 2 types of stem cells which can be obtained from bone marrow?

Answer 11

Haematopoietic stem cells
Mesenchymal stem cells

Question 12

What do haematopoetic stem cells give rise to?

Answer 12

Blood cell lineages - can be used for some leukaemias.

Question 13

What do mesenchymal stem cells give rise to?

Answer 13

Cartilage
Bone
Fat
Muscle
Haematopoetic support cells
Astrocytes

Can be used in bone fracture, cartilage repair, and tissues such as CNS and heart.

Question 14

Pros and cons of mesenchymal stem cells?

Answer 14

Pros:
Versatile.
Can be harvested from donor and implanted back into donor following expansion and differentiation in vivo so no immunocompatability issues.
Process is rapid (within a month).

Cons:
Stem cells are sparse (1 in 100,000).
Can potentially propagate/transmit harmful mutations/disease if used for allogenic transplants - require robust screening.
Numbers and potency diminish with age - as we get older our tissues don’t repair as effectively or quickly.

Question 15

What stem cells does umbilical cord blood give?

Answer 15

HSC and some MSC.
These cells have greater plasticity and lower immunological risk than HSC/MSC obtained from adult sources.

Question 16

How are umbilical cord stem cells obtained?

Answer 16

The blood is collected from the umbilical cord after birth so there is no risk to the baby and minimal ethical issues (as long as there is informed consent). The placenta and cord are then discarded as clinical waste.

Alternatively, it is possible to collect them in utero via an ultrasound guide needle but there are more issues with ethics and there is some risk to the baby (but it is a routine procedure).

Question 17

What are embryonic stem cells?

Answer 17

Cells which exist briefly in the natural development of the embryo (inner cell mass of blastocyst) which have potential to generate any cell type in the body.

Question 18

What is the main ethical concerns with ES cells?

Answer 18

It involves the destruction of the embryo which can deter donors, and involves cloning (therapeutic NOT reproductive).

Question 19

What are some disadvantages of using embryonic stem cells?

Answer 19

Difficult to accurately predict or control differentiation
Stability (mutations/tumours) unknown
Transmission of heritable diseases unknown but can be reduced with screening.

Question 20

What are induced pluripotent stem cells?

Answer 20

Differentiated cells which have been reprogrammed to become pluripotent stem cells, allowing them to be used as disease models or used in therapy.

Question 21

How are induced pluripotent stem cells formed?

Answer 21

Transcription factors associated with pluripotency stimulate introduction of certain genes (e.g., c-Myc, Klf4, Oct4, Sox2) which induce reprogramming to a more immature phenotype.

Question 22

What factors are necessary for growth of stem cells?

Answer 22

Defined conditions – for example, growth factors and hormones expected or known to favour growth and differentiation to a particular cell type. These can be added individually or in combination to culture medium.

Bioreactors (mass production/scaleup of cell numbers)

Physical forces/3D interactions/scaffolds
o cell numbers, cell-cell, cell-matrix/scaffold interactions
o Mechanical forces (bone, muscle tissues compared to liver or gut)
o oxygen tensions (lower in most tissues than external environment)

Gene transfection – inducing expression of proteins like transcription factors

Question 23

Where do all embryonic stem cells for use in labs and therapeutics have to be sourced from?

Answer 23

UK Stem Cell Bank run by the National Institute for Biological Standards and Control.

Question 24

What are the different types of cloning?

Answer 24

Cell cloning - Creation of a line of cells genetically and phenotypically identical to the originating cell.
Therapeutic cloning - Reprogramming the nucleus of an adult cell by transfer to the cytoplasm of an enucleated oocyte (somatic cell nuclear transfer) and isolating ES cells after formation of blastocysts in vitro.

Reproductive cloning - Reprogramming the nucleus of an adult cell by transfer to the cytoplasm of an enucleated oocyte (somatic cell nuclear transfer) and re-implanting the embryo to enable formation of a viable foetus. This is banned.

Question 25

How might reproductive cloning be achieved without the use of an oocyte?

Answer 25

Identification and purification of cytoplasmic/nuclear re-programming factors could revert mature cells into stem cells and transform into the desired cell type. Without use of an oocyte, this could negate some ethical issues.

Question 26

What are organoids?

Answer 26

3D cell culture systems/models where stem cells are cultured in environments with defined cytokines, growth factors, and ECM, and self-organise to form or mimic elements of tissues and organs.

Question 27

Other than ethics, what is another issue of reproductive cloning?

Answer 27

Can be successful, but fraught with challenges including low success rates and significant genetic/developmental abnormalities. In the case of Dolly, 238 embryos were created to produce only one viable one. However, this could be used for conservation of endangered species.

Question 28

What is transgene technology?

Answer 28

Introducing human genes into animal embryos to produce therapeutic proteins (e.g., to encourage production of clotting factors in milk of sheep),

Question 29

How are synthetic embryos produce

Answer 29

By mixing embryonic stem cells and related stem cells which self-organise to form something which imitates the early stages of the embryo in vitro.

Question 30

What are the different types of grafts and their pros and cons?

Answer 30

Autografts – using tissues from the patient’s own body for transplanting into another site in the same patient e.g. bone, skin, blood vessels etc
o good clinical outcomes, little rejection
o - collection limited, creates two wound sites, risk of pain and infection

Allografts – using tissue from a donor (living or dead) and transplanting into another patient e.g. kidney, heart, lungs, liver, bone marrow, cornea
o + can restore normal function
o - life-long immunosuppressant therapy, shortage of donors, disease history

Xenografts –using tissues and organs from animals for transplantation into human e.g. primate, monkey and porcine organs and tissues
o + availability
o - rejection, disease transmission (zoonoses), ethics

Lab-made materials & devices – e.g. artificial hearts, heart valves, prosthetic hips, kidney dialysis, liver support devices etc.
o + fills immediate short-term needs
o - material fatigue, toxicity/corrosion, infection risks

Question 31

What is the issue with MSCs in trial?

Answer 31

Encouraging pre-clinical animal data does not seem to transfer to human clinical studies because:
* Immune compatibility – often the mice containing human cells in clinical trials have been bred or altered to remove their immune system, which may not be the best way to study them.
* Dosing – dosing for mice and humans are vastly different, and mice receive much higher doses per kg.
* Fitness of culture adapted MSCs – in animal trials, the cells are extracted then almost immediately reinserted. However, in humans, there can be weeks of expansion and cryopreservation where the cells are sat on a plastic petri dish rather than in the body and this can affect the cell itself.

Question 32

What are some of the key differences between murine pre-clinical studies and industry sponsored phase III MSC clinical trials?

Answer 32

Murine:
Overwhelmongly synergistic - genetically similar/identical cells and therefore immunocompatible.
Fresh cells.
50mil cells/kg IV.
Positive clinical outcomes.

Human:
11:8 allogenic:autolougous cells
Thawed cells
2mil cells/kg IV
Less sueccessful outcomes.

Question 33

What is Alofisel?

Answer 33

An adipose MSC treatment used for Crohn’s related entercoutaneous fistular disease - abnormal connection that develops between the intestinal tract or stomach with the skin leading to the contents of the stomach or intestines leaking through the skin.

120 million allogenic expanded addipose stem cells (eASCs) in 24ml are delivered at the cutaneous site of disease (intrafistular) to produce a local mass effect which fills the gap caused by the fistula.

Question 34

What are the different mechanisms of actions of MSCs? (7)

Answer 34

Differentiation – directly change into the tissue of interest to replace cells.

Paracrine effects – secrete cytokines and can affect inflammation which impacts their immunomodulatory function, as well as promote tissue rescue/repair. .

Exosome secretion – can secrete vesicles containing proteins, mRNA, and miRNA to target cells which can contribute to cell differentiation.

Cell-cell effects – MSCs directly interact with their environment and surrounding cells either by transfer of organelles or molecules by tunnelling nanotubes. Stem cell proteins can stimulate endogenous cell differentiation.

MSC-cell fusion – can fuse with existing cells and transfer genetic material causing them to change their responses.

Mitochondrial transfer – suspected but not well studied.

Efferocytosis – can trigger apoptosis which induces efferocytosis (the effective clearance of apoptotic cells by professional and non-professional phagocytes). The process is mechanically different from other forms of phagocytosis and involves the localization, binding, internalization, and degradation of apoptotic cells. The process of efferocytosis has secondary regulatory mechanisms for the immunomodulation of inflammation or a disorder.

Question 35

What are some examples of cytokines secreted by MSCs and their effects?

Answer 35

VEGF and PDGF promote angiogenesis
SDF and IGF promote mitogenesis
PGE and other cytokines involved in inflammation.

Question 36

What is MSCs anti-inflammatory effect?

Answer 36

Cross-talk with macrophages to cause production of IL-10 (anti-inflammatory cytokine).
Also, their apoptosis or display of surface markers of cell injury such as phosphatidylserine can trigger efferocytosis, which causes an anti-inflammatory and immunosuppressive effect.

Question 37

What are some examples of advanced therapy medicinal products?

Answer 37

• Gene therapy medicines - contain genes that lead to a therapeutic, prophylactic, or diagnostic effect. Involve the insertion of ‘recombinant’ genes into the body, to treat genetic disorders, cancer or long-term diseases.
• Somatic-cell therapy medicines - contain cells or tissues that have been manipulated to change their biological characteristics or cells or tissues not intended to be used for the same essential functions in the body. They can be used to cure, diagnose, or prevent diseases.
• Tissue-engineered medicines - contain cells or tissues that have been modified so they can be used to repair, regenerate, or replace human tissue.

Question 38

What is limbal stem cell deficiency (LSCD)?

Answer 38

A condition caused by ocular burns which destroy the limbus. Results in bulbar conjunctival cells invading the conrneal surface in an attempt to reform the epithelium, but resulting in neovascularisation, inflammation, an stromal scarring and opacity.

Question 39

How is HOLOCLAR used to treated LSCD?

Answer 39

Uses ex vivo autologous human corneal epithelial cells containing stem cells to engineer tissues that will replace the epithelial cells in the damaged cornea.

1. Extract limbeal stem cells.
2. Determine amount of holoclones (most proliferative clonogenic keratinocytes) by measuring expression of Np63a, the gene coding for transcription factor 63 which correlates with clinical success.
3. Grow cells in lab and culture them on fibrin matrix.
4. Remove conjunctival cell/white layer covering corena.
5. Graft limbic cell covered fibrin matrix onto eye.
6. Replenished limbal cells can now renew outer layer of cornea and patient can see again.

Question 40

What is the process of HOLOCLAR?

Answer 40

1. Limbal biopsy from healthy eye
2. Enzymatic dissociation
3. Limbal stem cells produced.
4. Primary culture expansion – cells are seeded onto irradiated mouse feeder cell layer (antibiotics present).
5. Cryopreservation
6. Thawing – cell suspension of epithelial cells Is obtained.
7. Secondary culturing - cells are plated on a fibrin matrix with irradiated mouse feeder cell layer (no antibiotics)
8. Discs of fibrin carrying autologous cells are prepared and packaged.
9. Cells grafted on to injured eye.

Question 41

What is stem cell tourism?

Answer 41

Where patients visit private clinics all over the world which advertise miraculous stem cell therapies for a wide range of incurable diseases.

Question 42

What are the main issues with stem cell tourism?

Answer 42

There is no regulation around this in many countries, so these clinics stem cell therapies have often not been proven for safety or efficacy, and have resulted in the deaths of many patients.

Question 43

What are some key characteristics of an unproven cell therapy?

Answer 43

• Unclear scientific rationale to suggest potential efficacy
• Lack of understanding on the mechanism of action and/or the biological function to support clinical use
• Insufficient data from in vitro assays, animal models and clinical studies regarding the safety profile to support the use in patients
• Lack of a standardized approach to confirm product quality and ensure consistency in cell manufacturing
• Inadequate information disclosed to patients to enable proper informed consent
• Use within non-standardized or non-validated administration methods
• Uncontrolled experimental procedures in humans

Question 44

What are some regulatory barriers to cellular based therapeutics?

Answer 44

• Concerns regarding evidentiary standards
• Need to navigate through convoluted regulatory pathways
• Time ‘lag’ between technological progressions and accompanying regulation
• Challenges in design of clinical trials – e.g. insufficient number of patients for conventional trials
• Ethics of randomising potentially terminal patients into placebo arms (when there appears to be a medicine targeted to their specific disease)

Question 45

What is PRIME?

Answer 45

A scheme run by the European Medicines Agency (EMA) to enhance support and expedite approval for the development of medicines that target an unmet medical need.

Question 46

What are some key considerations for ATMP regulation?

Answer 46

• Is the product an ATMP? Depends on the mode of action.
• Is the product for a rare disease? If so, may be eligible for orphan medicinal product designation:
  o intention to treat, prevent, or diagnose a disease that is life threatening or chronically debilitating
  o prevalence of the condition in the EU of not more than 5 individuals in every 10,000 members of the population, or marketing of the medicine would not generate sufficient returns to justify the investment needed for development
  o no satisfactory method of diagnosis, prevention, or treatment of the condition or, if such a method exists, the medicine must be of significant benefit to those affected by the condition.
• Is the product for paediatric use? – Requires paediatric investigation plan for clinical studies in children.
• Has the product been demonstrated to be safe, efficacious and of suitable quality for use in humans? Info obtained from progressive non-clinical studies (e.g., animal models of disease) and human clinical trials.

Question 47

How does ATMP product development differ to that of normal medicines?

Answer 47

Product development for normal medicines:
1. Nonclinical studies - safety and moA tested in animals/in vitro
2. Phase I clinical trials – safety and pharmacokinetics determined.
3. Phase II clinical trials – efficacy (proof of concept) and doses determined.
4. Phase III clinical trials – efficacy determined.
5. MAA awarded
6. Post approval safety – Post MA studies and real world evidence

Product development for ATMPs:
1. Nonclinical studies – safety and moA test in animals/in vitro
2. Phase I/II combination clinical trials – efficacy (proof of concept) and safety/dose finding
3. Phase III clinical trials – larger efficacy trial.
4. MAA awarded
5. Post approval safety – Post MA studies and real world evidence

Question 48

Why did Holoclar not have to undergo the usual testing for ATMP regulations?

Answer 48

It has already been in use for many years and therefore had a culmination of data proving the efficacy and safety, as well as long-term effects of the medicine.
They did have to later their manufacturing process to make it GMP compliant, but was awarded a conditional MA in 2015.

Question 49

What does a marketing authorisation (MA) require?

Answer 49

Regulatory review of quality, safety, and efficacy data generated during clinical development (prior to MA). Clinical development and product manufacturing must comply with standards and requirements, such as principles of good clinical practice and Good Manufacturing Practice.

Question 50

Who regulated medicinal products in the UK?

Answer 50

European Medicines Agency (EMA) - provides MA for European market.
MHRA