Lectures 19 & 20 - Stem cells

Question 1

Define a totipotent cell

Answer 1

have unlimited capability. Can develop to all postembryonic tissues and organs.

Question 2

Define a pluripotent cell

Answer 2

stem cells capable of giving rise to most tissues of an organism

Question 3

Define a multipotent cell

Answer 3

stem cells that are specialized to give rise to a few particular cell types.

Question 4

What is complicated in the stem cell research field?

Answer 4

Theres a lack of specific stem cell markers

no single one, need to do at least two three markers before you can be confident a stem cells been identified

may still even need to test properties e.g. self-renewal, to check they’re stem cells

Question 5

What can be said about adult stem cells?

Answer 5

-very limited symmetric self-renewal in vitro
- not necessarily from an adult organism, mean tissue specific stem cells
undifferentiated cell occurring in a differentiated tissue
e.g.s of tissue - Bone marrow, blood, skeletal muscle
- usually quite rare in a tissue

Question 6

What are embryonic stem cells

Answer 6

• derived from a group of cells called the inner cell mass, which is part of the early (4- to 5-day) embryo called the blastocyst

-Once removed from the blastocyst, the cells of the inner cell mass can be cultured into embryonic stem cells,
which are pluripotent

Question 7

What discovery of stem cells occurred in 1981

Answer 7

derived mouse embryonic stem (ES) cells from the inner cell mass of blastocysts

establish culture conditions for growing pluripotent mouse ES cells in vitro

(Evans, Kaufman, and Martin)

Question 8

After the initial discovery of mouse embryonic stem cells in 1981, what were the next stem cells discoveries?

Answer 8

1992: Neural stem cells are identified in the adult human brain

1996: The first mammal cloned from adult cells was Dolly the sheep, using Somatic Cell Nuclear Transfer (SCNT)

2006-2007: Yamanaka and Yu produced ES-like cells from human fibroblasts and are called induced pluripotent stem cells (iPSC)

Question 9

What are iPSCs

Answer 9

induced pluripotent stem cells

Question 10

Explain self-renewal symmetric cell division

Answer 10

one stem gives rise to two identical daughter cells

Question 11

Explain asymmetric cell division

Answer 11

a stem cell gives rise to one stem cell and one progenitor cell (two daughter cells not same)

Question 12

What is terminal symmetric cell division?

Answer 12

A progenitor gives rise to two progenitors (no stem cells left to differentiate)

Question 13

What are the different ways to generate an asymmetric division?

Answer 13

1. factor in the cytoplasm of the dividing stem cell, gathered at one end of the dividing cell, and will end up in the progenitor cell. e.g. cell polarity regulator found only on stem cell half, it will not differentiate.
2. environment/niche tells the cell either to differentiate or stay as a stem cell. Stem cell divides into two daughter cells (symmetric) but then one cell migrates/changes environment which provides a signal that the cell needs to differentiate itself

Question 14

What are features of the Hematopoietic stem cells (HSC)

Answer 14

• one of the first adult stem cells to be discovered with a well characterised lineage
• go from Long-term HSCs (LT-HSCs) that can self renew to short term (ST-HSCs) that can only renew a few times
• ST-HSCs then start to differentiate into a multipotent progenitor (MPP)
• MPP goes to CLP (common lymphoid progenitor) and CMP (common myeloid progenitor)
• these will in turn, change into all the different cell types of that progeny

Question 15

Explain the difference in marker expression between LT-HSCs, ST-HSCs and MPPs

Answer 15

LT –> ST = CD34 low –> high
ST –> MMP = FLK2 low –> high

Question 16

Explain features of neural stem cells (NSC)

Answer 16

Give rise to particular cells of the neural tissue: oligodendrocytes, astrocytes, and neurons

don’t give rise to microglia, endothelial cells or blood vessels of the brain

found along the ventricles of the developing brain

originally discovered in the subventricular zone of the adult brain

found in many regions of the adult brain but not all equivalent (some latent and some active) - sometimes problem to show multipotentcy in cell culture

Question 17

Explain NSCs are seen in the subventricular zone of the adult human brain

Answer 17

NSCs positive for marker GFAP

Question 18

Where does NSC give rise to new neurons in rodents

Answer 18

olfactory bulb, have to migrate to this
granule cell layer of dentate gyrus

Question 19

What function does neurogenesis play in the healthy brain?

Answer 19

Olfactory bulb:

Neurogenesis in the olfactory bulb has not yet been shown in human adults, only rats

papers that showed uses for neurogenesis:

-ongoing integration of new and different smells Gheusi et al (2000)

-blocking olfactory neurogenesis had no effect on simple olfactory discrimination and memory tests Imayoshi et al (2008)

Dentate gyrus:

-0.02% (human) new neurons every day

new neurons:

• increase memory capacity Becker S (2005)
• reduce interference between memories Wiskott et al (2006)
• role in emotional control and affective behaviour Samuels and Hen (2011) Kheirbek et al (2012)

Question 20

How is neurogenesis regulated?

Answer 20

Events that increase neurogenesis:
exercise, learning, enriched environment, seizures, oestrogen, odours, calorie restriction

Events that decrease neurogenesis:
stress, age, inflammation, alcohol, lack of sleep

Injuries and diseases: ischemia, epilepsy, meningitis, neurodegenerative diseases

Drugs and treatments: opiates, anti-depressants (Prozac), vs irradiation

Question 21

Explain features of gut stem cells (GSCs)

Answer 21

A population of adult stem cells

found in small intestine

give rise to different progenitors and to the differentiated cell types of the gut: Paneth, goblet, endocrine, columnar

found at the bottom of the crypt in the gut

Question 22

What is a niche

Answer 22

Microenvironment around stem cells that provides support and signals regulating self-renewal and differentiation

Question 23

What are the different ways that niches can interact with stem cells?

Answer 23

Direct contact with niche sells
Soluble factors that diffuse through tissue
Through an intermediate cell

Question 24

Explain the LIF signalling pathway

Answer 24

LIF = leukemia inhibitory factor

signalling pathway that affects stem cells

important as LIF is important in culturing embryonic stem cells

LIF acts on its receptor and activates different signalling pathways ending with phosphotylation and activation of transcription factors leading to activation of different genes and self renewal

one pathway LIF activates is: STAT3 –> Kif4 –> SOX2

Question 25

Explain the Wnt signalling pathway

Answer 25

In absence of Wnt: b-Catenin is sequestered in the cytoplasm my the APC complex through phosphotylation and ubiquination by the proteasome When Wnt is present: it is linked the receptor frizzled The APC complex is disassembled b-Catenin is not sequestered and can accumulate translocates to nucleus and activates the transcription Wnt target of genes

Question 26

Explain the notch signalling pathway

Answer 26

Notch is at the membrane, binds to ligand called delta jagged Enzymes ADAM/TACE cleave notch into three The notch intracellular domain ends up in the cytoplasm and translocates to the nucleus and forms a complex with other proteins, activating notch target genes

Question 27

Explain the sonic hedgehog signalling pathway

Answer 27

key pathways for didget and limb generation in developmet aswell as brain development Hedgehog is secreted by neighbouring cell and then binds to a receptor complex patch and through different activaton of signalling pathways end up with activation of gli In the precence of hedgehog Gli can bind to CBP in the nucleus and activate transcription of different genes (one of the target genes is Wnt)

Question 28

What different therapeutic strategies can use stem cells

Answer 28

Tissues/cells for transplantation Toxicity testing of drugs Identify drug targets and test potential therapeutics study cell differentiation

Question 29

What are the sources of adult stem cells

Answer 29

different tissue - either from a living or deceased donor depending on the source e.g. brain only after death of donor, skin and umbilical cord can be from living donors when you get peice of tissue, adult stem cells are very rare, and difficult to identify, isolate and purify so need to have a big chunck of tissue (sometimes not possible) or optimise the techniques to amplify them in vitro for the cells to proliferate

Question 30

What are the sources of embryonic stem cells

Answer 30

From in vitro fertilisation - allow fertilisation of egg and sperm, then development to blastocyst stage can isolate some of the ICM stem cells and derive the embryonic stem cell from there OR Somatic cell nuclear transfer (SCNT) take an egg cxell but remove nucleus from egg, as well as a donor cell, nucleus from donor cell goes into the egg. In the egg there are some factors that reprogramme the nucleus of the somatic cell to a totipotent state, creating an artificial embryo that you then allow to develop to blastocyst stage and then again harvest from ICM

Question 31

Explain a recent SCNT that was successful

Answer 31

2018 - Zhen Liu et al. - cloning of macaque monkeys by somatic cell nuclear transfer - managed to do it in primates almost 20 years after originally done - far from having technique as clinical strategies

Question 32

Explain the induction of pluripotent stem cells

Answer 32

Somatic cells are obtained from adult organism the reprogramming factors (Oct4, Sox2, Klf4, c-Myc) are introduced into the cultured somatic cells the cells are grown under ES cells conditions. After 2-3 weeks, iPS (induced pluripotent stem cells) cells emerge These iPSs may be differentiated into various cell types for regenerative medicine applications

Question 33

Cite two experiments that showed the induction of pluripotent stem cells without need for Oct4

Answer 33

Velychko et al. (2019) An et al. (2019)

Question 34

Explain disadvantages of iPSC

Answer 34

Low efficiency Genomic insults (mutations, insertions/deletions, chromosomal rearrangements)

Question 35

What are the three different techniques for getting pluripotent stem cells

Answer 35

ES cell lines Somatic cell nuclear transfer IPS cell lines ES and somatic involve the generation and destruction of an embryo IPS advantage as doesn't require embryo use

Question 36

Can all pluripotent stem cell generation techniques (and adult stem cells) have application to human cells

Answer 36

Yes

Question 37

Are all pluripotent stem cell generation techniques (and adult stem cells) prone to tumour formation

Answer 37

No - adult stem cells are not as much

Question 38

Can all pluripotent stem cell generation techniques (and adult stem cells) produce all cell types

Answer 38

No - adult cells cannot

Question 39

Do all pluripotent stem cell generation techniques (and adult stem cells) have easy/large access

Answer 39

NO - only ES cells from in vitro fertilization

Question 40

Can all pluripotent stem cell generation techniques (and adult stem cells) be genetically matched to a patient

Answer 40

NO - ES cells from in vitro fertilization cannot be

Question 41

Can all pluripotent stem cell generation techniques (and adult stem cells) be for use if embryos

Answer 41

NO - only ES cells from IVF and ES cells from somatic cell nuclear transfer

Question 42

Are all pluripotent stem cell generation techniques (and adult stem cells) likely to contain DNA abnormalities

Answer 42

NO - ES from IVF is NOT likely to contain abnormalities

Question 43

What is the proposed new definition for stem cell therapy?

Answer 43

type of cell therapy in which therapeutic efficacy is exclusively attributed to the potency (function) of donor stem cells, presented in any quantity and purity.

Question 44

What were the problems with defining stem cell therapy

Answer 44

Problems of quantity, purity, and function : - cannot rely just on the presence of stem cells in transplanted cell suspension/ tissue (ex: blood transfusion) - can not rely purely on degree of stem cell enrichment (purification) in transplanted cell suspension/ tissue (ex: HSC transplantation in leukemia with bone marrow transplant) - we should solely rely on anticipated mechanism of therapeutic action, which should be exclusively attributed to stem cells (not to progenitor or mature cells)

Question 45

What was one of the first stem cell treatments

Answer 45

Bone marrow transplant (leukemia) 2 types : 1. Allogeneic - with a donor. Have patient with disease, needs bone marrow transplant which comes from donor 2. Autologous - patients bone marrow harvested before chemo, bone marrow frozen whole patient had therapy and manipulated, bone marrow re-infused after chemo

Question 46

Explain in more detail the autologous transplant process

Answer 46

1. collection of stem cells from patients bone marroe 2. processing of bone marrow in lab (to purify and concentrate) 3. Cryopreservation to preserve 4. Chemotherapy and/or radiation therapy given to patient 5. Reinfusion - Thawed stem cells reinfused into the patient

Question 47

Explain differences between Allogenic and autologous stem cell therapy

Answer 47

Allogenic: - tissue matching required - tissue rejection - slower engraftment - slower immune recogition - possibility of diseases from donor e.g. HIV, Hepatitis Autologous: - tissue matching not required - no tissue rejection - faster engraphment - faster immune recognition - no possibility of diseases

Question 48

Explain combined stem cell therapy with gene therapy

Answer 48

Usual Direct delivery: - Therapeutic gene is packaged into a delivery vesicle such as a retrovirus - injected into the patient What you can do is: cell based delivery: - instead of directly delivering gene - in vitro, inject this therapeutic gene, allow to differentiate and proliferate if needed - deliver to stem cells that will be injected into the patient - either adult SCs from patient or ES cells from lab

Question 49

Explain an example in mouse models of personalised medicine using iPS cells

Answer 49

Mice with sickle cell anaemia caused by genetic mutation 1. skin cells collected 2.reprogram into ES like-iPS cells using Oct4, Sox2, KIf4, c-Myc viruses 3. mutation corrected 4. Genetically correct iPS cells differentiated into blood stem cells 5. transplanted into mouse mouse recovered

Question 50

Explain drug screening using human iPS cells

Answer 50

Adult stem cells can be differentiated into lots of different cell types e.g. three germ cell layers - mesoderm - endoderm - ectoderm all the cells that these differentiate into can be use to test for drugs either to show efficacy or to show toxicity

Question 51

Explain manipulation of endogenous stem cells as a therapeutic

Answer 51

gene vectors for gene therapy targeting stem cells so they will produce new differentiated cells that dont carry that disease marker can also be done by inserting proteins or particular small molecules

Question 52

Explain the advantages and disadvantages of gene therapy as a mo0de of delivery for stem cell therapy

Answer 52

A: Ability to directly reprogram stem cells in situ D: Dependent on development of safe and efficient delovery vectors that can cross the BBB precise control of gene expression may be challenge

Question 53

Explain the advantages and disadvantages of proteins as a mode of delivery for stem cell therapy

Answer 53

A: - direct and transient delivery to stem cell population - more control over course of administration D: - BBB may prevent systemic administration of particular proteins - side effects

Question 54

Explain the advantages and disadvantages of small molecules as a mode of delivery for stem cell therapy

Answer 54

A: - high throughput screening makes it easy to identift small n olecules capable of modulating stem cell behavour desired - high purity - can pass through BBB D: - Side effects

Question 55

Explain the strategy for restoring stem cell function when there is a genetic modifiction

Answer 55

Somatic cells located close to the niche are reprogrammed by factors into de novo stem cells

Question 56

Explain the strategy for restoring stem cell function when there is a epigenetic modifiction

Answer 56

Progenitors or differentiated cells can be dedifferntiated into stem cells ans the determination

Question 57

Explain the strategy for restoring stem cell function when there is a transcription factor not working properly

Answer 57

inject factors that will affect the stem cells directly to function normally, and then these can give rise to correct differetiated progeny

Question 58

Explain the strategy for restoring stem cell function when there is a problem in the niche

Answer 58

factors can directly target the niche and restore to normal so stem cells function as normal in the niche

Question 59

Explain the strategy for stem cells to repair Glial degeneration (demyelinating disease)

Answer 59

in damage, Schwann cells die or are destroyed and theres not enough myelin - replace cells, need to be in the right place (so can interact with correct cells) and of right phenotype - means that myelin is replaced

Question 60

Explain the strategy for stem cells to repair paracrine systems (e.g. parkinsons)

Answer 60

Intact: neurons secrete dopamine into environment of other neurone In parkinsons: dopaminergic neurones die so less dopamine secreted repair: other cells secreting dopamine so that neurone can have dopamine it needs to function properly, needs to be dopaminergic cells but doesn't need to be in the same place, don't need to have specific connection with target neurone

Question 61

Explain the strategy for stem cells to repair selective degeneration (ALS, Huntington, ataxia)

Answer 61

-replacement of single phenotype (specific cell that was lost needs to be replaced) - needs to be in the right place and connect with right neurones

Question 62

Explain the strategy for stem cells to repair global degeneration (trauma, stroke)

Answer 62

- replacement of multiple phenotypes (multiple cell types) - need to connect to each other and to the neurones left

Question 63

Explain features of parkinsons

Answer 63

Movement disorder 2nd most common neurodegenerative disorder - degeneration of neurones in the substantia nigra - degeneration of dopemanergic neurones that secrete dopamine in the striatum

Question 64

What are current available treatments for parkinsons

Answer 64

Pharmacological/chemical therapy: - loses its efficacy in several years, side effects Deep brain stimulation: - variable effect: reduce motor symptoms, but serious - adverse events are often associated with the surgical procedure Neural protection with trophic factors: - side effects Cell therapy: - with human foetal dopaminergic neurons (cells from several foetuses needed to treat one patient), inconsistent results due to variable sources of cells (only in animal models and clinical trials)

Question 65

Explain transplant of human embryonic mesencephalon in parkinsons

Answer 65

- reverse some motor symptoms - lower doses of medication (or discontinuation)

Question 66

Explain limitations to transplant of human embryonic mesencephalon in parkinsons

Answer 66

- immune rejection - overgrowth, tumour formation - developmental stage of graft - 90% of cells die during transplant - purity (no contamination with proliferating non-neural cells) - A9 substancia nigra phenotype - durability of the effects - patient selection (only classical Parkinson, early in disease) - graft-induced dyskinesia - pathogenetic process can affect transplanted neurons

Question 67

What molecule was used in rat models to mimic parkinsons so that stem cell transplants could be tested (cite aswell)

Answer 67

6-OHDA - injected into the substancia nigra (Guerra-crespo 2011)

Question 68

Explain direct conversion of differentiated cells to neurones

Answer 68

direct conversion from fibroblast (or other cells) to neurone, without going through stem cell stage a few different methods have been developed, use different factors to convert

Question 69

What are the challenges of direct conversion of differentiated cells to neurones

Answer 69

- ability to improve the reprogramming/direct conversion - combine efficient differentiation protocols with the precise modification of specific genome sequences

Question 70

Summariese the different methods to treat parkinsons with stem cells

Answer 70

Foetal brain, dopaminergic progenitors, inject ES cells differentiated into dopaminergic neurones Induced pluripotent stem cells (iPSCs) again differentiate direct conversion from fibroblast to dopaminergic neurone

Question 71

What are some ethical points that have been raised about the use of stem cells

Answer 71

- some sources of stem cells destroy embryos - uses of stem cells e.g treat life threatening disease = good, to test cosmetics = bad? - what are the rights of people who give embryos for research or donors of stem cells e.g if researcher finds disease in person should they know? - should their be rules in place for what can be done with cell lines? - shoud cross-species experiments be allowed?