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BIOL3043 - Cellular and molecular pathology > Lectures 19 & 20 - Stem cells > Flashcards

Lectures 19 & 20 - Stem cells Flashcards

1
Q

Define a totipotent cell

A

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

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2
Q

Define a pluripotent cell

A

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

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3
Q

Define a multipotent cell

A

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

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4
Q

What is complicated in the stem cell research field?

A

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

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5
Q

What can be said about adult stem cells?

A

-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

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6
Q

What are embryonic stem cells

A
  • 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

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7
Q

What discovery of stem cells occurred in 1981

A

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)

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8
Q

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

A

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)

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9
Q

What are iPSCs

A

induced pluripotent stem cells

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10
Q

Explain self-renewal symmetric cell division

A

one stem gives rise to two identical daughter cells

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11
Q

Explain asymmetric cell division

A

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

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12
Q

What is terminal symmetric cell division?

A

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

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13
Q

What are the different ways to generate an asymmetric division?

A
  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
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14
Q

What are features of the Hematopoietic stem cells (HSC)

A
  • 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
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15
Q

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

A

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

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16
Q

Explain features of neural stem cells (NSC)

A

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

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17
Q

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

A

NSCs positive for marker GFAP

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18
Q

Where does NSC give rise to new neurons in rodents

A

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

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19
Q

What function does neurogenesis play in the healthy brain?

A

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)
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20
Q

How is neurogenesis regulated?

A

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

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21
Q

Explain features of gut stem cells (GSCs)

A

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

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22
Q

What is a niche

A

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

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23
Q

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

A

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

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24
Q

Explain the LIF signalling pathway

A

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

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25
Q

Explain the Wnt signalling pathway

A

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

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26
Q

Explain the notch signalling pathway

A

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

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27
Q

Explain the sonic hedgehog signalling pathway

A

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)

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28
Q

What different therapeutic strategies can use stem cells

A

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

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29
Q

What are the sources of adult stem cells

A

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

30
Q

What are the sources of embryonic stem cells

A

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

31
Q

Explain a recent SCNT that was successful

A

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
32
Q

Explain the induction of pluripotent stem cells

A

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

33
Q

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

A

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

34
Q

Explain disadvantages of iPSC

A

Low efficiency

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

35
Q

What are the three different techniques for getting pluripotent stem cells

A

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

36
Q

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

A

Yes

37
Q

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

A

No - adult stem cells are not as much

38
Q

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

A

No - adult cells cannot

39
Q

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

A

NO - only ES cells from in vitro fertilization

40
Q

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

A

NO - ES cells from in vitro fertilization cannot be

41
Q

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

A

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

42
Q

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

A

NO - ES from IVF is NOT likely to contain abnormalities

43
Q

What is the proposed new definition for stem cell therapy?

A

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.

44
Q

What were the problems with defining stem cell therapy

A

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)
45
Q

What was one of the first stem cell treatments

A

Bone marrow transplant (leukemia) 2 types :
1. Allogeneic - with a donor. Have patient with disease, needs bone marrow transplant which comes from donor

  1. Autologous - patients bone marrow harvested before chemo, bone marrow frozen whole patient had therapy and manipulated, bone marrow re-infused after chemo
46
Q

Explain in more detail the autologous transplant process

A
  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
47
Q

Explain differences between Allogenic and autologous stem cell therapy

A

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

48
Q

Explain combined stem cell therapy with gene therapy

A

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

49
Q

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

A

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

50
Q

Explain drug screening using human iPS cells

A

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

51
Q

Explain manipulation of endogenous stem cells as a therapeutic

A

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

52
Q

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

A

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

53
Q

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

A

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

54
Q

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

A

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

55
Q

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

A

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

56
Q

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

A

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

57
Q

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

A

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

58
Q

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

A

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

59
Q

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

A

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

60
Q

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

A

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

61
Q

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

A

-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

62
Q

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

A
  • replacement of multiple phenotypes (multiple cell types)
  • need to connect to each other and to the neurones left
63
Q

Explain features of parkinsons

A

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

64
Q

What are current available treatments for parkinsons

A

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)

65
Q

Explain transplant of human embryonic mesencephalon in parkinsons

A
  • reverse some motor symptoms
  • lower doses of medication (or discontinuation)
66
Q

Explain limitations to transplant of human embryonic mesencephalon in parkinsons

A
  • 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
67
Q

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

A

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

68
Q

Explain direct conversion of differentiated cells to neurones

A

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

69
Q

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

A
  • ability to improve the reprogramming/direct conversion
  • combine efficient differentiation protocols with the precise modification of specific genome sequences
70
Q

Summariese the different methods to treat parkinsons with stem cells

A

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

71
Q

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

A
  • 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?

BIOL3043 - Cellular and molecular pathology (11 decks)

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