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Flashcards in lecture 9 Deck (39):
1

What is a stem cell?

Two unique defining attributes:
1. the ability to differentiate into many different cell types
2. the capacity for self renewal

2

Where has a lot of the knowledge of stem cells come from?

- the study of embryonic cells - early stages of development
- generally the capacity to proliferate at very early stages is very strong i.e. high cell cycle rate/rate of proliferation
- slows down as the organism develops into a larger mass of cells that has a more complex structure
- depends on the species
- mammalian cells divide slower than invertebrate

3

At which stage in development do you begin to see 'structure'?

- blastocyst
- prior to this: morula relatively amorphous, 2/4/8 cell stage just like bunches of grapes - nothing to differentiate between them

4

What is the structure of the blastocyst?

- inner cell mass
- blastocyst cavity
- all surrounded by trophoblast

5

What generally forms the placenta?

- trophoblast cells

6

What cells give rise to the embryo?

- inner cell mass (essentially)

7

What happens at gastrulation?

- concentration gradients of certain factors
- massive amount of migration
- fold
- balloon inwards
- formation of spinal cord and brain (neurulation)

8

What is the movement of the blastocyst?

1. 7 days: blastocyst starts to implant in wall of uterus
2. 8 days: trophoblast takes root in wall of uterus
3. 9 days: amniotic cavity grows
4. 10-11 days: embryo fully implanted

9

What is a key event in early development?

"It is not birth, marriage, or death, but gastrulation which is truly the most important time in your life" - lewis wolpert (1986)
- gastrulation is a crucial time in the development of multicellular animals
- during gastrulation, several important things are accomplished:
1. the three primary germ layers are established: ectoderm, mesoderm, endoderm.
2. The basic body plan is established, including the physical construction of the rudimentary body exes.
3. Cells move into new positions, allowing them to interact with new neighbouring cells. This leads to inductive interactions, which are the hallmark of neurulation and organogenesis.

10

What happens to stem cells in the inner cell mass?

- commit to become specified to one of the three germ layers: endoderm, mesoderm, ectoderm
- however if you take a cell out of the ICM prior to gastrulation you can get them to differentiate into (almost) any cell type

11

What is important in getting stem cells to differentiate?

- understanding the requirements of stem cells in terms of the chemicals/mediators that induce specific differentiation

12

What are the major fates of the three germ layers?

Ectoderm:
- skin cells of epidermis
- neuron of brain
- pigment cell

Mesoderm:
- cardiac muscle
- skeletal muscle
- tubule cell of kidney
- red blood cells
- smooth muscle

Endoderm:
- lung cell (alveolar cell)
- thyroid cell
- pancreatic cell

(also germ cells produce sperm and egg)

13

What is homeostasis? How do stem cells contribute to homeostasis?

- The ability to regulate internal conditions, usually by a system of feedback controls
- stabilise health and functioning, regardless of the outside changing conditions
- one piece of homeostasis is the constant or periodic generation of new cells to replace old, damaged, and dying cells
- adult stem cells fulfill this role through the process of regeneration

14

How are stem cells activated in AMI?

- ischemic injury to the myocardium causes the release of chemokines (e.g. G-CSF, SCF, SDF-1)
- mobilise quiescent stem cells from the bone marrow to peripheral circulation
- move through the blood to the site of injury through chemotaxis
- populate area of injury and attempt to cover lost tissue
- capacity of stem cells to regenerate tissue is limited
- experimentally there are conditions that seem to make this better

15

How are stem cells activated in brain injury?

- different types of nerve cells: e.g. microglia (immune-type cell that mainly sits in CNS), glial cells, neurons
- importantly - neural stem cells
- in human brain it was only 16 years ago that it was proven that the human brain has adult human stem cells (only make up a few thousand of the billions and billions of nerve cells in the human brain)
- only sit in very defined locations in the brain - not randomly distributed throughout

Hypoxia or some other trauma:
- reactive microglia (developmentally related to macrophages) start releasing chemokines and cytokines after injury
- triggers a number of other cell types e.g. astrocytes
- nerve stem cells begin to divide much more rapidly - rapid response
- huge activation of stem cells in their localised region that then differentiate and migrate towards the site of injury

- unfortunately brain injury when it's relatively severe, there is no capacity to regenerate to an extent that allows complete recovery
- polemic seems to be that the capacity of neural stem cells to repopulate is just not enough to recover all the connections correctly
- functional aspects not well understood, nor do they seem to occur perfectly

- hope is that we can develop some technology that improves the capacity of neural stem cells/ the brain to recover

16

What are the major stem cell types?

- there are four main types of naturally occurring stem cells that are being investigated for their potential use in research and medicine
- they differ in their degree of differentiation and ability to self-renew
-- embryonic stem cells (ES cells)
-- embryonic germ cells
-- adult stem cells
-- umbilical cord and placenta stem cells (thought to be useful for prospective therapy)

17

What are embryonic stem cell lines?

- cell lines isolated from a blastocyst and maintained as an ongoing/frozen line
- can be grown indefinitely
- importantly these cells can be used for scientific experiments and in the case of mice, can be genetically modified and an ES cell derived mouse generated

18

What are the commitment options available to a cell?

- totipotent - every single differentiated cell type in the body
- pluripotent - embryonic stem cells
- multipotent
- oligopotent
- unipotent - can only differentiate to one cell type

- cord blood is a combination of pluripotent and multipotent stem cells

19

Compare adult and embryonic stem cells

Adult stem cells
- typically reside in or near their tissue
- are capable of giving rise to functional cells in their tissue (but not other tissue types)
- are typically found only in tissues that undergo regular turnover
- decrease in both number and activity as one ages

embryonic stem cells
- created from inner cell mass cells that exist only in very early embryos
- are capable of giving rise to all of the cell types in the body including non-regenerative
- can be divided many times (possibly indefinitely) in culture to make many cells

20

What are umbilical cord and placenta stem cells?

- isolated immediately following birth
- more flexibility = some pluripotent characteristics
- research is limited but growing
- can be easily isolated and banked (c.f. bone marrow)

21

What lessons about stem cells can we learn from the haemopoietic system?

- pluripotent haematopoietic stem cell (HSC) resides in bone marrow
- differentiates into either myeloid stem cells or lymphoid stem cells: each of these has a more restricted 'fate' but still capable of self-renewing
- evident that very specific factors guide these stem cells to differentiate into specific cell types
- well defined and understood systems

22

What are stem cell niches?

- in their natural state, stem cells exist in a microenvironment supporting their maintenance and normal function – this is the 'stem cell niche'
- e.g. in bone marrow, HSCs need to be right next to stromal cells

23

What is plasticity?

- the capacity of a stem cell to divide and how many different types it can divide into
- plasticity correlates with potency

24

What does plasticity relfect?

Developmental origins
- adult stem cells derived from different organisms could potentially, under the right conditions, differentiate into different cell types of organs
BUT only in organs related to them developmentally

i.e. ectodermal origin stem cells to ectodermal organs e.g. CNS, epidermis etc

25

What is somatic cell nuclear transfer?

- sometimes referred to as "therapeutic cloning)
- asexual reproduction
- no sperm involved
- transfers nucleus from a mature cell into a donor egg
- requires electric or chemical stimulus to begin dividing
- functionally different from regular fertilised egg
- proven in 1997 - Dolly the sheep

26

What is the purpose of SCNT?

- find cures and therapies for diseases
- awaken the natural capacity for self-repair that resides in our genes

27

What are the potential results of SCNT?

- patients will receive own stem cells to treat disease
- no need for donor match
-- like transplantation, but without rejection

28

Describe the experiment that led to the formation of Dolly.

1. mammary cell donor - cultured mammary cells are semistarved, arresting the cell cycle and causing dedifferentiation
2. egg cell donor - nucleus removed
3. cells fused - nucleus from mammary cell
4. grown in culture
5. early embryo implanted in uterus of a third sheep
6. embryonic development
lamb "dolly" genetically identical to mammary cell donor

29

How would SCNT treat disease?

- donor nucleus
- take skin cells from patient (with. eg. diabetes)
- produce somatic cell nuclear transfer clone
- produce lots of cells under synthetic conditions
- get them to transform (somehow) into pancreatic stem cells and transplant them into patient
- i.e. cure diabetes

30

What is another stem cell therapy?

- take HSC from patient
- make stem cells and transplant back into patient

31

What is the problem with stem cell use in humans thus far?

- well documented and published papers of patients who have undergone experimental stem cell therapies
- conditions weren't well understood
- a small number generated tumours that were derived from the stem cells
- link between a number of the pathways in stem cells and cancer

32

How can stem cells potentially be used for regenerative medicine?

- with stem cell therapy (embryonic or adult), there is enormous promise of treating diseases previously thought to be unmanageable

33

What is the main ethical/moral issue associated with stem cell use?

- the question is not whether or not to use stem cells
- the question is whether to use adult or embryonic stem cells

34

What is the goal of molecular reprogramming?

- manipulate a somatic cells potency
- i.e. artificial generation of stem cells
- paper in 2006 found 4 factors that when expressed could induce formation of a pluripotent stem cell from a fibroblast

35

What are iPSs? How could they be used in treatment?

- induced pluripotent stem cells
- take somatic cell from patient, induce pluripotency using particular factors and grow these - produce lots of the cell type of interest and potentially transplant back into patient
- done experimentally quite readily
- perhaps genetically modify stem cells before replacing

36

What is stress stimulus-triggered acquisition of pluripotency?

- three stressors - a bacterial toxin that perforates the cell membrane, exposure to low pH and physical squeezing - were each able to coax the cells toward pluripotency
- 30x more effective than iPS

37

What are the advantages and problems of different stem cell therapies based on cell type?

ES
+
- grow well
- pluripotent

- non-self
- directed differentiation
- ES contamination in product

SCNT
+
- self

- inefficient
- oocyte supply

iPSC-self (therapeutic cloning)
+
- grow well
- pluripotent
- self

- directed differentiation
- labor intensive
- inefficient

neonatal (e.g. cord blood)
+
- availability
- could be self

- growth
- numbers

Adult stem cells
+
- limited plasticity
- could be self

- grow poorly
- accessibility-numbers

38

What is the coolest thing ever

synthetic heart (kinda)

39

What is the risk of stem cells? How can this also be useful?

- a lot of the pathways active in stem cells are also active in cancer cells
- existence of cells called tumour-initiating cells
- behave like stem cells
- aren't genetically completely normal
- give rise to tumours
- understanding how these differentiate from normal stem cells will hopefully enable us to understand how to treat cancers more efficiently

currently:
- use drugs that kill tumour cells but not cancer stem cells
- tumour shrinks but grows back

future:
- drugs that kill tumour stem cells
- tumour loses its ability to generate new cells