Cell Sources for Regenerative Medicine Flashcards
1
Q
Types of germ cells
A
Sperm
Egg
2
Q
Examples of endoderm cells
A
Pancreatic
Thyroid
Lung (alveolar)
3
Q
Examples of mesoderm cells
A
Cardiac muscle Skeletal Kidney tubule Red blood Smooth muscle
4
Q
Examples of endoderm cells
A
keratinocytes
neuronal
pigment
5
Q
Define cell differentiation
A
Progression from naive/unspecialised cellular state to a more specialised state/fate
- might be accompanied by morphology change
6
Q
Define cell potency
A
Describes developmental potential of a cell
7
Q
Define Totipotent/Pluripotent/Multipotent
A
Stem cells which can adopt multiple fates
8
Q
Examples of totipotent cells
A
Zygotes
Embryonic Stem Cell Colonies
9
Q
Examples of terminally differentiated cells
A
Cartilage
Bone
Skeletal Muscle
10
Q
Properties of terminally differentiated cells
A
Low/no developmental potential
Limited/no capacity for self-renewal
11
Q
Define reprogramming
A
Going from terminal differentiation to potent cells
- changing phenotype of cells
- manipulation in the lab
12
Q
Describe self-renewal
A
- ensures stem cell populations and potency are accordingly maintained undifferentiated
- ensures cell products still have multiple cell fates
13
Q
What are the 2 aspects of self renewal?
A
Balancing act between:
- mitogenesis/growth (making more cells)
- differentiation or specialisation (exiting the cell cycle and growth is attenuated)
14
Q
2 methods of self-renewal
A
1) Asymmetric Division
2) Stochastic Mechanisms
15
Q
Asymmetric Division
A
- every division gives rise to 1 differentiated daughter cell and 1 undifferentiated self renewed stem cell
16
Q
Stochastic Mechanisms
A
- each time a stem cell divides, 2 daughter cells are produced with the potential to yield a differentiated cell or a stem cell
- can produce either type of cell depending on signals that the body gives (has it just been injured so needs clotting factors for example)
17
Q
Define commitment
A
Stem cell commits to a differentiation pathway
- may still have several steps before terminal differentiation
18
Q
Define early progenitor
A
Multipotent cell in the early part of the pathway
- reduced self-renewal
19
Q
Define late precursor
A
Oligopotent cell
In mid-to-final stages of pathway
- may be undergoing changes to morphology
- progressively limited capacity for self renewal
20
Q
Define terminally differentiated
A
Adult mature cell
At end of its differentiation pathway
Usually no capacity for self-renewal
Specialized morphology & function
21
Q
What is the differentiation pathway?
A
Stem cell -> Early Progenitor -> Late Precursor -> Terminally Differentiated
22
Q
What makes up the human genome?
A
- 23 pairs of chromosomes
- 1 inherited from each parent
- 22 autosomes
- 2 sex chromosomes
23
Q
What are housekeeping genes?
A
Genes that are common to all cells
All cells need them
- govern basic cell function/structure/function
24
Q
What are lineage specific or determinant genes?
A
- specific to certain cells
- impact cell fate
- turn on a genetic programme for cell function/cell form
- tend to be transcription factors and genome regulators
- code for proteins that regulate entire networks of genes known as transcription factors
25
What are lineage markers?
- activity is restricted to a cell type of some sort of specific function to that cell
- at bottom of hierarchy
- requires lineage determinant to turn it on at the top of hierarchy
26
Flow of information in biological systems
DNA codes for RNA (transcription)
RNA is functional to make a protein or a non-coding protein (translation)
Post translational modifications and protein folding makes proteins functionable
27
Define progressive canalisation
Irreversible differentiation pathway
As a cell commits to 1 pathway it cannot come back on an opposing pathway (and regain potency)
In the case of multipotent pathways, it gets successively confined to a particular path
28
How do multipotent pathways work?
- give rise to multiple fates
- bifurcations in pathways represent developmental decisions so progenitors are prompted towards 1 particular fate
- inhibition of other fate to ensure no misexpression of genes
- 1 pathway turned on, others are shut down
29
What does the term 'primed' mean?
Gene is not turned on but has the capacity to be turned on
30
What happens during interphase?
DNA decondenses into a mass of chromatin
| In each chromosome mass territory
31
What do transcription factors do?
- induce gene transcription (DNA -> RNA)
| - can also silence gene transcription
32
What is epigenetic regulation
- how the genome is tagged to be active/inactive
- or how the genome is organized (active vs. inactive regions)
- how the genome is organised in a cell type will impact which ones can be turned off/on and therefore determines cell fate & potency
- the 6th level of controlling genetic info. (top of hierarchy)
- can turn cells on and off or silence them
33
What is in vitro differentiation?
- how we derive different cell types in a laboratory setting
34
Steps of in vitro differentiation
- alter cell culture conditions (can drive expansion or particular fate)
- selection for a particular cell type (using FACS or genetic modification to sort cells)
- expansion of desired cell type (with rounds until numbers achieved of population)
- controlled differentiation to specific cell type
- final selection (FACS, morphological features)
35
How does altering cell conditions change cell fate?
- change media (supplements, growth factors, drugs, nanoparticles)
- substrate or scaffold
- physical/mechanical stimuli
36
What is FACS?
Fluorescence Assisted Cell Sorting
37
How is in vitro differentiation done in a lab?
- take a stem cell and grow it under conditions that promote self renewal and do not promote differentiation
- remove factors that promote self-renewal and replace with factors that promote differentiation
- select cells
- repeat steps 2 and 3 until desired cells are achieved in number desired
38
What is an induction signal?
- mechanical or chemical stimuli outside of the cell or at the cell surface
- signal propagates through the cell and terminates at the nucleus changing gene expression via transcription factors
- promotes specific differentiation
- originates outside the cell
- comes about through autocrine/paracrine/endocrine signaling from neighboring cells and ECM (especially mechanical stimuli)
- propagates through cytoplasm, triggering protein-protein interactions
39
What is cell sequestration?
Certain proteins or enzymes may get sequestered (relocated) to a different part of the cell or structure
- due to cell shape changing throughout differentiation
40
What is cell polarisation?
- redistribution of cell contents
- can lead to differentiation following a cell division
- e.g. if a cell elongates, forms a apical-basal polarisation, may split so one daughter cell gets all factors and other gets none
41
Embryo Cell Development
Day 8 - individual cells
Day 8-16 - cells compact
Inner-cell mass between 64-128 cells which goes on to make the embryo, outer cells make placenta (first differentiation step)
42
First mammalian differentiation event
8 cells = apical basal polarisation
16 cells = emergence of inner cells
32 cells = outer/inner
Blastocyst = fates fixed
- takes place during preimplantation development
- establishes outer trophectoderm forming the placenta and inner cell mass = embryo proper
- inner cells are able to keep their pluripotency
- outer cells only exposed to other inner cells
- embryonic stem cells come from inner cell mass
43
What is the source of embryonic stem cells?
- derived from inner cell mass of pre-implantation embryos
- expanded out using culture techniques
- form round colonies in culture
- artefact as we have are artificially holding them in time, this doesn't naturally happen
- control is an issue as can make so many different developmental decisions = risk of cancer as lots of opportunities for mistakes
44
Foetal and Adult Stem Cells
- early progenitor cells
- more limited than embryonic stem cells in terms of capacity for self-renewal and potency
- usually only differentiate into cells which inhabit the niche they develop
- in vivo found in niches (micro-environments) involving other cells with they interact with
- kept in a quiescent state
- less chance of developmental mistakes = cancer
- but cannot make as many different types of cells as limited potency
45
Haematopoietic Stem Cell Niches
- endosteal niche = quiescent HSCs associated with osteoblasts (exit cell cycle)
- perivascular niche (active HSCS, self-renewal)
- mesenchymal stem cells = in bone marrow medulla interacting with stromal cells (bone, cartilage, fat fates)
46
What are the growth characteristics of mouse embryonic stem cells in vitro?
- form tight rounded colonies
47
What are the growth characteristics of human embryonic stem cells in vitro?
- form flat loose aggregates
48
What factors aid in stem cell self renewal of mouse embryonic stem cells vs. human embryonic stem cells?
```
Mouse = Leukaemia inhibitory factor which keep mouse ES in embryonic form
Human = different set of molecules + media conditions maintain these
```
49
Nuclear Reprogramming
- reprogramming somatic cells by SCNT (somatic cell nuclear transfer)
- Dolly the Sheep
- nucleus of unfertillised oocyte removed
- nucleus from adult terminally differentiated cell transferred into enucleated oocyte
- stimulation of oocyte with an electric pulse kicks of development
- oocyte with transferred nucleus activated
- undergoes epigenetic reprogramming in new environment, forms a blastocyst according to implanted nucleus genetic info
- embryo is transferred into surrogate to gestate
- also known as reproductive cloning
50
Ethics & Regulation of nuclear reprogramming
- creates an embryo so not ethically neutral means of producing cells
- presents a need for a source of oocytes
- most countries prohibit or limit use
- UN ban all forms of human cloning
- many countries enabled exceptions to allow for research but under tight restrictions only
51
Which genes can be turned on to induce a pluripotent state?
- Sox2
- Pou5f1
- Oct4
- c-Myc
52
2 models of iPS cells being used in therapeutics
iPS = induced pluripotent stem cells
1) disease modelling using patient's iPS cells to tailor drug therapy
2) genetically modified iPS cells differentiated into HSCs to correct a genetic defect (e.g. sickle cell anaemia)
53
Third dimension of cell differentiation in vitro
- ES in LIF-free culture in hanging drops to facilitate clustering
- cells interact, get same compaction event as you do in embryo 8 -> 16 -> 32 cell transition
- this spurs differentiation
- clustered ES cells form embryoid bodies (3D Induction)
- cells cultured out in the presence of specific growth factors or by co-culture to promote differentiation
- disaggregration of embryos followed by selection using FACS
- repeat steps 3-4 until you arrive at cell fate desired
54
What is co-culturing?
Culture 2 or more different types of cells together
| - in order to cells to signal with each other and end up with a specific phenotype
