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Tissue Engineering & Regenerative Medicine > Cell Sources for Regenerative Medicine > Flashcards

Cell Sources for Regenerative Medicine Flashcards

1
Q

Types of germ cells

A

Sperm

Egg

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2
Q

Examples of endoderm cells

A

Pancreatic
Thyroid
Lung (alveolar)

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3
Q

Examples of mesoderm cells

A 
Cardiac muscle
Skeletal
Kidney tubule
Red blood
Smooth muscle
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4
Q

Examples of endoderm cells

A

keratinocytes
neuronal
pigment

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5
Q

Define cell differentiation

A

Progression from naive/unspecialised cellular state to a more specialised state/fate

  • might be accompanied by morphology change
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6
Q

Define cell potency

A

Describes developmental potential of a cell

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7
Q

Define Totipotent/Pluripotent/Multipotent

A

Stem cells which can adopt multiple fates

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8
Q

Examples of totipotent cells

A

Zygotes

Embryonic Stem Cell Colonies

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9
Q

Examples of terminally differentiated cells

A

Cartilage
Bone
Skeletal Muscle

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10
Q

Properties of terminally differentiated cells

A

Low/no developmental potential

Limited/no capacity for self-renewal

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11
Q

Define reprogramming

A

Going from terminal differentiation to potent cells

  • changing phenotype of cells
  • manipulation in the lab
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12
Q

Describe self-renewal

A
  • ensures stem cell populations and potency are accordingly maintained undifferentiated
  • ensures cell products still have multiple cell fates
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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)
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14
Q

2 methods of self-renewal

A

1) Asymmetric Division

2) Stochastic Mechanisms

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15
Q

Asymmetric Division

A
  • every division gives rise to 1 differentiated daughter cell and 1 undifferentiated self renewed stem cell
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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)
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17
Q

Define commitment

A

Stem cell commits to a differentiation pathway

- may still have several steps before terminal differentiation

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18
Q

Define early progenitor

A

Multipotent cell in the early part of the pathway

- reduced self-renewal

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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

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20
Q

Define terminally differentiated

A

Adult mature cell
At end of its differentiation pathway
Usually no capacity for self-renewal
Specialized morphology & function

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21
Q

What is the differentiation pathway?

A

Stem cell -> Early Progenitor -> Late Precursor -> Terminally Differentiated

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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
Q

What are lineage markers?

A
  • 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
Q

Flow of information in biological systems

A

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
Q

Define progressive canalisation

A

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
Q

How do multipotent pathways work?

A
  • 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
Q

What does the term ‘primed’ mean?

A

Gene is not turned on but has the capacity to be turned on

30
Q

What happens during interphase?

A

DNA decondenses into a mass of chromatin

In each chromosome mass territory

31
Q

What do transcription factors do?

A
  • induce gene transcription (DNA -> RNA)

- can also silence gene transcription

32
Q

What is epigenetic regulation

A
  • 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
Q

What is in vitro differentiation?

A
  • how we derive different cell types in a laboratory setting
34
Q

Steps of in vitro differentiation

A
  • 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
Q

How does altering cell conditions change cell fate?

A
  • change media (supplements, growth factors, drugs, nanoparticles)
  • substrate or scaffold
  • physical/mechanical stimuli
36
Q

What is FACS?

A

Fluorescence Assisted Cell Sorting

37
Q

How is in vitro differentiation done in a lab?

A
  • 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
Q

What is an induction signal?

A
  • 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
Q

What is cell sequestration?

A

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
Q

What is cell polarisation?

A
  • 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
Q

Embryo Cell Development

A

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
Q

First mammalian differentiation event

A

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
Q

What is the source of embryonic stem cells?

A
  • 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
Q

Foetal and Adult Stem Cells

A
  • 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
Q

Haematopoietic Stem Cell Niches

A
  • 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
Q

What are the growth characteristics of mouse embryonic stem cells in vitro?

A
  • form tight rounded colonies
47
Q

What are the growth characteristics of human embryonic stem cells in vitro?

A
  • form flat loose aggregates
48
Q

What factors aid in stem cell self renewal of mouse embryonic stem cells vs. human embryonic stem cells?

A 
Mouse = Leukaemia inhibitory factor which keep mouse ES in embryonic form
Human = different set of molecules + media conditions maintain these
49
Q

Nuclear Reprogramming

A
  • 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
Q

Ethics & Regulation of nuclear reprogramming

A
  • 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
Q

Which genes can be turned on to induce a pluripotent state?

A
  • Sox2
  • Pou5f1
  • Oct4
  • c-Myc
52
Q

2 models of iPS cells being used in therapeutics

A

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
Q

Third dimension of cell differentiation in vitro

A
  • 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
Q

What is co-culturing?

A

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

Tissue Engineering & Regenerative Medicine (4 decks)

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