lecture 15: stem cells – basic concepts Flashcards
1
Q
What happens between blastocyst and foetus stage?
A
- cell determination
- cell proliferation
- cell differentiation
- patterning and morphogenesis
- programmed cell death
2
Q
What happens during embryogenesis?
A
- cells become restricted in their developmental capacity
- Morula
- trophectoderm
- inner cell mass → ES cells
- primitive endoderm
- parietal endoderm
- visceral endoderm
- primitive ectoderm
- epiblast
- definitive endoderm → liver, pancreas, lung
- mesoderm → blood, heart, skeletal muscle
- ectoderm → CNS, skin
- germ cells
- epiblast
- primitive endoderm
3
Q
How do determination and differentiation occur?
A
- result not from changes in genes, but from changes in gene expression (exception; immune system and gametes)
- results from alterations in chromatin structure and transcription factor expression
- often quite permanent and heritable through many cell divisions
4
Q
What is determination?
A
- occurs prior to overt differentiation – a heritable change in a cell’s developmental potential-operationally defined
- may not be able to visually see differences in the cell
- e.g. multipotent cardiovascular progenitors
5
Q
What is differentiation?
A
- result of changes in gene expression
- cell acquires correct shape polarity, orientation with respect to neighbours, appropriate organelles and proteins which enable it to carry out metabolic signalling, transport or contractile functions required in a particular tissue
6
Q
What is transdifferentiation?
A
- de-differentiation
- cell normally committed to one lineage is switched to a different lineage pathway
- many known examples from disease states – intestinal metaplasia of the oesophagus, squamous metaplasia in the respiratory tract or bladder
- may be induced experimentally by ectopic expression of master regulator transcription factors
7
Q
What are possible examples of transdifferentiation between cells in two closely related lineages?
A
- oval cell progenitor → hepatic oval cell
- bile duct
- hepatocyte
- pancreatic oval cell → hepatocytes
- two very closely related tissues in development
- distinct in terms of function but capable of interconversion
8
Q
What is intestinal metaplasia?
A
- damage to oesophageal epithelium through acid reflux from the stomach leads to conversion of squamous epithelium into intestine
- the condition is a precursor to oesophageal adenocarcinoma
9
Q
What is developmental capacity?
A
- a multipotent cell can give rise to several types of mature cell
- a pluripotent cell can give rise to all types of adult tissue cells plus extraembryonic tissue: cells which support embryonic development
- a totipotent cell can give rise to a new individual given appropriate maternal support
- restricted up to about 4- to 8-cell stage of development
10
Q
What are adult tissues?
A
- continuously renewing - bone marrow, skin, gut
- conditionally renewing - liver, kidney
- non-renewing - cardiac muscle
11
Q
What is cell turnover in the adult body?
A
- we lose 20 billion cells per day
- the lining of the intestine is replaced every four days
- every 4 weeks a completely new epidermis is generated
- some tissues turn over slowly - hepatocytes live for 300 days, cardiomyocytes 0.5% annually
- needs to be done very precisely
12
Q
What is a stem cell?
A
- a primitive cell which can either self renew (reproduce itself) or give rise to more specialised cell types
- stem cell is the ancestor at the top of the family tree of related cell types
- one blood stem cell gives rise to red cells, white cells and platelets
13
Q
Where are tissue stem cells located?
A
- skin: replaced by stem cells deep in the tissue - the basal layer, cells in the middle undergoing maturation process, stratum corneum at the top
- hair: follicle, region called the bulge is where stem cells live and are responsible for this constant turnover
- intestine: paneth cells
- blood
14
Q
What are stem cells?
A
- capable of self renewal or differentiation
- may give rise to transit amplifying cell compartment-committed cells with limited division capacity
- often lacking in specialised organelles, and show high nucleus/cytoplasm ratio
- long lived – express telomerase
- slowly dividing
- few in number
- may be restricted spatially to specific zones or niches
- respond to signals which will regulate their growth and proliferation, enabling them to meet changing demands
- e.g. when someone is undergoing chemotherapy
- often own bone marrow will be ablated
- has to be replaced or the patient will die
- can be done by infusing just a few stem cells
- will grow back and repopulate the entire blood forming system → stem cells, mature cells, etc
- dramatic example of how a normally quiescent cell can undergo this massive degree of expansion
15
Q
What are tissue stem cells?
A
- proper tissue organisation and response to demands of growth or repair require that there be restrictions on developmental potential of adult stem cells
- these limits are strictly imposed by powerful molecular restraints on gene expression and are heritable during many rounds of cell division
- an adult stem cell may show relaxation of these restrictions in an altered environment, possibly accounting for plasticity
- even so, plasticity is observed usually at low frequency
16
Q
What is proof of stem cell isolation?
A
- a single cell can repopulate a tissue and give rise to differentiated progeny as well as more stem cells
- identified in transplantation assays with marked cells
- critical that descendants of stem cell are shown to be functional
17
Q
What are markers of specific differentiation stages in cell lineages?
A
- transcription factors
- cell surface molecules (e.g. CDs)
- cytostructural molecules e.g. intermediate filaments specific functional gene products
- specific functional gene products
18
Q
What does a stem cell hierarchy look like?
A
- the best studied mammalian stem cell system is the blood forming system
- haematopoietic stem cell hierarchy showing transcription factors that control key decision points
- top: Long term repopulation HSC
- capable of very extensive self renewal and all the different lineages
- three seperate lineages:
- megakaryocytes
- granulocytes, neutrophils, macrophages,
- lymphoid cells
- particular transcription factors that regulate fate choice
- important not just for an academic understanding but also for understanding disease
- by the time you get to the bottom cells don’t have much proliferative capacity
19
Q
What characterises distinct stages of haematopoiesis?
A
- cell surface markers
- proteins that are more less specifically expressed at different stages of this lineage
- enable us to know exactly where we are in the heirarchy
- defined by monoclonal antibodies
- can be separated using cell sorter
20
Q
What is the discovery of novel stem cell populations?
A
- recent findings show that tissues formerly thought to be static in adult life contain stem cell populations
- examples include the heart and the central nervous system
- much investigation is currently directed at understanding the role of these stem cells in normal physiology and disease
21
Q
Where are neural stem cells located?
A
- neurons are born constantly throughout life in specific brain regions
- basically two stem cell populations in the mammalian CNS:
- subventricular zone
- hippocampus
- constantly ticking off
- rostral migratory stream up to olfactory bulb to replace olfactory neurons → rare example of a neural population that is continuously being lost
- ones in hippocampus
- maybe involved in learning and memory
- a lot of excitement → can we affect these stem cell populations with drugs to enhance memory processes, are they involved in disorders of memory and ageing
22
Q
What is adult neurogenesis?
A
- occurs in subventricular zone and hippocampus
- new neurons from the SVZ wind up in the olfactory epithelium
- hippocampal neurogenesis may have a role in learning and memory
23
Q
How do stem cells function in the gut?
A
- the four types of differentiated cell in the adult gut are formed constantly from stem cells in the crypts
- goblet cells → produce mucin
- enteroendocrine cell → produce gut hormones
- paneth cells → innate immunity, produce defensins and lysozyne
- enterocytes → absorptive cells
- these cells are constantly being lost
- crypt is deep inside
- extrinsic signals and networks of transcription factors regulate gut cell differentiation
- Wnt signalling → if derailed will give rise to gut cancer
- Notch signalling
- these control the behaviour of the stem cells
- particular transcription factors that are critical to these fate decisions
- beginning to identify what regulates those choices in terms of external signals and transcription factors
24
Q
What regulates stem cells in the hair follicle?
A
- extrinsic signals involved in regulation of stem cell growth and differentiation in the hair follicle
- now know the signalling pathways that regulate hair cells as they transition from quiescent through to burst of proliferation/maturation and ultimately formation of the hair shaft
- extrinsic signalling from surrounding cells regulates stem cell proliferation
- cells that are at the top of the heirarchy are only slowly ticking over
- next level down are the ones that proliferate rapidly
- regulation in the niche → sources of specific signals that will keep those stem cells very carefully regulated → BMP and wnt signalling
- quiescent stem cells that are not dividing represent a tissue reserve that is activated during damage
- active stem or progenitor cells are responsible for homeostasis under normal conditions
- used to think that it was the top of the heirarchy stem cells that were responsible for physiological turn over, current thinking is they are more reserve
- really it is the intermediate cells that are responsible for day to day homeostasis
- can proliferate enough to keep things on track most of the time
- only when there is damage that the top dogs are really pulled into the game
25
Q
What is a conditionally renewing cell population?
A
- liver
- a cell population that under normal circumstances is pretty quiet
- but when there is damage or demand for proliferation due to injury it can kick in again
- the liver is a conditonally renewing tissue with facultative stem cells
- liver can regenerate by proliferation of hepatocytes, or from bipotential stem cells found in the biliary tree
- if you take a healthy animal/human and chop out two thirds of its liver, the rest of the liver will grow back
- happens by proliferation of the mature cells themselves in healthy individuals
- stem cells sit in the termini of the bile ducts in a structure called the canal of hering
- only kick into action when stimulated by rare pathological circumstances in which the hepatocytes are unable to proliferate/repopulate of their own accord (e.g. cirrhosis, viral infection)
26
Q
What can be used to mark putatative stem cells in bile duct termini in normal liver?
A
- monoclonal antibody
27
Q
What occurs to stem cell populations in biliary atresia?
A
- population expands
- congenital blockage of the extrahepatic bile duct leads to accumulation of bile in the liver and cirhosis
- bipotential biliary cells proliferate in an attempt to repair the damage
28
Q
What controls the formation of hepatocytes and bile duct from liver stem cells?
A
- many signalling systems
- in most forms of liver disease this repair doesn’t function well
- exploit this process so that instead of getting scar tissue we get healing and regeneration
29
Q
What are two/three types of human pluripotent stem cells?
A
- nowadays there are three sources
- embryonic stem cells
- zygote
- blastocyst
- ICM
- tissue cultures
- pluripotent stem cells
- contribution to germ line (mice)
- induced pluripotent stem cells
- yamanaka factors
- fibroblast
- tissue culture
- pluripotent stem cells
- teratoma formation
- derivation of SCNT
- take an egg remove the genetic material
- put an adult cell nucleus back in
- egg can reprogramme adult cell nucleus back to an early developmental state
- generate a blastocyst
- isolate ICM cells etc
- pluripotent stem cell lines
- interesting applications in terms of making custom made tissues from stem cells of individual patients
- can help bypass issue of rejection
- multiple refinements to the procedure enabled ES generation from a small number of oocytes
- cloning: a powerful tool to study cellular reprogramming
30
Q
What are properties of pluripotent stem cells?
A
- grow indefinitely in vitro
- maintain normal genetic makeup
- cloned lines capable of differentiation into a wide range of somatic and extraembryonic tissues in vivo and in vitro - at high frequency and under a range of conditions
- capable of colonising all tissues including germ line after blastocyst injection to give chimaeric offspring
31
Q
What is the blastocyst stage of development?
A
- body plan not yet apparent
- many cells will not form new human, but will give rise to tissue such as placenta which support pregnancy
- embryo does not yet necessarily represent a unique individual (twins can form up to 14 days)
- no precursors of nervous system present yet
- not possible to predict whether embryo will be able to develop to term
32
Q
How do we get the establishment of ES cells?
A
- inner cell mass
- ES colony 10-15 days later
- if you do this process correctly you can essentially keep these cell lines going forever
33
Q
What is biological proof of pluripotency?
A
- formation of germ line chimaeras is a rigorous demonstration of pluripotency for mouse ES cells
- human ES cells: ability to form teratomas containing tissues representatitve of all three embryonic germ layers
- teratomas contain structures resembling early embryos
- i.e. these cells are able to recapitulate process of early development
- spontaneous ES cell differentiation in vitro
- nerve and muscle cells are found in a complex mixture of many cell types
34
Q
What are seven signalling systems that control animal development?
A
- Wnt
- Hedgehog
- Notch
- TGFβ
- nuclear rec
- JAK-state
- tyr kin
- developmental mechanisms are relatively conserved from an evolutionary stand point
- what we learn from fruit flies/zebra fish etc does apply in a big sense to humans
- +/- 1 or 2
35
Q
How does ES cell differentiation occur?
A
- goes through germ layer intermediates
- can recapitulate these signalling pathways in a culture dish to get what we want
36
Q
What is the route to cardiac progenitors?
A
- know these processes pretty well
- so relatively easy to generate human heart muscle in a dish from embryonic cells
37
Q
How has SCNT been used?
A
- SCNT and reprogramming
- a cat cloned by nuclear transplantation
- many species of mammal have now been cloned
- can cloning technology be used to surmount immunological barriers to stem cell transplantation?
- very difficult to do in humans → very difficult to do due to its inefficiency, obtaining eggs in humans is a very invasive/risky/difficult process, not really amenable to a large scale
38
Q
What is reprogramming to pluripotency?
A
- induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors
- what the egg does is not a miracle it’s a biochemical reaction
- iPSCs
- somatic cells “reprogrammed” by viral transfection
- ES-specific transgenes introduced into host cells Oct-4, Sox2, Klf-4, c-Myc
- subset of cells: ES-like colonies = iPS cells
- avoids use of embryos
39
Q
What are applications of iPSC?
A
- research: disease modelling
- therapy: tissue matching
40
Q
How do pluripotent stem cells have important applications in biomedical research?
A
- basic studies of early human development and its disorders- birth defects, childhood cancers
- functional genomics in human cells
- discovery of novel factors controlling the tissue regeneration and repair
- in vitro models for drug discovery and toxicology
- e.g. modelling the long Q-T syndrome with human iPSC
- congenital type 2 LQTS: model for LQT caused by heart failure, cardiac hypertrophy or drugs
- approaches to human functional genomics → complex or GWA study traits
41
Q
What is functional genomics of human ES cells?
A
- there are differences between mice and humans
- we can make targeted genetic modifications in human ES cells to create disease models. We can study the efefcts of the mutations of development and physiology of specific cell types
- we can use the differentiated cells to develop and screen new medicines
42
Q
Why have the human cerebral cortex in a dish?
A
- human cortical development differs signficantly from other mammals
- ES and iPS cells can be used to model human cortical development
- schizophrenia, autism and epilepsy are disorders of brain development
- iPSC from patients with these diseases can be used to recapitulate key events in pathogenesis
- integration of neural progenitors from human ES cells into mouse cerebral cortex: implications for brain repair in childhood
43
Q
How will stem cell research revolutionise medicine?
A
- powerful new tools to study human biology in health and disease
- normal human cells to study in the laboratory – use to develop new drugs. alternative to animal models or direct tests on human guinea pigs
- cells for replacement therapy in devastating conditions involving cell loss or injury
- new understanding of the body’s natural healing process, how and why it fails, and how to improve healing
