lecture 15: stem cells – basic concepts

Question 1

What happens between blastocyst and foetus stage?

Answer 1

• cell determination
• cell proliferation
• cell differentiation
• patterning and morphogenesis
• programmed cell death

Question 2

What happens during embryogenesis?

Answer 2

• 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

Question 3

How do determination and differentiation occur?

Answer 3

• 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

Question 4

What is determination?

Answer 4

• 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

Question 5

What is differentiation?

Answer 5

• 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

Question 6

What is transdifferentiation?

Answer 6

• 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

Question 7

What are possible examples of transdifferentiation between cells in two closely related lineages?

Answer 7

• 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

Question 8

What is intestinal metaplasia?

Answer 8

• 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

Question 9

What is developmental capacity?

Answer 9

• 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

Question 10

What are adult tissues?

Answer 10

• continuously renewing - bone marrow, skin, gut
• conditionally renewing - liver, kidney
• non-renewing - cardiac muscle

Question 11

What is cell turnover in the adult body?

Answer 11

• 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

Question 12

What is a stem cell?

Answer 12

• 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

Question 13

Where are tissue stem cells located?

Answer 13

• 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

Question 14

What are stem cells?

Answer 14

• 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

Question 15

What are tissue stem cells?

Answer 15

• 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

Question 16

What is proof of stem cell isolation?

Answer 16

• 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

Question 17

What are markers of specific differentiation stages in cell lineages?

Answer 17

• transcription factors
• cell surface molecules (e.g. CDs)
• cytostructural molecules e.g. intermediate filaments specific functional gene products
• specific functional gene products

Question 18

What does a stem cell hierarchy look like?

Answer 18

• 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

Question 19

What characterises distinct stages of haematopoiesis?

Answer 19

• 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

Question 20

What is the discovery of novel stem cell populations?

Answer 20

• 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

Question 21

Where are neural stem cells located?

Answer 21

• 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

Question 22

What is adult neurogenesis?

Answer 22

• 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

Question 23

How do stem cells function in the gut?

Answer 23

• 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

Question 24

What regulates stem cells in the hair follicle?

Answer 24

• 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

Question 25

What is a conditionally renewing cell population?

Answer 25

* 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)

Question 26

What can be used to mark putatative stem cells in bile duct termini in normal liver?

Answer 26

* monoclonal antibody

Question 27

What occurs to stem cell populations in biliary atresia?

Answer 27

* 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

Question 28

What controls the formation of hepatocytes and bile duct from liver stem cells?

Answer 28

* 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

Question 29

What are two/three types of human pluripotent stem cells?

Answer 29

* 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

Question 30

What are properties of pluripotent stem cells?

Answer 30

* 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

Question 31

What is the blastocyst stage of development?

Answer 31

* 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

Question 32

How do we get the establishment of ES cells?

Answer 32

* inner cell mass * ES colony 10-15 days later * if you do this process correctly you can essentially keep these cell lines going forever

Question 33

What is biological proof of pluripotency?

Answer 33

* 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

Question 34

What are seven signalling systems that control animal development?

Answer 34

* 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

Question 35

How does ES cell differentiation occur?

Answer 35

* goes through germ layer intermediates * can recapitulate these signalling pathways in a culture dish to get what we want

Question 36

What is the route to cardiac progenitors?

Answer 36

* know these processes pretty well * so relatively easy to generate human heart muscle in a dish from embryonic cells

Question 37

How has SCNT been used?

Answer 37

* 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

Question 38

What is reprogramming to pluripotency?

Answer 38

* 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

Question 39

What are applications of iPSC?

Answer 39

* research: disease modelling * therapy: tissue matching

Question 40

How do pluripotent stem cells have important applications in biomedical research?

Answer 40

* 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

Question 41

What is functional genomics of human ES cells?

Answer 41

* 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

Question 42

Why have the human cerebral cortex in a dish?

Answer 42

* 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

Question 43

How will stem cell research revolutionise medicine?

Answer 43

* 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