lecture 17: neural stem cells and their niches

Question 1

What is the functional definition of stem cells?

Answer 1

• properties of a stem cell:
  
  • self-renew
  • generate all the cell types of the tissue (multipotent)
• allows for normal tissue homeostasis
• the decision between self-renewal and differentiation must be tightly controlled
• self-renew - if uncontrolled could lead to tumours
• differentiation - too much could lead to loss of stem cells

Question 2

What are neural stem cells?

Answer 2

• can self renew and differentiate to three major cell types:
  
  • neurons
  • glia
  • oligodendrocytes

Question 3

What are glial cells of the nervous system?

Answer 3

• oligodendrocytes: insulate CNS neurons
• astrocytes: provide trophic support to CNS neurons and involved in inflammation
• schwann cells: insulate PNS neurons, derived from neural crest
• all have different origins in the developing nervous system

Question 4

What are cell types within the NS?

Answer 4

• neurons (both central and peripheral nervous system)
• oligodendrocytes (myelinating cells of the CNS)
• schwann cells (myelinating cells and glia of the PNS)
• astrocytes (glia of the CNS)
• neural crest (stem cells that form PNS and other non-neural types)
• radial glia (adult neural stem cells)
• ependymal cells (line central canal)

Question 5

What are neurospheres?

Answer 5

• aggregates of neural stem cells cultured in suspension
• neurosphere + laminin substrate neural media GF (-)
  
  • neurons
    
    • β_III-Tubulin
    • NCAM
    • NESTIN
    • MAP2AB
• neurosphere + fibronectin substrate, neural media GF (+) (EGF/bGFG/PDGF-AA [20ng/ml]), neural media GF (+) (T3 [30nM])
  
  • astrocytes
    
    • GFAP
  • oligodendrocytes
    
    • O4

Question 6

Of what do neurospheres consist?

Answer 6

• a heterogeneous population of neural stem cells and progenitor sub types

Question 7

What are sources of neural stem cells?

Answer 7

• pluripotent stem cells (embryonic and iPS)
• foetal tissue: neuroepithelial cells of the neural tube
• adult brain: subventricular zone of ventricles, hippocampus

Question 8

What can be done with pluripotent stem cells?

Answer 8

• in vitro differentiation of stem cells into cell type of interest
• embryonic stem cell colony
  
  • endoderm: hepatocytes, islet cells
  • mesoderm: cardiomyocytes, muscle, kidney cells
  • ectoderm: neural tissue (neuroectoderm), skin

Question 9

What is the maintenance and expansion of neural stem cells?

Answer 9

• monolayer
• mechanical dissociation
  
  • pieces are cultured in suspension in Neural Basal Media with growth factors, bFGF and EGF, required for maintenance of neural stem cells
• cluster of neural stem and progenitor cells in neurosphere
• rosette structures: similar to neuroepithelial cells of the embryonic neural tube

Question 10

What are some of the signals involved in directing formation of neural progenitors from ES cells?

Answer 10

• ES cell → BMP → extraembryonic endoderm
• noggin ⊣ BMP
• next is neural progenitor

Question 11

What are neural inducing factors for embryonic stem cells?

Answer 11

• noggin
• dickkopf
• FGF2
• retinoic acid
• HESC colony → noggin 14 days → neural induction
• genes expressed in neural stem cells: Pax6, Sox1, Sox2, Sox3, Nestin, CD133

Question 12

To what does neural induction default?

Answer 12

• forebrain progenitors

Question 13

What proteins are expressed in different regions of the brain?

Answer 13

• FOXG1
  
  • Tel of forebrain
• SIX3
  
  • Ros Di, Tel of forebrain
• PAX6
  
  • all of forebrain (tel, ros di, cau di)
• OTX1
  
  • part of Mes (midbrain)
  • all of forebrain
• OTX2
  
  • all of mid brain and forebrain
• IRX3
  
  • from cau di to met (hind brain)

Question 14

In the presence of noggin, how will human ES cells progress (roughly)?

Answer 14

1. human ES cells
2. neurospheres
3. dorsal anterior cell types

Question 15

What happens with addition of exogenous factors (e.g. Shh, Wnt, RA, FGF8) during neural induction?

Answer 15

• ventral posterior cell types
• e.g. sonic hedgehog → ventral GAD67+ neurons

Question 16

Summary of induction of pluripotent stem cells

Answer 16

• induction of pluripotent stem cells to neural requires inhibition of BMP and Nodal (or downstream SMAD) signalling
• expression of Sox2 and Pax6 are expressed in neural stem cells
• differentiation of hPSC-derived neural stem cells defaults to forebrain cortical neurons
• derivation of specific neuronal cell populations of other lineages in culture may require supplementation of factors involved in neurogenesis patterning and cell fate

Question 17

What are iPSCs?

Answer 17

Question 18

What is the structure of neural tube along the dorsal-ventral axis?

Answer 18

Question 19

What is the ventricular zone of the neural tube?

Answer 19

• consists of dividing neuroepithelial cells (foetal neural stem cells)
• one cell layer thick
• nuclei move within cell as they divide
• mitosis occurs closest to lumen of neural tube
• not present in the adult nervous system (becomes the ependymal layer)

Question 20

What are symmetric versus asymmetric cell divisions of neuroepithelial cells?

Answer 20

• symmetric cell division (horizontal)
  
  • divide to give rise to self i.e. mitotic neuroepithelial cell
  • forms the ventricular zone
• asymmetric cell division (vertical)
  
  • divide to give rise to post-mitotic progenitor cell
  • forms the intermediate/mantle zone

Question 21

How is the spinal cord organised?

Answer 21

• ventricular zone: dividing germinal neuroepithelial cells (adult - ependymal layer)
• intermediate/mantle zone: grey matter (cell bodies of neurons and glia)
• marginal zone: white matter (axonal connections)

Question 22

summary of foetal neuroepithelial cells

Answer 22

• foetal neuroepthelial cells can be isolated from developing neural tube and cultured in vitro to form neurospheres
• fate of neural stem cells tends to be restricted to region of neural tube where isolated from, especially along the anterior-posterior axis
• foetal neural stem cells have been used in clinical trials for cell replacement (e.g. Parkinson’s disease)
• highly ethical because obtain tissue from aborted foetuses
• often used in research to study properties of neural stem cells

Question 23

What is identification and assessing properties of adult stem cells?

Answer 23

• a lack of definitive markers has hampered unequivocally identifying stem cells in many adult tissues
• in most cases, adult stem cells turnover slowly
• gold standard: testing self-renewal and multipotency in vitro and in vivo
• strategy 1:
  
  • in vitro/transplantation combination
  • stem cells are identified on the basis of a molecular marker and followed by in vitro culture or in vivo transplantation in recipient animal
• strategy 2:
  
  • genetic marking in situ
  • marker introduced into stem cell, allows visualisation of the modified SC and its clonal offspring over time
• can’t do step 2 without step 1

Question 24

How do neuroblasts migrate?

Answer 24

• radially, along the radial glial processes, to the outer cortical plate zone

Question 25

What is corticogenesis?

Answer 25

• neuroepithelial cells within ventricular zone become:
  
  • radial glial cells: extend processes into cortical plate
  • neuroblast (short neuronal precursor cell): migrates to cortical plate
  • intermediate progenitor cell: proliferate in subventricular zone and also give rise to migrating neuroblasts

Question 26

What is the adult subventricular zone (SVZ) niche?

Answer 26

• niche:
  
  • stem cell in contact with progenitor and niche cells as well as basal lamina
  • influenced by axons and blood vessels
• SVZ astrocyte /stem cell → self-renewal → transit amplifying cell → neuroblast
• ciliated cells line walls of ventricles
• blood bessels
• basal lamina

Question 27

What do neuroblasts from the SVZ do?

Answer 27

• neuroblasts from the SVZ of the brain ventricles migrate along the rostral migratory stream to the olfacto bulb where they differentiate into neurons
• SVZ astrocytes (B cells) in this region are stem cells which generate migrating neuroblasts (A cells) via a rapidly dividing transit-amplifying cell (C cells)
• chains of neuroblasts travel along the rostral migratory stream to the olfactory bulbs and differentiate to neurons
• signals released from axons (pink) regulate proliferation and survival in this region
• a specialised basal lamina (BL) extends from perivascular cells and contacts all cell types
• endothelial cells, blood vessels (BV) and the BL are all likely key components of the niche

Question 28

What is the adult SubGranular Zone (SGZ) niche?

Answer 28

• SGZ astrocytes divide to generate intermediate precursor cells (type D) which progressively generate more differentiated cells D1 D2 D3, which mature into granule neurons
• niche: stem cells in direct contact with progenitor and differentiated cells, blood vessel, basal lamina and influenced by neurons

Question 29

What is known of adult stem cells undergoing cell division?

Answer 29

• can undergo symmetric or unidirectional cell division when required for tissue regeneration or repair
• some progenitor cells retain the ability to return to stem cells or act as stem cells when required

Question 30

How do different daughter cells arise?

Answer 30

• anchored to niche cells e.g. to basal lamina between stem cells and stroma or between stem cells and blood vessels
• unequal distribution of determinants

Question 31

What is the definition of a stem cell niche?

Answer 31

• describes the microenvironment in which stem cells are found and which interacts with stem cells to regulate stem cell fate
• ‘niche’ can be in vivo or in vitro stem cell microenvironments
• stem-cell populations are established in ‘niches’ - specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair
• the niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation
• it constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms
• yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets
• the interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics
• the simple location of stem cells is not sufficient to define a niche
• the niche must have both anatomic and functional dimensions

Question 32

What are possible signalling factors and interactions regulating a stem cell niche?

Answer 32

• possible interactions:
  
  • cell-cell interactions between stem cells
  • cell-ECM interactions
  • cell-BL interactions
  • cell-BV or nerve cell interactions
  • via
  • adhesion molecules, gap junctions, receptor-signalling molecules, hemi-channels (allow communication between cell and the interstitial fluid environment)
• diffusible factors:
  
  • growth factors , cytokines, ECM proteoglycans (can sequester molecules and inhibit signalling or release when needed)
  • physiochemical nature of the environment: - pH, metabolite or ion concentration, e.g. ATP or oxygen tension
• note: the stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood

Question 33

What happens to adult stem cells during ageing?

Answer 33

• in an organ or tissue, generally adult stem cells remain in an undifferentiated state throughout adult life
• when cultured in vitro they appear to undergo an ‘ageing’ process i.e. their morphology and prolifertive capacity are altered
• as the adult ages, the niche atrophies and stem cells deteriorate over time and exhibit a decline in proliferation rate - why?
• a clue came from studying stem cells in Drosophila
• a key-self-renewal factor expressed by neighbouring niche cells decreases over time
• by genetically activating signals, stem cells can retain activity in ageing fly and mouse
• see Method to Delay Aging of Stem Cells Developed
• see: Toledano H et al., the let-7-lmp axis regulates ageing of the drosophila testis stem-cell niche

Question 34

What are challenges of stem cells?

Answer 34

• differentiation of neural stem cells to a specific cell lineage
• isolation and purification of cells
• integration, migration and function of cells in vivo

Question 35

What are issues with transplantation using stem cells and/or their derivatives?

Answer 35

• do they survive?
• do they integrate?
  
  • embryonic microenvironment
  • adult microenvironment
  • diseased microenvironment
• are they tumourgenic?
• do they function?
• the microenvironment influences the fate of stem cells