Download neural or other stem cells can not be used for (neural) cell

Neural stem cells:
Biological characterization and potential applications
2017
Emília Madarász
Inst. of Experimental Medicine of Hungarian Academy of Sciences
Terminally differentiated cells
Axon
Symmetric mitosis
Asymmetric mitosis
Sejttest
+
+
Dendrit
Agykérgi piramis-sejt
Funder cells: new phenotype
Amplifying cells
Inner cell mass
trophoblasts
Fertilized oocyte
Day 3
Early blastocyte (day 5)
Embryonic stem cells, tissue stem cells, induced pluripotent stem cells
In vivo
Inner cell mass (ICM)
oviduct
Embedding
trophoectoderm
blastocyte
trophoblasts
In vitro
Early blastocyte
Embryonic stem cells (ESCs)
ES cell lines
Tissue stem cells
ICM
Uterus wall
Day 32;
6 mm
Day 13
0.4 mm
Day 18
1.25 mm
Induced pluripotent stem cells (IPSCs):
introduction of „stemness genes” to non-differentiated tissue cells
Heterogenous tissue stem cell populations from the beginning of embryo development
ESTABLISHMENT OF BODY AXES
14 day human embryo
ICM
epiblast
hypoblast
Neural plate
nodus
Primitive streak
Body axis
Primitive streak
Body axis
Neural tube
Roof plate
Alar
plate
Basal
plate
~16 day after fertilization
Differential gene expression determines:
Dopaminerg idegsejtek
HC: hippocampus
ST: striatum
SN: substantia nigra (A8, A9)
VTA: ventral tegmental area (A10)
B.olf.: Bulbus olfactorius (A16)
• the boundaries of brain regions
• the neurotransmitter-phenotype of neurons
• the position of fibre tracks
Noradrenerg idegsejtek
LC: locus coeruleus
T: thalamus
Hy: hyppocampus
LTA: lateral tegmental
area
Szerotonerg idegsejtek
SCN: suprachiasmatic nucleus
T: thalamus
HC: hyppocampus
LTA: lateral tegmental area
R: raphe magok
Kolinerg idegsejtek
Septum medialia: Ch1
nucl.fasc. diagonalis: Ch2, Ch3
nucl. basalis Meynerti : Ch4
nucl. pedunculopontis: Ch5
nucl. tegment.
Laterodorsalis:Ch6
medialis habenula : Ch7
Nucl. parabigeminal : Ch8.
Ventral layers of the spinal cord:
Motoneurons, g-efferents,
vegetative „motoneurons”
Symmetric mitosis
Asymmetric mitosis
+
+
Pia - ECM
Radial neuroepithel cell
Lateral
induction / inhibition
ventricle
Radial glia
ECM
Symmetric
asymmetric
mitoses
G.C. Schoenwolf , 2001
Neuronal
precursor
Primary germinative zone
Notch
Hes
ErbB2
BLBP
Delta-1
Mash/Ngn1,2
NRG-1
1. Notch/Delta system
1.
3.
2.
Differentiating cells
Non-differentiating
cell
…differences between neighbouring cells caused by stochastic
events and intrinsic or extrinsic factors are stabilized or amplified
through Notch and Delta signals…
Notch/Delta system
Lateral inhibition (Drosophila proneural cluster)
Embryonic cell migration from the primary germinative zone
17 day human
Migrating neuronal
precursors
radial glia
Rakic P. J. Comp. Neurol. 1972, 145: 61-84
Halfter, W. et al.
J. Neurosci. 2002;
Neuronal precursors derived from the primary germinative zone migrate along the radial glia cells
The secondary germinative zone derives from the primary zone:
Astroglia,
Oligodendroglia
Local interneuron
Small projecting neuron
Projecting neuron
Radial glia
SVZ
VZ
ependyma
Primary germinative layer
prenatal
Secondary germinative layer
postnatal
E10.5
E1
0,
5
E1
0,5
Alvarez-Buylla 1998
E10,5
E10,
5
Adult
Shuurmans 2004
The forebrain cortex composed by neurons derived from both,
the primary and secondary germinative zones
Adult-hood neurogenic zones
ependyma
agyszövet
kamra
Forebrain subventricular zone (SVZ)
nmfhé
Hippocampus subgranular zone (SGZ)
Embryonic neuroectodermal stem cells
Neural groove Neural plate
Neural crest
= Neuroepithelial
stem cells
17 day human
Embryonic/fetal neural tissue stem cells
Neural tube
Primary neural stem cells
= radial glia cells in the
ventricular zone
Secondary neural stem cells
= neural stem cells in the
subventricular zone
E 14.5 mouse forebran
Adult-hood neural stem cells
subventricular zone
subgranular zone
In case of injury, the subependymal zone can produce cells along the entire neuroaxis
Large number of different neural stem cell populations exist synchroneously
Characterization of the different neural stem cells is poor
Terminally differentiated neurons
X
?
Radial glia-like neural stem cells
Embryonic neuroectodermal stem cells
Amplifying progenitor →
Stem cell 4
?
Lateral
Induction/inhibition
Amplifying progenitor →
Stem cell 3
?
?
Stem cell 1’
Amplifying progenitor →
Stem cell 2
Radial glia
1 self-renewing + 1 more differentiated progeny
Symmetric mitosis
Asymmetric mitosis
+
+
In response to tissue damages, large-scale cell production occurs in the CNS
Jorfi et al., 2015. J.Neur.Engin
Resident progenitors are
scattered in the fibre
tracks
Zadori et al., 2011
Their fate????
mainly scar formation
Baumann 2007. Phys.Rev.
Neuron characteristics
Arborizing processes  extremely large cell surface in comparison to volume
• special intracellular transport machinery
• local substance production and protein synthesis
• large number of adhesion points
(pre- and postsynapses, astroglia-neuron connections, oligodendroglia tight junctions)
Rapid information forwarding to distant cell parts
• axon – dendrit polarity
• voltage-dependent ion channels and special ion pumps: characteristic types and arrangements
Specialised secretory cell
• more than one secreted material (neurotransmitter, neuromodulator, neuropeptide)
• different release mechanisms (normal exocytosis, synaptic release)
Specialised chamical receptor cell
• characteristic receptor areas in the cell membrane (postsynapses)
The neuronal surface is a mozaic of specialised membrane areas
The terminally differentiated neuron is incapable
•
•
to divide
to migrate
Cell motility composed by elongation and generation of processes
The process-endings and synapses possess self-standing regulatory mechanisms: plasticity
of process elongation and synaptic reorganization
Stereotype anatomical structure
Bascet cell;
rat hippocampus CA1 region
axon
s.r.
s.p.
dendrite
100 mm
s.o.
Zemankovics Rita rekonstrukciója; MTA KOKI
1013 neurons x 104 synapses = 1017 neuron-neuron connections
J. Spacek; http://synapses.mcg.edu/atlas/
The axon growth cone follows extracellular instructions
Phase-contrast video-microscopy
Jürgen Löschinger felvétele
Electronmicroscopy on growing neurites
*: growth cone; elongating process
Growth cones attach preferably to neighboring processes:
The neurites compose fascicles
The processes of cortical neurons guide the thalamic processes
toward the future cortical targets
Zoltán Molnár
„Timed” receptor-ligandum interactions
forbid defined process-elongation routes
Slit ↔ Robo
P2
P17
Stanfield; O’Leary; 1985-95
Retrográd labelling from the medulla :
P2: the entire layer V
P17: the sensomotory cortex only
Double labelling on P2 and on P17 :
Not the cells, only the non-appropriate processes do disappear during development:
Developmental elimination of excess (non-used) processes
Kémiai szinapszis
1949!
target
Growth cone
BDNF
Protein synthesis
exocytosis
ECM
GABA
GABAA
GABAB
Propagated waves [Ca2+]I
release
[Ca2+]I
phosphorylation
Protein production
release
Jelitai, Madarasz; 2006
Synapses can be formed and maintained between synchronuosly active partners
„Fire together, wire together”
Corpus geniculatum laterale:
Layering disappears, if the n. optici
are synchroniously stimulated
Giant depolaririzing potentials (GDP)
Ben-Ari, 2007
-
Generated in divers spots of the developig brain
Spread along fasciculated processes
Help to generate synapses of syncronuosly active axons
Play fundamental role in the formation of large projecting
fibre tracks
ES cells, embryonic tissue stem cells and IPSCs are not to be used for direct
cell therapy!
High probability of non-controlled proliferation → tumour formation;
Unknown tissue instructions → unknown routes of cell development
RA 9
Schlett, 1997. J. Neurosci.Res.
synaptophysin
E9
D 21
*
kamra
SVZ
NeuN-bIII tubulin
40 mm
Demeter et al., 2004. Exp. Neurol.
Brain injury → neural stem cell proliferation
Transient and „abortive”:
Does not result in neural tissue
regeneration
Zheng et al., J Neurotrauma. 2013
neuron fromation starts, but.....
Neurogenesis and neuronal regeneration in status epilepticus
Rotheneichner et al., Epilepsia, 2013
The conditions for neural tissue formation are missing in the adult/diseased brain
IPSCs
Reprogramming
Indukált pluripotens őssejtek (IPSC)
Kfl, Oct4, Nanog, Sox2, Myc
Epigenetic effects: growth factors (?)
ES-like cells, with ES-like troubles
IPSC
Terminally non-differentiated!
IPSCs derived from different tissue progenitors posses equal developmental potential?
Advanteges:
Derivation from the own body;
Personal cytogenetics → improved drug-testing;
Far in future, potential own tissue implantation, cell therapy
The formation and maintenance of the functional nervous
tissue resulted by continouos selection mechanisms
Cell genesis: surviving cells are selected from an excess of generated cells
Cell migration: permissive, attractive and repulsive signals select further
the survivng progenitors and ingrowing axons
Signals of the functioning tissue (secreted molecules and cell to cell
connections) keep alive the cells
The environment selects !
Only defined regions (Hyppocampus g. dentatus granule cell layer, bulbus olfactorius, SVZ)
of the adult CNS provides conditions for survival and development of stem cells and
novel neuronal circuits
The environmental conditions for survival and proper development of neural stem cells
are far from known, yet.
For the time being,
neural or other stem cells can not be used for (neural) cell replacement therapy
Stem cells can be used for:
In vitro
• drug testing;
• assessing individual drug reaction of own-derived iPSC generated neurons
• academic studies of neuronal development and circuit formation
• investigation of conditions for neurite regeneration
• surface optimalization of intracerebral prostheses
In vivo
• dampening of local inflammatory processes
• stimulating inherent regenerative processes
Mesenchymal stem cells (MSCs) – in promissing clinical trials
MSCs from cordblood, cord, bonemarrow
Not cell replacement ! → inhibition of inflammation,
stimulation of inherent tissue regeneration
http://www.clinicaltrials.gov
Thank you for your attention !