2. overview
Introduction
Location of neural stem cell (NSC)
Development of NSC
Cell signalling pathway for NSC
Markesr’s of NSC
Factor affecting growth and multiplication of NSC
Isolation and culture of NSC
NSC for Therapeutic Use
Conclusion
4. The term“neural stem cell” is used loosely to describe cells
that:
Can generate neural tissue or are derived from the nervous
system
Have some capacity for self-renewal, and
Can give rise to cells other than themselves through
asymmetric cell division
7. The differentiation of the neuroepithelial stem cells into
neurons and glial then proceeds in a temporal specific
manner and in specific region of the developing neural
tube (McConnell,1995; Rao, 1999)
For decades, it was believed that most neurons in the adult
central nervous system (CNS) were terminally
differentiated and usually not replaced when they died
It has been established that active neurogenesis, a process
of generating functionally integrated neurons from
undifferentiated, multipotent progenitor cells (Gage, 2000;
Temple, 2001)
8. The concept of NSC plasticity and of their dependence on
environmental cues is strengthened by transplantation and
manipulation studies in vivo (Gage et al., 1995)
This plasticity may perhaps outstretch the brain
boundaries, so that NSCs (neuroectodermal in origin) can
give rise to cells that normally derive from germ layers
other than the neuroectoderm (Jiang et al., 2002)
In addition, NSCs have been utilized as one of the
potential sources for the cell replacement therapy of CNS
disorders (Bjorklund et al., 2000; Falk, 2009)
9. Alternative sources of NSC progenitor cell for
cell therapy
Bone marrow
Blood cord
Hair follicles
Skin
Amniotic placental fluid
Human foetus
10. Location of (NSC)
NSC have been assumed to exist only in the embryonic
nervous system at the beginning of neural tube formation
NSC are present in the central nervous system (CNS), in the
peripheral nervous system (PNS) and in neural crest cell
populations
Germinal regions persist in the adult mammalian brain that is
capable of generating new neurons: the subventricular zone
(SVZ), and the subgranular zone (SGZ)
11. The spinal cord Progenitor cells in the mammalian fetal CNS
are considered to be concentrated in seven major areas:
Olfactory bulb
Ventricular zone (VZ)
Subventricular zone (SVZ) of the forebrain
Hippocampus
Cerebellum
Cerebral cortex
Spinal cord
Progenitor cells number and pattern of development vary in
different species (Gage, 2000)
14. When the neural plate first emerges, does it consist solely of
stem cells ?
Analysis of adherent clone production suggests stem cells are
prevalent at early stages
In spinal neural tube from embryonic day 8 (E8) rat, over 50%
of the viable cells at 24 hours are stem cells (Kalyani et al.,
1997, 1998; Qian et al., 2000)
In telencephalon of E10 mouse, estimates of stem cells range
from 5 to 20% (Kilpatrick and Bartlett, 1993)
15. But the frequency of stem cells declines rapidly (Kalyani et al.,
1997, 1998)
Stem cells seem to be much rarer when neurosphere production
is used as the assay: only 0.3% of E8.5 mouse anterior neural
plate cells make neurospheres
Perhaps neurosphere-generating cells are a subpopulation of
early stem cells
16. If stem cells are, or rapidly become, a subset of early neural
progenitor cells in vivo, how are they distributed ?
Clonal studies suggest that most glial, both astrocytes and
oligodendrocytes, originate from stem cells (Rao, 1999)
Neurospheres generated from different CNS regions express
region- specific markers
Regulatory sequences control region-specific expression of the
transcription factor Sox2, so that expression is seen in
telencephalic but not spinal cord stem cells (Zappone, 2000)
17. Neural stem cells cultured from early to mid-gestation give rise
to more neurons than those cultured at later periods (Qian et al.,
2000)
Nestin is an intermediate filament expressed by the
neuroepithelial cells of the neural tube (Lendahl et al., 1990)
Nestin-positive cells make contact with the ventricular surface
and have radially oriented processes
18. Developmental changes in stem cells are accomplished by
changes in their growth factor
Thus signaling molecules, such as FGF, bone
morphogenetic proteins and Noggin can influence neural
stem cells from neural induction
Growth factor concentrations vary during development of
NSC (Wolpert, 1994)
19.
20. Cell signalling pathway for neural stem cells
Notch signaling pathway
It has been found that Notch signaling plays a significant role
in neurogenesis in both embryonic and adult brains (Hitoshi et
al., 2002; Yoon et al., 2008)
Upon activation of Notch by its ligands, the Notch intracellular
domain (NICD) is released from the membrane and
translocates to the nucleus
It was reported that subsets of GFAP+ cells function as neural
stem cells in the adult SVZ (Imura et al., 2003)
22. WNT Signaling Pathway
The Wnt family of signaling proteins participates in multiple
developmental events during embryogenesis
Mitogenic stimulation
Prevent differentiation
Cell fate determination
Removal of Wnt1 results in severe defects of the midbrain,
cerebellum and spinal cord
23. Marker’s of Neural Stem Cells
Cell specific markers are a valuable tool in tracing neural
stem cells during development
A reliable marker should identify NSC not only in the
embryonic brain but also in the adult brain
Markers have been described which are either cell surface
proteins such as CD133, Nestin, an intermediate filament
molecule, or Musashi, an RNA binding protein
24. Nestin
Highly accepted marker for NSC (Frederikson et al., 1988;
Naresh k. et al., 2012 )
Nestin expression, neither restricted to the embryonic CNS, nor
the progenitor cells of neurons, but can be found in the PNS
Nestin seems to play a role in the structural organisation of
cells where it probably participates in remodelling processes
and isolated from human fetal striatum and from rat brain
(Michalczyk and Ziman, 2005,Li et al. 2005, Zhanget al. 2006)
25. Sox2
Sox2 is a “founder member” of the Sox gene family
Sox2 can also re-establish pluripotency in terminally
differentiated cells reprogramming them to induced pluripotent
stem cells (iPS) (Silva J et al.,2009)
Sox2 express in the developing central nervous system (CNS)
(Ellis et al., 2004, Collignon etal., 1996)
Regulate the Notch pathway which is responsible for
maintenance of neural stem cells (Bani-Yaghoub et al., 2006)
26. RNA-binding protein Musashi-1
Musashi is an evolutionarily conserved family of RNA-binding
proteins that is expressed in the nervous system (Okano et al.,
2002)
Level of expression is selectively higher in NSCs than in neural
precursor cells (Kaneko et al., 2000)
Musashi-1 protein has been found to function in cooperation
with Musashi-2 protein to activate Notch signaling
27. Pax 6
Members of Pax family proteins are HD (homeodomain)
containing transcription factors
Transcription factor Pax6 plays an important role in fate
determination of neural progenitor cells in animal models
(Gehring and Ikeo, 1999)
Pax6 is expressed in ventral region (Spinal Cord) and
plays crucial roles in generation of ventral neurons
(Kuldeep k. et al., 2014)
28. CD 133
Widely used as a marker for identification and isolation of
neural precursor cells from normal brain or tumor tissue (Sun
et al.,2009)
CD133 five membrane domain glycoprotein and is expressed
on immature hematopoietic and progenitor cells (Uchida et al.,
2000)
When these CD133+cells were isolated, they were able to form
Clonal neurospheres and produce new tumours after serial
transplantation (Yuan et al., 2004; Li et al., 2005)
30. Factor Affecting Growth And Multiplication Of
Neural Stem Cells
The most commonly used methods for the isolation and culture
of stem cells use serum-free culture medium supplemented
with various hormones and nutrients and mitogenic growth
factors EGF or FGF-2 (Bottenstein and Sato, 1979, Naresh k.
et al., 2012 )
EGF has been used to culture forebrain stem cells as
neurospheres from embryonic and adult mouse (Reynolds et
al., 1992)
A combination of EGF and FGF-2 is needed to culture
embryonic and adult mouse spinal cord, striatum and
subventricular zone (SVZ) derived progenitor cells as
neurospheres (Gritti et al.,1999; Kuldeep k. et al., 2014)
31. Neurospheres, are kept proliferating by adding growth
factors (EGF), (bFGF) and (LIF) (Palmer et al., 2001)
It also suggest signaling of FGF together with Wnt
signaling regulates late features of the dorsal
telencephalon (Gunhaga et al., 2003)
35. Neural Stem Cells for Therapeutic Use
In the nervous system, replacement of neurons is often
considered to be the main goal of cell therapy
But cells, including stem cells, are already being used as gene
delivery tools and for rescuing neurons rather than replacing
them
NSC can be genetically transduced currently, by the most
efficient and popular way of introducing genes into NSC is by
means of mutagenesis
36. Clinical trials of neural stem cells
Stem cell
source
Disease Delivery Year References Current
state of the
art
Fetal neural
stem cells
Batten disease,
or neuronal
ceroid
lipofuscinosis
Brain
neurosurgery
2006 Taupin
P.(2006)
phaseI
ongoing
Embryonic
stem cell-
oligodendroc
yte
progenitors
Spinal cord
injury
Spinal cord
injection
2009 Alper
J.(2009)
Withdrawn
because of
risk of
economic
failure
Fetal neural
stem cells
(8-week-old
fetus)
Amyotrophic
lateral
sclerosis
Multisite
injection into
the
spinal cord
2010 Raore
B.(2011)
ongoing (14
patients
transplanted)
37. Stem cell source Disease Delivery Year References Current state of
the art
Human
embryonic
stem
cells
Stroke Brain
neurosurgery
2010 Stroemer
P.(2009)
Under way
Genetically
modified
human
neural stem
cells (Seung
U. Kim,
University
of British
Columbia)
Glioma Intravenous
delivery
2010 Aboody
KS.(2008)
Recruitment
of
patients
Neural fetal
stem cells
(Stem
Cell Factory,
Hospital S.
Maria,
Amyotrophic
lateral
sclerosis
Multisite
injection
into the
spinal cord
2012 Vescovi
AL.(1999)
Recruitment
of patients
39. Drug Treatments in Development
Major Depressive Disorder
Alzheimer’s Disease
Stroke
Traumatic Brain Injury
Post-Traumatic Stress Disorder
Neurodegeneration
40. CONCLUSION
The development of methods to establish NSC lines in vitro has
been one of the main goals of researchers since the discovery of
active neurogenesis in the adult mammalian CNS
Current preclinical studies strongly suggest that the therapeutic
efficacy of stem cell transplantation
For clinical application, it is important that these protective strategies
are proven safe and effective in humans
Several clinical trials using human embryonic stem-derived NSCs or
fetal NSCs are currently under way
41. Our greatest limitation in treating many neurodegenerative disorders is
the lack of understanding of what causes the onset or drives the
progression of sporadic and idiopathic pathologies
They show tropism towards brain pathology, which appears to be
mediated at least in part by chemokines
We will be benefit from repetive and unconvential concept and
unexpected result that will lead us to future discoveries that we cannot
imagine today
42.
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