Stem cells potential in dentistry - Semmelweis...
Transcript of Stem cells potential in dentistry - Semmelweis...
Stem cells – potential in
dentistry
Dr. Gábor Varga
Department of Oral Biology
2015
Sections of tooth undergoing development.
LAMINA BUD STAGE CAP STAGE BELL STAGE ERUPTION
Tooth development
Tucker, A., and Sharpe, P.
The cutting-edge of mammalian development; how the embryo makes teeth.
Nature reviews 5, 499, 2004.
Neuronal development:
a link to tooth development
Control of tooth shape –
ectomesemchymal influence
Barx1 gene expression is strongly related to molar formation
a) its suppression leads to incisor formation in molar area)
b) its ectopic expression in incisor area leads to molar formation)
Control of tissue differentiation –
inductive action of mesenchyme
Morphogenesis of tooth
Tooth development is driven by
communication between cells using signal
molecules activating specific receptors
Molecular components of control
A model of the molecular regulation of
tooth development from initiation to
crown morphogenesis Epithelium
Mesenchyme
Definition of stem cell
• Unlimited self-renewal
• Capability to differentiate and form tissues
- Embryonic stem cells - pluripotent
- Adult/postnatal stem cells - multipotent
Totipotent cells
zygote
Embryo
Blastocyst
embryoblast
Adult
Developmental
scheme from zygote
to adulthood
Ethical and immune
problems
Totipotent cells
zygote
Embryo
Blastocyst
embryoblast
Stem cells from embryonic origin Stem cell
culture
Adult
Stem cell isolation
from embrionic origin
Totipotent cells
zygote
Embryo
Blastocyst
embryoblast
Stem cells from differentiated tissues
Stem cell culture
Adult
Stem cell isolation
from differentiated
tissues
Stem cell culture
Cell transplantation for therapy
Drug development Studies on mechanism of action
Basic research Studies on embrionáal and tissue differentiation
Bone marrow Neuronal tiss. Heart tiss. Insulin production Bone and tooth
Potential utilization of stem cells
Stem cells are continuously present in the
cervical loop of mouse incisors
Stem cells
RESULTS I: COLONY FORMING OF PDLSC CULTURES
The plated periodontal ligamentum derived cells form colonies. This
photo shows 14 days old colonies
Colony formation and DNA synthesis of
DPSC and BMSC cells
Gronthos S. et al. PNAS. 2000; 97:13625-13630
Cultures PDLSC cells show typical fibroblast-like morphology
RESULTS II: MORPHOLOGY OF CULTURED PDLSC CELLS
day 6 40x day 6 100x
day7 200x day 13 200x day7 200x day7 200x day 11 200x
LIPID accumulation
Different mineral formation of DPSCs and BMSCs in vitro
DPSC
BMSC
L-ascorbate-2-
phosphatet+dexamethason
+inorganic phosphate
L-ascorbate-2-
phosphatet+dexamethason
Gronthos S. et al. PNAS. 2000; 97:13625-13630
STRO-1 immunostaining was observed in the primary DPSC cultures (5th passage, 5 week old)
anti-STRO-1
anti-STRO-1 anti-STRO-1
control (w/o primary antibody)
STRO-1 mesenchymal stem cell marker in DPSC cultures (400x magn.)
RESULTS III: IMMUNOCYTOCHEMISTRY
HA-TCP-bound DPSC and BMSC cells behave in differnt
manner when transplanted in vivo to mouse
DPSC:
•Odontoblast-like cells
•dentin
BMSC:
•osteocytes
•Lamellar bone
•Hemopoetic structures Gronthos S. Et al. PNAS. 2000; 97:13625-13630
A PDLSC cells in periodontal regenration –
an animal model
A – PDL B – attachment on tooth surface C – Alveolar bone
Anti-human mitochondrium labelling
Seo B-M. et al. Lancet. 2004; 364:149-155
Dentin Bone
Bone marrow or pulp tissue collection
Stem cell culture
Implantation to mouse Scaffold
structure
Stem cells of dental and bone origin
Bone marrow collection
Bone regeneration
Stem cell culture
Scaffold material
1 – Stem cells for bone regeneration
Dentin regeneration
Stem cell culture
Pulp tissue collection
Scaffold material
2 – Pulp and dentin regeneration
Periodontal tissue
regeneration
Stem cell culture
Periodontal tissue collection
Scaffold material
3 – Periodontal regeneration
Stem cell culture
Stem cell collection
Differentiation to cementoblasts or osteoblasts
4 – Support for implantation
Regeneration following implantation
COMPLETE TOOTH-
regeneration
Stem cell culture
Stem cell collection
Differencition to epithelial- mesenchimal complex
5 - Complete tooth regeneration
Importance of cells,
scaffold and
bioactive molecules
Tooth
development
needs epithelial-
mesenchymal
intercations
Process of tooth replacement in mouse Ikeda et al., (2009) Fully functional bioingeniered tooth replacement as an organ replacement therapy. PNAS USA. 106, 13475-
13480.
The new tooth with green fluorescent label is visible in mouse
mouth
Process of tooth replacement in mouse Ikeda et al., (2009) Fully functional bioingeneered tooth replacement as an organ replacement therapy. PNAS
USA. 106, 13475-13480.
C) Eruption
D) Histochemical analysis
E-F) Fluorescent label
G) Oral photo
H) Mikro-CT picture
Three key elements for Regenerative
Dentistry
Tucker, A., and Sharpe, P.
The cutting-edge of mammalian development; how the embryo makes teeth.
Nature reviews 5, 499, 2004.
Neuronal development:
a link to tooth development
hDPSC cell morphology during culture, and
osteogenic differentiation A B
C D
E F
Király et al., Neurochem Int, 2009
Neural induction was carried out first by demethylation, then by simultaneous PKC
and cyclic AMP pathway activation, finally by a neurodifferentiation coctail.
Neural differentiation by a new three-step
protocol
Changes in DPSC cell morphology and cell
viability during neuronal differentiation
D
A
C
E
D
F
A B
Király et al., Neurochem Int, 2009
10
m
Cells after 10 days of differentiation display multi- and
bipolar neuron-like structures
Differentiated cells:
Complex neurite-like structures
Vimentin (200 bp)
Nestin (220 bp)
A B C D E F
NSE (329 bp)
NF-M (333 bp)
N-tubulin (243 bp)
GFAP (266 bp)
β-actin (234 bp)
NGN2 (196 bp)
*
0,1
1,0
10,0
100,0
Rela
tive n
orm
aliz
ed q
uantity
*
A B C D E F A B C D E F A B C D E F
A B C D E F A B C D E F A B C D E F
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Changes in gene
expression
Immuncytohemistry after neuronal differentiation (A) neuron specific microtubule marker N-tubulin, (B) neuronal NF-M
and (C) glial GFAP intermediate filament (D) NeuN neuronal nuclei
protein. Nuclei are visualized by DAPI
A B
C D
Király et al., Neurochem Int, 2009
Patch clamp recordings to provide evidence
for functional neurons
-500
-300
-100
100
300
500
-10 0 10 20 30 40 50
Tra
ce
(pA
)
Time (ms)
A
-500
-400
-300
-200
-100
0
-70 -50 -30 -10 10
V [mV] I [pA]
Na current
TTX blocking
B C
VD sodium current
TTX
TEA
KDR current
Király et al., Neurochem Int, 2009
• Our novel neuroinductive protocol resulted in
the appearance of neuron-like cells in DPSC and
PDLSC cultures.
•Patch clamp analysis indicates the functional activity of
voltage-dependent sodium and potassium channels in the
differentiated cells.
• During induction a time dependent alteration of the gene
expression pattern of the gene transcripts was seen:
vimentin and nestin decreased, while NSE and NF-M.
increased during neuronal induction. (mRNA values were
normalized to the expression of RPLP0).
• We also observed an increase in immunoreactive cells
for of N-tubulin and Neurofilament-M, both of them are
regarded as neurospecific markers.
In vivo utilization of differentiated hDPSCs in
rat brain damage
Király et al., unpublished, 2010
Three key elements for Regenerative
Dentistry by tissue engineering
Thank you for your attention