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

*

* *

*

*

*

* *

*

*

* *

*

*

* *

* *

*

*

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