Stem cell researches in regenerativedentistry and the future

Haruyoshi YAMAZA, D.D.S., Ph.D.

USJI Seminar, Washington D.C.September 10, 2012

Haruyoshi YAMAZA, D.D.S., Ph.D.

Section of Pediatric Dentistry

Kyushu University Faculty of Dental Science

What are Stem Cells?

Definition of Stem Cells

Self-renewal Capability

Multipotency

Self-renewal

Multipotency

Neural Cells

Stem Cells ProgeniesProgenitors

Self-renewal

Specialized Cells

Hepatocytes

Hematopoietic Cells

Endothelial Cells

Myocytes

What are Stem cells?• Kinds of stem cells

– Embryonic stem (ES) cells

– Induced Pluripotent Stem Cells (iPS Cells)

Fertilized Egg Blastocyst(Inner Cell Mass [Embryo])

ES Cells

Gene Transfection

– Somatic Stem cells• Hematopoietic Stem Cells (HSCs)• Mesenchymal Stem Cells (MSCs)

Gene Transfection

Somatic Cells

Genes

iPS Cell

Cell source of Somatic Stem Cells

• Bone Marrow

• Peripheral Blood

• Brain/Spinal Cord

• Blood Vessel

• Muscle• Muscle

• Skin/Enteric Epithelium

• Retina

• Liver/ Pancreas

• Adipose Tissue

• Umbilical Cord/Umbilical Cord Blood

• Dental Tissues

Stem Cells from Craniofacial Region• Dental pulps• Apical papilla• Dental follicle• Gingiva• Periodontal ligaments• Jaw bone

• Dental pulp stem cells (DPSCs)• Stem cells from apical papilla (SCAP)• Dental follicle progenitor cells (DFPCs)• Gingiva-derived mesenchymal stem

cells (GMSCs)• Periodontal ligament stem cells

(PDLSCs)• Orofacial bone/bone-marrow-derived

MSCs (OMSCs)

Cell Source

Dental Pulp Tissues of Human Exfoliated Deciduous Teeth

SHED(Stem Cells From Human Exfoliated Deciduous Teeth)

Clinically and Biologically Discarded Samples

Accessible and Feasible Cell Source

Miura et al., PNAS, 2002

Clonogenic Potential

SHED display stem cell properties

CFU-F Assay

CD146 CD73 CD105 SSEA4

CD34 CD45 CD14

MSCMarkers

ES cellMarkers

Cell Surface Marker Expression

Flow Cytometric Assay

Proliferative Potency andSelf-renewal Capacity

BrdU Incorporation Assay

CD34 CD45 CD14

HSCMarkers

SHED display stem cell properties

Multidifferentiation Potential

Osteogenic Assay

Alizarin Red Staining

Chondrogenic Assay Adipogenic Assay

hAggrecan

hSox9

hGAPDH

RT-PCR Oil Red-O Staining

RT-PCR

hRunx2

hALP

hOCN

hGAPDH

hGAPDH

hPPARγ2

hLPL

hGAPDH

RT-PCR

Multipotency intoMesenchymal Lineage Cells

Collection ofExfoliated

Deciduous Tooth

Cryopreservation inLiquid Nitrogen (LN)Storage in

FreezingMedium

LN Tank

Tissue Thawing25-30 months

A scheme of the cryopreservation and isolation of SHED

Kyushu University Hospital

Remove ofDental Pulp Tissue

Cell Isolation(Enzyme treatment)

CFU-F Formation

SHED-Fresh SHED-Cryo

MSC markers by flow cytometric analysis

200

100

STRO-126.1%

CD7394.0%

200

100

0

CD14691.9%

200

100

00

SHED-Cryo possess MSC properties

0100 101102 103104

0100 101102 103104

0100 101102 103104

CFU-F

0

50

100

150

1

Colony Numbers(/1x106)

ns

0

50

100

1

BrdU+ Cells(%)

ns

BrdU

Multipotency: Osteogenic induction

Alizarin Red ALP

RUNX2

Specific gene expressionof osteoblast

0

1

2

1

ALP Activity(U /Total Protein)

0

50

100

1

ns ns

Alizarin Red+Area (%)

ALP

OCN

GAPDH

Multipotency: Adipogenic induction

Oil red O

LPL

0.4

Oil Red-O(OD405)

ns

Specific gene expressionof adipocyte

LPL

PPAR2

GAPDH

0

0.2

1

Neural cell differentiation

0

30

60

1

\

0

30

60

1

\

NFM+ Cells (%) III+ Cells (%)

NFM /DAPI III/DAPI

ns ns

Multipotency: Neural and Endothelial cellsdifferentiation

NFM /DAPI III/DAPI

Endothelial cell differentiation

0

30

60

1

\

CD31 /DAPI

0

30

60

1

\

CD34 /DAPI

ns ns

CD31+ Cells (%) CD34+ Cells (%)

Neurofilament M Tubulin βIII

in vivo tissue regeneration andself-renewal assays

Magnetic Sorting ofHuman CD146+ Cells

in vivo tissue regeneration and self-renewal assays

A scheme of the transplantation of SHED-Cryo intocalvarial bone defect of immunocompromised mice

Expansion ofSHED-Cryo

Mixture ofSHED-Cryo & HA/TCP

HA/TCP Carriers

Bone tissue engineering

SHED-Cryo & HA/TCP

Generation ofCalvarial Bone

Defect

Implantation ofSHED-Cryo/HA/TCP

Complex

MicroCTHistology

H&E stainingImmunofluorescence

P P

HA/TCP

12 weeks

Calvarial Regeneration in the critical bone defect modelEdge part Middle part

TRAP staining

Summary

• SHED-Cryo maintained the properties as MSCs such self-renew and multipotency.

• SHED could induce bone regeneration in calvaria-defectmice models.

• SHED are an ideal source for clinical banking of stem cells.

• SHED has great benefits in regenerative medicine,especially bone graft into the cleft of upper jaw onpatients of cleft lip and palate.

Future direction

Neural Cells(Alzheimer's disease)

Hepatocytes(Cirrhosis of Liver)

Hematopoietic Cells

SHED

Exfoliated Deciduous TeethEpithelial Cells(Skin Scar)

Isolation

Differentiation&

Transplantation

Hematopoietic Cells(Leukemia)

Endothelial Cells(Atherosclerosis)

Myocytes(Muscular Dystrophy )

Osteoblasts(Osteogenesis Imperfecta )

Isolation

SystematicInfusion

Acknowledgement

• Dr. Takayoshi Yamaza, D.D.S., Ph.D.Molecular Cell Biology and Oral AnatomyKyushu University Faculty of Dental Science

• Dr. Ma Lan, D.D.S.Pediatric DentistryPediatric DentistryGraduate School of Dental Science, Kyushu University

• Prof. Kazuaki Nonaka, D.D.S., Ph.D.Pediatric DentistryKyushu University Faculty of Dental Science

• Staffs in Pediatric Dentistry of Kyushu University

Thank you!Thank you!