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!