Pharma&Biotech BioResearch Characteristics of Dental Pulp Stem Cells 15 October 2013 / Speaker: Dr. Isabella Drewelus 16 October 2013 / Speaker: Andrew Winner Lonza Walkersville, Inc., Walkersville, MD 21793 / October 2013 Lonza
Agenda Introduction to Dental Pulp Stem Cells Review of oral stem cells DPSC Isolation and Cell Culture DPSCs vs. Bone Marrow derived MSCs Intro Materials and Methods Results Conclusions 2
Oral Cavity Stem Cells Multiple cell types Vary in multipotency and gene expression Image Courtesy of sciencedirect.com 3
Stem Cells from Apical Papilla (SCAP) Isolated from the apical tip of the tooth Found just underneath the enamel organ Some Cementum Matrix formation No recorded myogenic or chondrogenic differentiation Image Courtesy of sciencedirect.com 4
Stem Cells from Exfoliated Deciduous Teeth (SHED) Also called milk teeth or baby teeth Easily obtained No known dentin-pulp complex formation 5
Dental Pulp Stem Cells DPSCs First described in 2000 by Dr. Song Tao Shi* Usually harvested from adult third molars ( Wisdom teeth) 20-30 million cells per tooth most bang for the buck Easier to obtain Most multipotent of oral cavity cells Very similar to BM derived MSCs Image Courtesy of sciencedirect.com *Shi, S. and Gronthos, S. (2003), Perivascular Niche of Postnatal Mesenchymal Stem Cells in Human Bone Marrow and Dental Pulp. J Bone Miner Res, 18: 696 704. doi: 10.1359/jbmr.2003.18.4.696 6
Dental Pulp Stem Cells DPSCs (Continued) Mesenchymal-like multipotent cells that can differentiate into several cell types Myocardiocytes, Neurons, Myocytes, Osteoblasts, Chondrocytes, Adipocytes and other bone/tissue in the oral cavity Phenotype SSEA4+, OCT3/4+, NANOG+, SOX2+, LIN28+, CD13+, CD105+, CD34-, CD45-, CD90+, CD29+, CD73+, STRO1+ and CD146- Morphology in culture Able to form embryoid bodies-like structures (EBs) in vitro Can differentiate into tissues that have similar characteristics to mesoderm, endoderm and ectoderm Form tube-like structures in a Matrigel assay 7
DPSC Differentiation: Compared to MSCs MSCs Osteocytes Chondrocytes Myocytes Adipocytes Neurons DPSCs Osteocytes Chondrocytes Myocytes Adipocytes Neurons Also: Astrocytes Stromal cells 8
Isolation of DPSCs* 1. Sterilized teeth are gauze wrapped tooth squeezed in a vise until cracked 2. Remove pulp with sterile tweezers digest with a solution of collagenase type I and dispase for 30-40 minutes 3. Suspend in culture medium, the centrifuge to pellet 4. Re-suspend the cell pellet in regular growth medium and pass the cells through a strainer to obtain single cell suspensions 5. Cells ready for culture *Gronthos, S. (2000), Postnatal human dental pulp stem cells in vitro and in vivo Proc. Natl. Acad. Sci. USA, 10.1073/pnas.240309797 9
Culture and Expansion of DPSCs Used Poietics DPSC Bullet Kit Growth Media Supplements Nearly identical to culturing MSCs Seeding density is 5,000-6,000 cells per cm 2 Fed 3-4 days after plating Near confluence by day 6 or 7 10
Agenda Introduction to Dental Pulp Stem Cells Review of oral stem cells DPSC Isolation and Cell Culture DPSCs vs. Bone Marrow derived MSCs Intro Materials and Methods Results Conclusions 11
Experimental Design Use flow cytometry to compare the expression of 5 known markers genes both DPSCs and Bone Marrow MSCs CD-166 (ALCOM): leukocyte cell adhesion molecule commonly used as a hematopoietic stem cell marker CD-105 (Endoglin): membrane glycoprotein commonly used to identify MSCs CD-45 is a protein tyrosine phosphatase, type C that is present on the surface of all hematopoietic cells CD-29 is part of the α3β1 complex that helps define neural lineage differentiation CD34 is a cell surface glycoprotein that is commonly used marker for hematopoietic cells 12
Periodontal Ligament Stem Cells (PDLSCs) Fibrous connective tissue Neural and vascular components No recorded myogenic or chondrogenic differentiation Weaker expression of Nanog than other oral cavity stem cells Image Courtesy of studiodentaire.com 13
Materials and Methods: Cells and Cell Culture Dental Pulp Stem Cells Poietics hdpsc (PT-5025) Poietics hdpsc BulletKit (PT-3005) 5 Day Culture Mesenchymal Stem Cells Poietics hmsc (PT-2501) Poietics hmsc GM BulletKit (PT-3001) 5 Day Culture 14
Materials and Methods: Flow Cytometry DPSCs and MCS both Antibody labeled for: CD-105, CD-166, CD-29, CD-34, CD-45, CD-90 Cells acquired on a BD FACSCanto II flow cytometer Analyzed using FACSDiva 6.1.3. Image Courtesy of Imperial College of London 15
Results: DPSCs (Figures A) Representative data of FACS performed on human DPSC at passage two. These cell tested positive for CD166, CD105 and CD29 but negative for CD45 and CD34. 16
Results: MSCs (Figures B) Representative data of FACS performed on human MSC at passage two. These cell tested positive for CD166, CD105 and CD29 but negative for CD45 and CD34. 17
Results (Continued) % Positive CD105 CD166 CD29 CD45 CD34 HDPSC 94.9 98.7 98.2 3 0 MSC 99.7 98.8 99.8 8.8-0.7 Table 1. Summary of Flow Cytometry Results Flow-cytometric analysis revealed human DPSCs were positive for markers CD166, CD105 and CD29, but negative for CD34 and CD45, suggesting that these cells are mesenchymal-like stem cells. 18
Agenda Introduction to Dental Pulp Stem Cells Review of oral stem cells DPSC Isolation and Cell Culture DPSCs vs. Bone Marrow derived MSCs Intro Materials and Methods Results Conclusions 19
Conclusions Stem Cells harvested from human dental pulp represent a less invasive and more affordable source of stem cells for research and possibly even future therapeutic purposes. Flow-cytometric analysis revealed human DPSCs were positive for markers CD166, CD105 and CD29, but negative for CD34 and CD45, suggesting that these cells are mesenchymal-like stem cells. 20
Poietics DPSC and hmsc Cat. No. Description Quantity PT-5025 Human Dental Pulp Stem Cells 1M >= 1 Million Cells / cryoamp PT-2501 Human Mesenchymal Stem Cells 1M >= 1 Million Cells / cryoamp PT-3005 DPSC BulletKit (Pt-3927 & PT-4516) 1 Kit PT-3001 MSCGM BulletKit (PT-3238 & PT-4105) 1 Kit 21
References 1) Gronthos, S. (2000), Postnatal human dental pulp stem cells in vitro and in vivo Proc. Natl. Acad. Sci. USA, 10.1073/pnas.240309797 2) Shi, S. and Gronthos, S. (2003), Perivascular Niche of Postnatal Mesenchymal Stem Cells in Human Bone Marrow and Dental Pulp. J Bone Miner Res, 18: 696 704. doi: 10.1359/jbmr.2003.18.4.696 3) Huang, GTJ. (2009) Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources J Dent Res. USA 2009 September; 88(9): 792 806 4) Pierdomenico, Laura et al. (2005) Multipotent Mesenchymal Stem Cells With Immunsupressive Activity can Be Easily Isolated from Dental Pulp. Transplantation 80(6): 836-842. 5) 3 Gluhak-Heinrich, J. et al. (2012) Bmp2 Is Required for Odontoblast Differentiation and Pulp Vasculogenesis JDR 91: 58-64 6) 4 Whitney GS, Starling GC, Bowen MA, et al. (1995). "The membrane-proximal scavenger receptor cysteine-rich domain of CD6 contains the activated leukocyte cell adhesion molecule binding site.". J. Biol. Chem. 270 (31): 18187 90. 7) 5 Kaplan R, Morse B, Huebner K, et al (September 1990). "Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain". Proc. Natl. Acad. Sci. U.S.A. 87 (18): 7000 4 8) 6 Goodfellow PJ, Nevanlinna HA, Gorman P, Sheer D, Lam G, Goodfellow PN (July 1989). "Assignment of the gene encoding the beta-subunit of the human fibronectin receptor (beta- FNR) to chromosome 10p11.2". Ann Hum Genet 53 (Pt 1): 15 22 22
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