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. 2000 Dec 5;97(25):13625-30.
doi: 10.1073/pnas.240309797.

Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo

S Gronthos  1 , M Mankani, J Brahim, P G Robey, S Shi
Affiliations

Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo

S Gronthos et al. Proc Natl Acad Sci U S A. .

Abstract

Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

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Figures

Figure 1
Figure 1
Colony-forming efficiency and cell proliferation in vitro. Representative high (A) and low (B) density colonies after 14 days. The morphology is typical of fibroblast-like cells (C). The incidence of colony-forming cells from dental pulp tissue and bone marrow at various plating densities indicates that there are more clonogenic cells in dental pulp than in bone marrow (D). The number of BrdUrd-positive cells were expressed as a percentage of the total number of cells counted for DPSCs and BMSCs (E). Statistical significance (*) was determined by using the Student's t test (P ≤ 0.05).
Figure 2
Figure 2
Immunophenotype of cultured DPSCs. Studies based on immunoperoxidase reactivity were performed on first passage cultures of DSPCs. Representative staining patterns are shown for: integrin β1 (A); CD44 (B); collagen type I (C); collagen type III (D); fibroblast growth factor-2 (E); osteonectin (F); osteocalcin (G); MUC-18 (CD146) (H); α-smooth muscle actin (I); osteopontin (J); and vascular cell adhesion molecule 1 (K). Endogenous alkaline phosphatase activity is shown in L.
Figure 3
Figure 3
Developmental potential in vitro. Adherent layers of cultured DPSCs (A and B), and BMSCs (C and D) are shown with Alizarin Red staining as a measure of calcium accumulation after 6 weeks of induction with l-ascorbate-2-phosphate and dexamethasone with inorganic phosphate (A and C). After 6 weeks in the same medium but without inorganic phosphate, lipid accumulation was noted in BMSCs (D), but not in DPSCs (B).
Figure 4
Figure 4
Developmental potential in vivo. Cross sections are representative of DPSC transplants (A, C, and D) and BMSC transplants, (B, E, and F) 6 weeks posttransplantation and stained with hematoxylin and eosin. In the DPSC transplants, the HA/TCP carrier surfaces (c) are lined with a dentin-like matrix (d), surrounding a pulp-like tissue with blood vessels (bv) and an interface layer of odontoblast-like cells (od) (A). A magnified view of the dentin matrix (d) highlights the odontoblast-like layer (od) and odontoblast processes (arrow) (C). Polarized light demonstrates perpendicular alignment (dashed lines) of the collagen fibers to the forming surface (D). In BMSC transplants, lamellar bone (b) is formed on the HA/TCP surfaces (c) and surrounds a vascular, hematopoietic marrow organ (hp) with accumulated adipocytes (a) (B). A magnified view shows that the new bone contains osteocytes (oc), embedded within the calcified matrix, and osteoblasts (ob) lining the bone surfaces (E). With polarized light, collagen fibrils are seen to be deposited parallel with the forming surface (dashed lines) (F).
Figure 5
Figure 5
In situ hybridization for the human-specific alu DNA sequence. Alu-positive cells in the pulp tissue (large arrow) and odontoblast-like layer (small arrow) adjacent to the dentin matrix (d) are easily recognized in 6-week DPSC transplants (A). Osteocytes encased in the new bone matrix (small arrow) and the osteoblasts lining the bone (b) surfaces (large arrow) show positive reactivity with the alu probe in the BMSC transplants (B). Hematopoietic elements (hp) in the marrow-like organ fail to show reactivity with the alu probe.
Figure 6
Figure 6
Expression of the human-specific DSPP, osteocalcin (OC), bone sialoprotein (BSP) mRNA in DPSC transplants. Transcripts for DSPP, BSP, OC, and glyceraldehyde-3-phosphate dehydrogenase were detected by reverse transcription–PCR by using total RNA isolated from 6-week-old DPSC transplants (A). DSPP-positive cells were also found in the pulp tissue and odontoblast layer (arrow) adjacent to the dentin matrix (d) by in situ hybridization (B). Specificity of the probe was verified by hybridization in the odontoblast layer (arrow) of human dental pulp (p) tissue (C). No reactivity of the DSPP-specific probe was detected in human bone, bone marrow, and muscle tissue (data not shown).
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