Characterization of human dental pulp stem cells

2.1. Proliferation Capacity of DPSCs In Vitro

We first identified putative human DPSCs by their ability to generate clonogenic cell colonies in vitro, a common feature displayed by various stem cell populations previously isolated from other tissues. To determine the colony-forming efficiency of DPSCs from whole pulp tissue, single-cell suspensions were prepared by collagenase-dispase treatment of pulp fragments, followed by filtration through a fine mesh to remove cell aggregates prior to seeding the cells at low plating densities (23). The resulting colonies consisted of adherent, fibroblasticlike cells, analogous to colony-forming units-fibroblastic (CFU-F), which form in vitro by osteogenic precursors known as bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells (24-26). An average incidence of approx 40 dental pulp-derived CFU-F were generated per 10,000 cells plated (23).

Recent cloning experiments have indicated that the majority (67%) of the individual colonies failed to proliferate beyond 20 population doublings. Therefore, as primary DPSC cultures are expanded over successive cell passages, progeny arising from the minor subfraction of highly proliferative colonies constitute the bulk of the cell population. This mirrors the growth patterns observed for individual CFU-F derived from primary BMSSCs following ex vivo expansion. Multicolony-derived DPSCs were consistent in their capacity to proliferate in vitro at an average 30% greater rate compared to that observed for BMSSCs.

To account for the rapid rate of proliferation demonstrated by DPSCs, we employed cyclic deoxyribonucleic acid (cDNA) microarray analysis to identify differences in gene expression profiles between DPSCs and BMSSCs (27). The high incidence of DPSCs undergoing S phase was recently correlated with high expression levels of the cell cycle activator, cyclin-depen-dent kinase 6 (CDK6) (27). The activation of CDK6 is mediated by D-type cyclins to promote the progression of cells through G1 to the start of DNA synthesis (28,29). In turn, d-cyclins are activated by various growth factors, such as mitogen IGF-2 (insulin-like growth factor 2), which was also found to be highly expressed by DPSCs compared to BMSSCs. Conversely, DPSCs expressed lower levels of insulin-like growth factor binding protein 7 (IGFBP-7) than BMSSCs (27), a factor known to bind to IGF-1 and IGF-2 that induces inhibition of cell growth (30).

The consequences of these and other differentially expressed genes regarding the growth and development of mineralized dentin and bone are currently under investigation. Studies thus far indicate that DPSCs maintain their high rate of proliferation even after extensive subculture beyond 40 population doublings. Taken together with their clonogenic nature, highly proliferative DPSCs satisfy two characteristics of postnatal somatic stem cells (1,3).

2.2. Phenotypic Analysis of DPSCs

Elucidating the gene and protein expression patterns of primitive DPSC populations and functional odontoblasts is pivotal for understanding the process of odontogenesis. Mineralized dentin is composed of a complex scaffold of extracellular matrix, consisting mainly of collagen type I and some noncollagenous glycoproteins (dentin matrix protein 1, collagen type I, osteonectin, osteopontin, bone sialoprotein, and osteocalcin) also commonly found in the matrix of bone (27,31-37).

These similarities are intriguing considering that, during embryogenesis, odontoblasts are derived from neuroectodermal mesenchyme, in contrast to osteoblasts of the axial and peripheral skeleton, which arise from mesoder-mal mesenchyme. However, two matrix proteins, dentin sialoprotein (DSP) (38) and dentin phosphoprotein (DPP), are thought to be expressed uniquely in dentin (39). Both DSP and DPP have been shown to be encoded by a single gene, known as dentin sialophosphoprotein (DSPP) (40), which is expressed following formation of the collagen-rich predentin matrix, prior to mineralization (41).

Importantly, primary DPSC cultures that had not been induced to differentiate were negative for the odontoblast specific marker DSPP, initially by in situ hybridization (23) and more recently by Western blot analysis using a human-specific DSPP polyclonal antibody developed by Dr. Larry Fisher (Fig. 1A). Immunohistological studies identified dentin-specific staining during early stages of ectopic mineralization by DPSCs in xenogeneic transplants (Fig. 1B). In sections of human pulp tissue, the DSPP antibody only reacted with cells in the mature odontoblast layer (61). These data suggest that the clonogenic dental pulp-derived cells represent an undifferentiated preodontogenic phenotype. This was also supported in animal studies that failed to detect DSPP messenger RNA (mRNA) expression in cultured dental papilla cells derived from rat incisors using reverse tran-scriptase polymerase chain reaction (RT-PCR) in contrast to the high expression of DSPP detected from freshly isolated odontoblast-pulp tissue (42,43).

To date, the precise anatomical location of DPSCs is largely unknown because of a lack of markers specific to the preodontogenic population. Cir-

Fig. 1. DSPP expression by DPSCs. (A) Western blot analysis of cultured DPSCs, human dentin, and mouse dentin with a rabbit polyclonal antihuman DSPP antibody. Ex vivo expanded DPSCs were transplanted with HA/TCP subcutane-ously into immunocompromised mice. (B) Immunoreactivity of DSPP antibody (arrow) with the dentin-pulp interface is shown in recovered 8-wk-old DPSC transplants.

Fig. 1. DSPP expression by DPSCs. (A) Western blot analysis of cultured DPSCs, human dentin, and mouse dentin with a rabbit polyclonal antihuman DSPP antibody. Ex vivo expanded DPSCs were transplanted with HA/TCP subcutane-ously into immunocompromised mice. (B) Immunoreactivity of DSPP antibody (arrow) with the dentin-pulp interface is shown in recovered 8-wk-old DPSC transplants.

cumstantial evidence suggests that preodontoblasts may originate from pericytes migrating from the pulpal endothelium (44). Extensive immunophenotyping of ex vivo expanded DPSCs demonstrated their expression of various markers associated with endothelial or smooth muscle cells such as vascular cell adhesion molecule-1(VCAM-1), CD146 (MUC-18), and a-smooth muscle actin (23). In addition, a-smooth muscle actin-positive cells have also been detected close to mineralized deposits in human dental pulp cultures (45). The expression of these perivascular markers implicates a possible niche for DPSCs in association with blood vessel walls. It is hoped further characterization of DPSCs using current molecular technology will provide novel markers useful in their identification in situ and isolation and purification ex vivo.

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