Differentiation potential of dpscs in vitro and in vivo

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3.1. Regenerating a Dentin-Pulp Complex In Vivo

Mineralization within the papal chamber is a frequent event that usually manifests as small calcified pulp stones because of caries, aging, trauma, and systemic conditions (46). Previous studies have established animal cell cultures from dental pulp tissue using a variety of culture methods and noted the ability of such cultures to form mineralized nodules in vitro (43,47-50). In analogy, human-derived dental pulp can also be cultivated in vitro and possesses the capacity to form mineralized deposits in the presence of inductive media containing ascorbic acid, dexamethasone, and an excess of phosphate (23,51,52). The use of methodology such as infrared microspec-troscopic examination and X-ray diffraction electron microscopic (EM) analysis has confirmed the dentinlike nature of the crystalline structures that comprise the mineralized nodules in vitro, which are distinct from the crystal structures of mineralized enamel and bone in vivo (47,49,51,52).

To determine the capacity of ex vivo expanded DPSCs to generate a functional dentin-pulplike tissue in vivo, we utilized an established transplantation system previously optimized for the formation of ectopic bone by cultured BMSSCs (53,54). Until recently, the ability to evoke ectopic dentin formation in vivo was only demonstrated successfully in animal models that utilized rodent or bovine developing papilla tissue (18-21). Similar studies using human intact developing dental papilla or adult dental pulp tissue failed to generate a mineralized dentin matrix or odontoblastlike cells following transplantation into immunocompromised mice (55,56). Previous reports showed that, unlike rodent-derived bone marrow stromal and dental pulp cells, human equivalents require a suitable conductive carrier, such as hydroxyapatite/tricalcium phosphate (HA/TCP) particles, to induce the formation of bone and dentin in vivo (19,54). HA/TCP and other biomaterials have also been used, with partial success, in the clinic to stimulate a pupal proliferation response to aid in the repair of damaged dentin (9,11).

We previously demonstrated that cultured adult human dental pulp cells are capable of generating a dentin-pulplike complex in vivo in conjunction with HA/TCP as a carrier vehicle (23). Typical DPSC transplants developed areas of vascularized pulp tissue surrounded by a well-defined layer of odontoblastlike cells, aligned around mineralized dentin with their processes extending into tubular structures. The odontoblastlike cells and fibrous pulp tissue in the transplants were shown to be donor in origin by their reactivity to the human-specific, alu cDNA probe (23). In addition, orientation of the collagen fibers in the dentin was characteristic of ordered primary dentin, perpendicular to the odontoblast layer. Backscatter EM analysis demonstrated that the dentinlike material formed in the transplants had a globular appearance consistent with the structure of dentin in situ (unpublished observations). Moreover, the presence of human DSPP detected in the transplants confirmed the ability of DPSCs to regenerate a human dentin-pulp microenvironment in vivo.

Studies explored whether DPSCs possess the ability to self-renew. To answer this question, we harvested primary DPSC implants at 2 mo posttransplantation and liberated the cells by enzymatic digestion for subsequent expansion in vitro. Donor human cells were isolated from the cultures by fluorescence activated cell sorting (FACS) using a human Pj-integrin-specific monoclonal antibody, then retransplanted into immunodeficient mice for 2 mo. Recovered secondary transplants yielded the same dentin-pulplike structures as observed in the primary transplants. Human DSPP protein was found in the dentin matrix by immunohistochemical staining, and in situ hybridization studies confirmed the human origin of the odontoblast-pulp cells contained in the secondary DPSC transplants (Fig. 2). Efforts are now under way to determine whether the self-renewing stem cell compartment is localized in the fibrous pulp tissue of the primary transplants.

The developmental potential of individual ex vivo expanded DSPC colonies were also assessed. Of the clones from the initial primary cultures, 25% demonstrated a reduced capacity to form ectopic dentin in vivo; 30% showed an increased capacity when compared to parental multicolony-derived cells. These data are suggestive of a hierarchy of pulp cell differentiation that corresponds to the variations seen in the proliferation rates and developmental potential between individual DSPC clones. Therefore, pulp tissue seems to harbor a rare population of high-proliferating cells with the ability to regenerate a dentin-pulp structure in vivo and the capacity for self-renewal.

3.2. Adipogenic Potential of DPSCs In Vitro

To determine whether DPSCs represent multipotent stem cells, we cultured the cells in various inductive media previously shown to promote the differentiation of adipocytes. The development of fat is not a feature of dental pulp, as opposed to the abundance of fat cells in bone marrow. Analogous to this, the dentin-pulp structures observed in DPSC transplants failed to support either a hematopoietic marrow or any fat cell development, commonly detected in BMSSC transplants following significant bone formation (23). In vitro studies also failed to induce adipogenesis in long-term DPSC cultures grown in the presence of the glucocorticoid dexamethasone, in con-

Fig. 2. Self-renewal capacity of DPSCs. Cell cultures were established from 3-mo-old DPSC primary transplants following collagenase/dispase treatment. Ex vivo expanded human cells were selected by FACS, then retransplanted into immunocompromised mice with HA/TCP. (A) Secondary transplants developed a dentin-pulp complex in vivo. (B) The dentin-pulp interface stained positive (arrow) for human DSPP protein. (C) Fibrous pulp tissue was positive (arrow) for the human-specific alu repetitive element by in situ hybridization.

Fig. 2. Self-renewal capacity of DPSCs. Cell cultures were established from 3-mo-old DPSC primary transplants following collagenase/dispase treatment. Ex vivo expanded human cells were selected by FACS, then retransplanted into immunocompromised mice with HA/TCP. (A) Secondary transplants developed a dentin-pulp complex in vivo. (B) The dentin-pulp interface stained positive (arrow) for human DSPP protein. (C) Fibrous pulp tissue was positive (arrow) for the human-specific alu repetitive element by in situ hybridization.

trast to the abundant clusters of lipid-laden adipocytes observed in corresponding BMSSC cultures. More recent studies using a potent cocktail of adipogenenic inductive agents (0.5 mM methylisobutylxanthine, 0.5 ^M hydrocortisone, 60 ^M indomethacin) (57) have demonstrated the presence of Oil red O-positive fat-containing adipocytes in DPSC cultures following several weeks of induction (Fig. 3A). This was also correlated with an upregulation of the early adipogenic master regulatory gene peroxisome

Dpsc Stem Cells
Fig. 3. Fat development in vitro. Histochemical staining of oil red O-positive (arrow) lipid-laden adipocytes in DPSC cultures following 5 wk of induction with 0.5 mM methylisobutylxanthine, 0.5 |M hydrocortisone, and 60 ||M indomethacin.

proliferator-activated receptor r2 (PPAR2) and the mature adipocyte marker lipoprotein lipase using RT-PCR (62). These observations highlight the plasticity of the DPSC population to develop into functional stromal cell types not normally associated with dental pulp tissue.

3.3. Neuronal Potential of DPSC

Dental pulp contains prominent nerve fibers that penetrate through the tubules alongside the odontogenic cellular processes and act as a protective system in response to degradation of the dentin layer (6). This system of nerve fibers in the dentin matrix allows teeth to receive external stimulation that acts through pain receptors. During development, dental nerve tissue and odontoblasts are both presumed to originate from migratory neural crest cells (6-8). Recent investigations have explored the possibility that DPSCs have the potential to differentiate into neural-like cells. Ex vivo expanded DPSCs were constitutively expressed nestin, an early marker of neural precursor cells, and glial fibrillary acidic protein (GFAP), an antigen characteristic of glial cells (Fig. 4A).

In accord with these findings, other investigators have identified the same markers in dental pulp tissue in situ (58,59). When DPSCs were cultured

Fig. 4. Neuronal differentiation in vitro. (A) Basal mRNA expression levels of (1) GFAP and (2) nestin transcripts in DPSCs cultured under normal conditions. (B) NeuN protein expression (arrows) following 2 wk of culture in neoronal inductive conditions: Neuroblast A medium (Invitrogen/GIBCO), 5% horse serum, 1% fetal bovine serum, transferrin 100 |g/mL, insulin 25 |g/mL, retinoic acid 0.5 |M, and BDNF (brain-derived neurotrophic factor) 10 ng/mL.

Fig. 4. Neuronal differentiation in vitro. (A) Basal mRNA expression levels of (1) GFAP and (2) nestin transcripts in DPSCs cultured under normal conditions. (B) NeuN protein expression (arrows) following 2 wk of culture in neoronal inductive conditions: Neuroblast A medium (Invitrogen/GIBCO), 5% horse serum, 1% fetal bovine serum, transferrin 100 |g/mL, insulin 25 |g/mL, retinoic acid 0.5 |M, and BDNF (brain-derived neurotrophic factor) 10 ng/mL.

under defined neural inductive conditions, there was enhanced expression of both nestin and GFAP. Morphological assessment of induced DPSCs identified long cytoplasmic processes protruding from rounded cell bodies, in contrast to their usual bipolar fibroblasticlike appearance. Moreover, DPSCs cultured under neural inductive conditions (60) were found to express the neuron-specific marker neuronal nuclei (NeuN) by immunohis-tochemical staining (Fig. 4B).

These preliminary studies provided the first experimental evidence that adult human DPSCs may possess the potential to differentiate into neural-like cells with expression of nestin, GFAP, and NeuN in vitro. Transplantation studies are now under way to determine the capacity of human DPSCs to form functional neuronal tissue following their transplantation into different brain sites in immunocompromised mice.

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