disorganized dentin-like structure, mimicking a structure generat- ed in reparative dentin formation after tooth injury [69]. Reparative dentin exhibits disorganized arrangement due to lack of signaling from inner enamel epithelium and basement membrane which results in multiple inducing molecules secreted from dentin matrix [71]. Lack of epithelium induction may explain why DPSC clones generated a disorganized structure. Like the renal capsule or subcutaneous regions, we used the intramuscular model to transplant DPSCs since we hypothesized that a high vascularized system in muscle may enhance the function of DPSCs [2,3,80]. In turn, IM transplantation of DPSCs generated well-organized but immature collagen-forming matrix. These results may be explained by several possibilities. First, each transplant environment has a distinct influence on differentiation of DPSCs. Secondly, the IM transplanted structure was more organized possibly because muscle fibers function as scaffold to stabilize and directly instruct transplanted cells toward the proper arrangement and orientation. Thirdly, the length of transplanta- tion in the IM injections was only 2 weeks, 3 weeks shorter than SC transplantation, and thus, longer IM transplantation may result in more mature matrix formation. To determine if longer transplantations are required for formation of more mature matrices, we performed IM and SC transplantations for 12 weeks. Surprisingly, longer in vivo exposure did not result in more mature matrix formation in the intramuscular transplants. However, longer subcutaneous trans- plantations with HAp/TCP resulted in more mature matrix formation in both DPSC and BMSC transplants but the morphology and matrix compositions differed. First, we observed many abundant small blood vessels in close proximity to odontoblast-like cells in DPSC transplants. Many of these microvessels were fenestrated and stained positive for VEGFR-3. We observed pericyte-like cells adjacent to these microvessels; some of which were donor DPSC-derived. This observation illustrates the perivascular nature of DPSCs and their potential to function as pericytes [81]. Although BMSC trans- plants also contained abundant microvessels, bone condensations were significantly distant to vessels. Interestingly, in BMSC transplantations we observed a large number of microvessels in areas rich in adipocytes. These cells may be from BMSC-derived or derived from host cells which were signaled by BMSCs to differentiate into adipocytes. Secondly, long-term DPSC transplantation resulted in a more organized mineralized matrix formed by elongated and polarized cells resembling odontoblast-like morphology which were in close proximity to microvessels. These matrices represent dentin-like structure with strong positive staining for dentin matrix proteins, DMP1, DSP, and BSP. In contrast, BMSC transplantation showed mineralized matrices with osteocyte-like cells in lacunae. Consistently, the matrices produced by BMSCs were positive for the bone protein, BSP, but not positive for the dentin protein, DSP. However, some studies reported that certain states of bone or tooth development can express low levels of dentin or bone proteins, respectively; therefore, we cannot distinguish bone or dentin structures by only protein expression [31,49]. As mentioned above, the combination of cell/tissue morphology and matrix protein expression is important to characterize the structure created by DPSCs and BMSCs [2,50,80]. Thirdly, as described previously in human DPSC in vivo transplantation, murine DPSCs can create collagen fibers arranging perpendicularly to the scaffold/matrix surface whereas BMSCs created collagen fibers running parallel to the scaffold/ matrix surface [2,3]. Lastly, the distribution and association of odontoblasts with microvessels resembles dentinogenesis whereas Neural Crest-Derived Dental Pulp Stem Cells the formation of bone condensation in avascular areas resembles early osteogenesis [57–59]. In summary, we showed dental pulp cells in mouse neonates with neural crest-related stem cell properties. Although some studies showed that both neural crest and non-neural crest cells can be found in dental pulp during tooth development, our stem cell conditions select for only the outgrowth of neural crest-derived stem cells (Figure 14A). Thus, future studies should aim at comparing the differentiation capacity, contribution and cross-talk of neural crest- and non-neural crest-derived cells from dental pulp. Upon transplantation, DPSCs formed dentin-like matrix composed of odontoblast-like cells and pericytes associated with microvessels in dental pulp-like tissue recapitulating dentinogenesis (Figure 14B). The mechanisms that control the differentiation fate of dental pulp stem cells are not completely elucidated but interactions with their niche are important for maintenance of stemness and induction/guidance for differentiation. In our transplantation model, interactions with microvessels seem pivotal for the differentiation of DPSCs into pericytes and odontoblasts. Since the dental pulp tissue is highly vascularized, it is probable that dental pulp stem cells reside in a vascular niche. To date multiple reports have revealed the presence of various adult stem cells residing within the vascular niches [70,82–84]. In addition, several recent studies have shown the existence of neural crest stem cells in various postnatal tissues [27,85,86]. Thus, future studies should also aim at understanding the interactions of neural crest-derived stem cells with their respective niches from multiple adult tissues. Understanding the mechanisms that control organogenesis and adult tissue homeostasis will be instrumental for tissue engineering and tissue regeneration. Thus, studying how neural crest-derived stem cells from multiple organs interact within each niche to remain either stem cells or differentiate may help elucidate the mechanisms for maintenance of tissue-specific stem cells and tissue- specific differentiation. This will broaden the potential clinical applications of DPSCs as a readily accessible source for tissue regeneration not only of pulp regeneration but possibly other neural crest derivatives. Materials and Methods Ethics Statement All mouse experiments and protocols were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) guidelines. Dental pulp isolation Dental pulp tissue were isolated in pools (5 mice per preparation) from 4–10 day-old neonatal Tie2-GFP (Jackson Laboratory) (n = 3 different preparations) and Wnt1-Cre/R26R- LacZ mice (n = 1 preparation) (a gift from Dr. Michael Cunning- ham, University of Washington) in accordance with approved Institutional Animal Care and Use Committee (IACUC) guide- lines. The molar teeth were separated from the mandible. Pulp tissue was gently isolated and kept in stem cell media described below. The tissue was washed with phosphate buffer saline (PBS) (HyClone) and digested with a 1.2 units/ml dispase II, 2 mg/ml collagenase type IV (Worthington) supplemented with 2 mM CaCl2 in PBS for 60 min at 37uC. Subsequently an equal volume of stem cell media was added to the digest prior to filtering through 70 mm nylon cell strainers (BD Falcon), and then centrifuging at 300 g for 10 min at room temperature. Cells were then resuspended in stem cell media and single cell suspensions were 2 plated at 1000 cells/cm . PLoS ONE | www.plosone.org 16 November 2011 | Volume 6 | Issue 11 | e27526