Neural Crest-Derived Dental Pulp Stem Cells Figure 14. Summary of results and working models of neural crest-derived stem cells isolated from murine dental pulp tissue. (A) Base on the Wnt1-Cre/R26R-LacZ mouse model, neural crest gene expression, and differentiation capacity, dental pulp stem cell (DPSC) populations contain a majority of neural crest-derived cells (approximately 90%) (shown in blue) while the remaining cell populations were derived from non- neural crest origin (shown in white). In our condition, only neural crest-derived cells can survive and proliferate. After in vitro differentiation, these cells can give rise to neural crest-lineages, neuronal-like, smooth muscle/pericyte-like, odontoblast-like cells, including classical mesenchymal cell lineages, osteoblast-like, chondrocyte-like, and adipocytes. Nonetheless, in this study the differentiation capacity of non-neural crest-derived mesenchymal stem cell (MSC)-like cells was not characterized. (B) In vivo models show the different capacity between DPSCs and bone marrow stromal cells (BMSCs). Based on the cell/tissue morphology and antibody staining, DPSCs formed dentin-like matrix composed of odontoblast-like cells and pericytes associated with microvessels (close distance about 9–12 mm) recapitulating dentinogenesis. On the other hand, the formation of bone condensation from BMSCs occurred in avascular areas (far distant, about 100–200 mm), resembling early osteogenesis. This model illustrates how two different types of mesenchymal stem cells derived from different origins form different niches and matrices when transplanted under the same conditions. Interestingly, these tissue-specific stem cells recapitulate formation of their own tissue (organogenesis). Understanding the mechanisms of this ‘‘memory’’ of their tissues of origin may be pivotal information for tissue regeneration and cell therapy. doi:10.1371/journal.pone.0027526.g014 Culture of DPSCs Cells (1000 cells/cm2) were cultured at 37uC under 5% O2 and 5% CO2 in stem cell media, containing a final concentration of 60% low-glucose DMEM (Gibco, Invitrogen), 40% MCDB201 (Sigma), 2% fetal calf serum (HyClone) selected previously for optimal growth of murine mesenchymal stem cells, insulin- transferrin-selenium(ITS) ( (Sigma), linoleic acid with bovine serum albumin (LA-BSA) (Sigma), 1029 M dexamethasone (Sigma), 1024 M ascorbic acid 2-phosphate (Sigma), 100 units/ ml penicillin with 100 mg/ml streptomycin (HyClone), and 16103 units/ml leukemia-inhibitory factor (LIF-ESGRO, Milli- pore), supplemented with 10 ng/mL EGF (Sigma) and 10 ng/mL PDGF-BB (R&D) [79]. Once adherent cells were more than 50% confluent, they were detached with 0.25% trypsin-EDTA (Invitrogen) and replated at a 1:4 dilution under the same culture condition with fresh media. Clonal culture of DPSCs Cultured DPSCs at passage 4–7 were plated at 50-100 cells/ cm2. Clones were derived from all three non-clonal lines but only clones from line 1 at passage 7 were used in the experiments described here. 24 h after plating, adherent cells were observed and single isolated cells were marked by circling the bottom of the plate with a lab marker. After 10 days, cell colonies were observed and isolated by using 868 mm cloning rings (Millipore). The clonal populations were expanded every 4 days and transferred to larger size culture dishes beginning with 96-well, 48-well, 24-well, 12-well, 6-well, 60 mm and 100 mm respectively, until finally being seeding in 150 mm culture plates by day 32. The DPSC clones were cultured at a 1:4 dilution until reaching the appropriate cell number for further experiments. The Wnt1-Cre/ R26R-LacZ derived DPSCs were cloned and expanded following the protocol described above. RT-PCR and Q-RT-PCR analyses DPSCs were extracted for total RNA by using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Quantity and purity of RNA was determined by 260/280 nm absorbance. First-strand cDNA was synthesized from 500 ng of RNA using the High Capacity cDNA synthesis kit from Applied Biosystems per manufacturer’s protocols using a randomized primer. RT-PCR and Q-RT-PCR protocol details and primers are included in Text S1 and Table S2, respectively. In vitro multi-differentiation The compositions of each differentiation media and cell densities are listed in Text S1 and Table S3. DPSCs were plated at corresponding cell density in 24-well plates (BD) and incubated overnight in stem cell media at 37uC under 5% O2 and 5% CO2. After 24 h, media was switched to corresponding differentiation media for 21 days with media change every 3 days. In addition to monolayer differentiation, for chondrogenic differentiation Wnt1- Cre/R26R-LacZ derived DPSCs were cultured in chondrogenic media by 3D-pellet culture technique. The 3D-pellet culture is one of widely used technique for chondrogenic differentiation [73]. Briefly, 26105 cells were transferred in a 15-ml polypropylene conical tube and centrifuged at 450 g for 10 min. The pellets were cultured at 37uC under 5% O2 condition in 1 ml of chondrogenic media for 21 days with media change every 3 days. After 21 days, the pellets were fixed 4% formaldehyde/PBS for 30 min and wash with PBS. Then, fixed pellets were embedded in optimal cutting temperature compound (OCT) (Tissue-Tek; Sakura Finetek), PLoS ONE | www.plosone.org 17 November 2011 | Volume 6 | Issue 11 | e27526