frozen with liquid nitrogen cooled isobutane and cut to 8–10 mm thick sections. Staining of DPSCs For immunocytochemistry, cells were fixed with ice cold methanol for 5 min, permeabilized with 1% BSA in 0.1% Triton-X 100 (Sigma)/PBS for 10 min, inhibited endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol for 30 min, and blocked non-specific binding sites with 10% goat or horse serum (Vector Burlingame, CA) for 1 h. All primary antibodies listed in Table S4 were used and incubated overnight at 4uC. Stained cells were incubated with a biotinylated antibody at 1:100 (Vector Burlingame, CA) for 1 h, washed and treated with the Vectastain ABC kit and 3, 39-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) substrate kit according to man- ufacturers protocol (Vector Burlingame, CA). For immunofluo- rescence, cells were fixed with 4% formaldehyde/PBS for 5 min, washed with 1% BSA in 0.1% Triton-X 100/PBS, and stained with primary antibodies as described above. Donkey-derived Alexa 488 or goat-derived Alexa 594-conjugated secondary antibodies (Invitrogen) were diluted at 1:800 and incubated for 1 h. Cells were stained with 49, 6-diamine-2-phenylindol (DAPI) at 1:1000 to visualize the nuclei. All antibodies were diluted in 1% BSA in 0.1% Triton-X 100/PBS. We used two types of controls, omitting the primary antibody and IgG isotype from the species made for the primary antibody (0.1 mg/ml) (Vector Burlingame, CA), were included for all staining. Toluidine blue and alcian blue were performed to determine proteoglycan producing cells in chondrogenic culture [87]. Oil Red O was used for characterizing lipid-containing cells in adipogenic culture [87]. X-gal was stained for determining the expression of LacZ gene in Wnt1-marked DPSCs [87]. Tooth histology and staining Mandibles were isolated from 4–10 day-old Tie2-GFP or Wnt1- Cre/R26R-LacZ mice and cut in halves after removing surrounding connective tissues. To preserve GFP, Tie2-GFP derived mandibles were fixed with 0.5% formaldehyde/PBS for 2 h at RT, and washed. The first wash was 30 min followed by 20-min and 10- min washes, respectively [88]. For Wnt1-Cre/R26R-LacZ derived mandibles, they were fixed with 4% formaldehyde/PBS for 2 h at 4uC, and wash. After washing, mandibles from both transgenic mice were immersed in 20% sucrose in PBS at 4uC overnight to preserve tissue morphology before embedding in OCT and frozen with liquid nitrogen cooled isobutane. Since those mandibles were incompletely calcified, we did not process decalcification. There- fore, we cut the tissues into 15–20 mm thickness to get a good morphology of tissue section. For immunofluorescence of tooth section, we did double staining between b-galactosidase or MSI1 and CD44. Briefly, tooth sections were fixed with 4% formaldehyde/PBS for 10 min at RT. Fixed sections were washed and permeabilized with 1% BSA in 0.1% Triton X-100/PBS. Then sections were blocked with 10% normal goat serum for 1 h at RT , and incubated with rabbit-anti-mouse-b-galactosidase or -MSI1 for 1 h at RT following three times of washing. Stained sections were subse- quently incubated with goat-derived Alexa 488 or goat-derived Alexa 647-conjugated secondary antibody (Invitrogen) at 1:800 dilution for 1 h at RT, following three times of washing. Then sections were incubated by the second set of primary antibody which is rat-anti-mouse-CD44 for 1 h at RT, washed, and incubated with goat-derived Alexa 594-conjugated secondary antibody (Invitrogen) at 1:800 dilution for 1 h at RT before washing three times. All immunofluorescence images described in Neural Crest-Derived Dental Pulp Stem Cells this manuscript was detected using a Zeiss Axiovert 200 fluorescent microscope (Thornwood, NY). Photographs were taken with an onboard monochrome AxioCam MRm camera and colored using Adobe Photoshop (San Jose, CA). Background was reduced using brightness and contrast adjustments, and color balance was performed to enhance colors. All the modifications were applied to the whole image using Adobe Photoshop. In vivo transplantation In accordance with approved IACUC protocols, 16106 of murine non-clonal DPSCs, clonal DPSCs, and BMSCs were separately transplanted into 1-month-old male Rag1 mice (Jackson Laboratory, Bar Harbor, ME, USA) by two methods (n = 3 mice/cell line); 1) intramuscular (IM) injection in the tibialis anterior, and 2) subcutaneous (SC) transplantation with hydroxy- apatite tricalciumphosphate (HAp/TCP) (Zimmer) in the dorsum. BMSCs were isolated and cultured as previously described [89]. Prior to transplantation, cells were labeled with the fluorescent membrane dye PKH-26 (Sigma) according to manufacturer’s instructions. Grafts were harvested after 2-wk and 12-wk IM, 5-wk and 12-wk SC transplantations. For SC-transplanted tissues, samples were fixed with 4% formaldehyde for 2 h then demineralized for 7 days in 10% EDTA at 4uC. Both IM- and SC-transplanted tissues were embedded in OCT, frozen with liquid nitrogen cooled isopentane and cut to 10–13 mm thick sections. Some SC-transplants were embedded in paraffin and cut to 5 mm thick sections. Sections were analyzed by H&E, Masson’s trichrome, immunohistochemistry, and immunofluorescence [87]. Supporting Information Figure S1 DPSC isolation and culture. (A and B) First and second developing molar teeth of 4–10 day-old neonatal mice were isolated from mandible. (C) DPSCs started to proliferate and form a small colony after 2 days in stem cell media under 5% O2 incubation. (D) DPSCs at day 10 formed larger colonies and were confluent. (E) The cell morphology of DPSCs in early cultures was heterogenous; most of which were spindle-shaped cells. T = teeth, and M = mandible. Scale bars indicate 200 mm. (TIF) Figure S2 Gene and protein expression of undifferenti- ated DPSCs. Immunocytochemistry showed positive staining of KLF4 to confirm the pluripotency gene Klf4 expression, and of neural crest-related proteins, MSI1 and SOX10. KLF4 and SOX10 showed nuclear and perinuclear staining. Perinuclear localization of KLF4 and SOX10 has been described in previous reports [7,8]. Staining with IgG isotype was used as negative control. RT-PCR also showed the expression of Sox10 in undifferentiated cells. RNA of salivary gland was used as positive control while the reaction without cDNA was used as negative control. Scale bars indicate 100 mm. (TIF) Figure S3 Down-regulation of Nanog and Klf4 after differentiation of DPSCs. Q-RT-PCR analyses showed Nanog and Klf4 were down-regulated after osteogenic and chondrogenic differentiation of DPSC line 1. RQ (Relative Quantification) values were normalized by the expression of mouse embryonic stem cells (mESCs). Similar results were observed for DPSC line 2. GAPDH was used for the internal control. Error bars represent 6SEM. (TIF) Figure S4 DPSC clonal isolation and gene profile. (A–D) At day 10, colonies derived from single cells can be visualized. (E PLoS ONE | www.plosone.org 18 November 2011 | Volume 6 | Issue 11 | e27526