When the expression of Caspase7 was compared between the spheroids from oral mucosa-derived cells and the spheroids from skin-derived cells, the expression was significantly higher in the spheroids from oral mucosa-derived cells regardless of the presence of additives (Fig.?3c). Assessment of stem cell marker manifestation in spheroids from dental mucosa- and skin-derived cells The expression of pluripotency-associated genes was analyzed using qRT-PCR. of spontaneous spheroid formation was tested. Next, the characteristics of spontaneous spheroids from oral mucosa- and skin-derived cells were compared with unique focus on the stemness and neuronal differentiation ability. Methods Dental mucosal cells were from the palate and buccal mucosa of C57BL/6J mice. Similarly, skin cells were from the back of the same mouse strain. Passage 2C3 cells were inoculated into the specific low-adherence tradition plates to form spontaneous spheroids. The effect of fundamental fibroblast growth element (bFGF), epidermal growth factor (EGF), and B27 product on spheroid formation YUKA1 and maintenance was assessed. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to investigate the manifestation of pluripotency markers, cell proliferation and apoptosis markers, and neurogenic differentiation markers. Results By using this tradition plate, spontaneous spheroid formation was feasible. This process depended on the presence of serum but was independent of the additives such as YUKA1 bFGF, EGF, and B27 product, although they improved the effectiveness and were essential for spheroid maintenance. This result was confirmed by the higher manifestation of Caspase7 in the spheroids cultured without the additives than that with the additives. The spheroids from oral mucosa-derived cells indicated stem cell markers, such as Sox2, SSEA1, Oct4, Nanog, and Nestin. YUKA1 The manifestation of Sox2 in spheroids from oral mucosal cells was higher than that in spheroids from skin-derived cells. Both spheroid-forming cell types experienced the ability to differentiate into neural and Schwann cells after neurogenic induction, although significantly higher MAP 2, MBP, Nestin, and Nurr1 gene manifestation was mentioned in the cells from oral mucosa-derived spheroids. Conclusions The results showed that spontaneous spheroids from oral mucosa-derived cells contain highly potent stem cells, which were as good as hSNFS skin-derived stem cells. The high manifestation of particular neuronal marker genes suggests an advantage of these cells for regeneration therapy for neuronal disorders. Electronic supplementary material The online version of this article (10.1186/s13287-019-1283-0) contains supplementary material, which is available to authorized users. N2 product (Invitrogen), 10% FBS, 100?U/ml penicillin, 100?g/ml streptomycin, and 0.25?g/ml amphotericin B (Biological Industries). The cells were differentiated for 1 or 2 2?weeks, and 50% of the medium was changed every 2?days. Immunofluorescence staining Immunofluorescence staining was performed as previously reported [9]. Primary antibodies focusing on the following proteins were used: SSEA1 (1:40, ab16285, Abcam), Sox2 (1:250, 97959, Abcam), Oct4 (1:250, ab19857, Abcam), Nanog (1:100, ab80892, Abcam), III-tubulin (1:250, ab87087, Abcam), Nestin (1:200, ab6142, Abcam), NEUN (1:100, ab177487, Abcam), MAP 2 (1:50, ab32454, Abcam), and S100 (1:100, ab52642, Abcam). The secondary antibodies used were as follows: goat anti-mouse IgM Alex Fluor 488 (1:200, ab150121, Abcam), goat anti-mouse IgG Alex Fluor 488 (1:500, ab150113, Abcam), and goat anti-rabbit IgG Alex Fluor 647 (1:200C1:500, ab150079, Abcam), and the nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, ab104139, Abcam) for 30?min at room temperature. To investigate the cell proliferation and apoptosis inside the spheroids, immunofluorescence staining for Ki67 and Caspase7 YUKA1 was performed. Spheroids were collected 3?days after seeding to the low adherent plate and solidified in iPGell (Genostaff, Tokyo, Japan) according to the manufacturers instructions, fixed with 4% paraformaldehyde in phosphate buffer, embedded in paraffin, and sectioned at a thickness of 8?m. The sections were permeabilized and clogged with 5% BSA, 5% donkey serum, and 0.5% Triton X-100 in PBS for 1?h. Main antibodies targeting the following proteins were used: Ki67 (1:100, ab15580, Abcam) and Caspase7 (1:100, ab69540, Abcam). After incubation with main antibodies over night at 4?C, the sections were washed with PBS for 3 times and incubated with the respective secondary antibodies. The secondary antibodies used were as follows: donkey anti-mouse IgG Alex Fluor 488 (1:250, ab96875, Abcam) and donkey anti-rabbit IgG Alex Fluor 647 (1:250, ab150075, Abcam), and the nuclei were counterstained with DAPI remedy. The fluorescence was observed having a phase-contrast microscope (KEYENCE BZ-X710, Keyence, Osaka, Japan) and analyzed by BZ-X Analyzer software. Quantitative reverse transcription polymerase chain reaction Total RNA was extracted from cells using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA), and cDNA was synthesized with PrimeScriptTM RT Expert Mix (Perfect Real Time) (TaKaRa, cat# RR036A, Kusatsu, Japan) according to the manufacturers protocol. qRT-PCR was performed at least in triplicate according to the manufacturers protocol. The data were quantified using the ??Cycle threshold method and were normalized against the levels of -actin. The primer sequences utilized for PCR are provided in Table?1. Table 1 Primer sequences for PCR Open in a separate window Statistical analysis Statistical analysis was performed using College students test. The results are offered as the mean??standard error of the mean (SEM) of a minimum of three experiments. Results Spontaneous spheroids from oral mucosa-derived cells The spheroid-forming ability of the oral mucosa-derived cells on the specific tradition dish was examined. In serum-free tradition medium, some.