Abstract
BackgroundOur group has developed a novel method for spontaneous spheroid formation using a specific low-adherence culture plate with around 90° water contact angle. In this study, this method was applied for oral mucosa-derived cells. First, the feasibility of spontaneous spheroid formation was tested. Next, the characteristics of spontaneous spheroids from oral mucosa- and skin-derived cells were compared with special focus on the stemness and neuronal differentiation capability.MethodsOral mucosal cells were obtained from the palate and buccal mucosa of C57BL/6J mice. Similarly, skin cells were obtained from the back of the same mouse strain. Passage 2–3 cells were inoculated into the specific low-adherence culture plates to form spontaneous spheroids. The effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and B27 supplement on spheroid formation and maintenance was assessed. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to investigate the expression of pluripotency markers, cell proliferation and apoptosis markers, and neurogenic differentiation markers.ResultsUsing this culture plate, spontaneous spheroid formation was feasible. This process depended on the presence of serum but was independent of the additives such as bFGF, EGF, and B27 supplement, although they improved the efficiency and were essential for spheroid maintenance. This result was confirmed by the higher expression of Caspase7 in the spheroids cultured without the additives than that with the additives. The spheroids from oral mucosa-derived cells expressed stem cell markers, such as Sox2, SSEA1, Oct4, Nanog, and Nestin. The expression of Sox2 in spheroids from oral mucosal cells was higher than that in spheroids from skin-derived cells. Both spheroid-forming cell types had the ability to differentiate into neural and Schwann cells after neurogenic induction, although significantly higher MAP 2, MBP, Nestin, and Nurr1 gene expression was noted in the cells from oral mucosa-derived spheroids.ConclusionsThe results showed that spontaneous spheroids from oral mucosa-derived cells contain highly potent stem cells, which were as good as skin-derived stem cells. The high expression of certain neuronal marker genes suggests an advantage of these cells for regeneration therapy for neuronal disorders.
Highlights
Our group has developed a novel method for spontaneous spheroid formation using a specific lowadherence culture plate with around 90° water contact angle
In serum-free culture medium, some of the oral mucosaderived cells attached to the culture dish and showed fibroblastic morphology at 24 h, and the cells became apoptotic after 72 h (Fig. 1a)
In serum-free culture medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and B27 supplement, oral mucosa-derived cells attached to the dish and showed some cell aggregates, but spheroid formation was not observed (Fig. 1a)
Summary
Our group has developed a novel method for spontaneous spheroid formation using a specific lowadherence culture plate with around 90° water contact angle. In this study, this method was applied for oral mucosa-derived cells. Rotation of culture medium has been used [7, 8] Because these methods depend on physical force and the cell-to-cell attachment is achieved by forced contact of the cells, we designated them as “mechanical spheroid formation” methods, and the process of spheroid formation is not selective for stem cells [9]. Other methods, including ours, do not use physical force, and spheroid formation occurs under static conditions. Most reported spontaneous spheroid formation is limited to neural stem cells and skin-derived stem cells [10, 11], and the detailed characteristics of spontaneous spheroids from oral mucosaderived cells have not been reported
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