Characterization of human dental pulp cells-derived spheroids in serum-free medium: Stem cells in the core

Abstract

Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration.

Notch/Wnt cross-signalling regulates stemness of dental pulp stem cells through expression of neural crest and core pluripotency factors

“…In vitro expansion of DPSCs is often inevitable due to the small amounts of tissue material and total cell numbers that can be collected from the human dental pulp, as compared to other stem cell sources. In this regard, despite ongoing progress in culture media formulations that do not contain foetal serum for maintenance of DPSCs (Bonnamain et al, 2013;Eubanks et al, 2014;Jung et al, 2016;Xiao and Tsutsui, 2013), currently the addition of FBS or related agents to the culture media permits easily overcoming the issue of initial cell expansion. However, it is becoming increasingly apparent that FBS-containing media also induce differentiation of DPSCs into a default osteo/odontogenic pathway (Pisciotta et al, 2012;Yu et al, 2010) and this may not be the best choice to generate certain cell lineages, particularly neural cells, from DPSCs (Jung et al, 2016).…”

“…It would also be very interesting to verify whether such preconditioning would also be effective for other non-mesenchymal cell lineages of interest, such as Schwann cells (Martens et al, 2014), neuron-like cells (Gervois et al, 2015) and hepatocyte-like cells (Atari et al, 2012). Finally, the adoption of this preconditioning strategy could also be applicable to differentiation protocols that do not rely on FBS (Eubanks et al, 2014;Xiao and Tsutsui, 2013), allowing for a faster translation to clinical therapy.…”

Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro

“…Within the last years, cells derived from pulp-tissue were investigated concerning their multipotent stem cell character as well as their ability to differentiate in diverse ways such as angiogenetic or osteogenic differentiation [24]. In this study, the possibility of human dental pulp cells (DPC) serving as potential progenitors for odontoblast formation and biomineralization initiators was investigated.…”

“…In this study, the possibility of human dental pulp cells (DPC) serving as potential progenitors for odontoblast formation and biomineralization initiators was investigated. Several studies displayed the high potential of DPC regarding this matter [22,24-26]. Besides the possibility to influence the specific differentiation of these cells, an odontoblast-like phenotype, structure, cell formation and differentiation behavior were shown in in vitro and in vivo setups [25-27].…”

“…The medium was changed twice per week. In preliminary tests it was possible to differentiate the cultivated human pulp cell spheres in different tissue specific ways to validate their multipotent stem cell character as described previously [23]. …”