Title: 0233 - 3D Culture to Maintain and Restore Mesenchymal Stem Cell Multipotency


Yumi Ohori (Presenter)
Tohoku University Graduate School of Dentistry

Kunimichi Niibe, Tohoku University Graduate School of Dentistry
Maolin Zhang, Tohoku University Graduate School of Dentistry
Xinchao Miao, Tohoku University Graduate School of Dentistry
Hiroshi Egusa, Tohoku University Graduate School of Dentistry


Objectives: Progress in mesenchymal stem cell (MSC) technologies has provided new approaches for regenerative prosthodontics, particularly in cases of missing teeth with severe bone resorption. Previous studies reported that bone marrow-derived mesenchymal stem/stromal cells (BMSCs) contain neural crest-derived stem cells, which would be a useful cell source for craniofacial tissue regeneration. In particular, low-affinity nerve growth factor receptor (LNGFR)+/thymus cell antigen-1 (THY-1)+ cells in human BMSCs (LT-BMSCs) have robust multilineage differentiation and self-renewal potency. However, conventional adherent culture of BMSCs results in a gradual loss of stemness. The objective of this study was to establish a three-dimensional (3D) culture method to prevent the loss of LT-BMSC multipotency over long-term culture.

Methods: Short-term (sLT-BMSCs: 5-9 passages) and long-term (lLT-BMSCs: 16-18 passages) adherent cultures of LT-BMSCs were prepared. After expansion, the cells were transferred to a culture flask for 21 days of shaking 3D culture in neural stem cell medium. Cell construct characteristics were investigated by RT-PCR and histochemistry. Multipotency was assessed by differentiation assays.

Results: Shaking 3D culture generated spheroids with high cellularity from both sLT-BMSCs and lLT-BMSCs. Although lLT-BMSCs showed impaired differentiation after adherent culture, 3D-cultured lLT-BMSC spheroids showed a marked recovery of differentiation toward osteoblast, chondrocyte and adipocyte lineages. Additionally, spheroid-derived cells showed high β3-tubulin expression after neuronal induction, and 3D-cultured sLT-BMSCs and lLT-BMSCs showed significant upregulation of VCAM-1 and PDGFRβ, implying high multipotency (ANOVA; p<0.01).

Conclusions: We established a method to restore and maintain the multipotency of LT-BMSCs, even after long-term proliferation, by generating 3D cell spheroids, which should have great clinical potential for alveolar bone augmentation in regenerative prosthodontics.

This abstract is based on research that was funded entirely or partially by an outside source:
Grand-in-Aid for JSPS Research Fellow Grant number JP18J21198

Disclosure Statement:
The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: NONE