Novel Mesenchymal Stem Cell Spheroids with Enhanced Stem Cell Characteristics and Bone Regeneration Ability

Abstract

Mesenchymal stem cells (MSCs) exhibit self-renewal, multi-lineage differentiation potential and immunomodulatory properties, and are promising candidates for cellular therapy of various tissues. Despite the effective function of MSCs, the gradual loss of stem cell characteristics that occurs with repeated passages may significantly limit their therapeutic potential. A novel 3D shaking method was previously established to generate MSC spheroids in growth medium (GM-spheroids) and successfully maintain the multipotency of expanded MSCs, yet the expression of MSC-related genes was still low. In this study, we used a neurosphere culture technique to optimize the shaking culture method using human bone marrow-derived MSCs (BM-MSCs). MSC spheroids generated in neurosphere medium (NM-spheroids) maintained high expression of MSC-related genes during 3 weeks of prolonged shaking culture. Moreover, NM-spheroids generated from expanded MSCs showed high viability, upregulation of MSC-related and immune-related genes, and recovery of differentiation potential in vitro. Expanded adherent MSCs, GM-spheroids, and NM-spheroids were transplanted into a rat femur bone defect model to investigate their therapeutic potential in bone repair. Adherent MSCs and GM-spheroids showed delayed bone healing. In contrast, NM-spheroids showed high transplantation efficiency and enhanced bone regeneration. These data suggest that NM-spheroids generated using modified neurosphere culture conditions under continuous shaking recovered their stem cell characteristics in vitro and enhanced bone regeneration in vivo. Therefore, NM-spheroids should have great clinical potential for bone and tissue regenerative therapies as a stem cell-based biomaterial therapy.

Application of Shear Stress for Enhanced Osteogenic Differentiation of Mouse Induced Pluripotent Stem Cells

“…Stem cell multipotency has been reported to be restored by mechanical stimuli in 3D cultures. A previous study demonstrated that shaking culture restored the multipotency of mouse 22 and human MSC spheroids after long-term expansion ex vivo 23 . In an iPSC study, mechanical stimulation using equiaxial stretching enhanced reprogramming e ciency, but did not enhance the transduction rate 24 .…”

Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells

“…Stem cell multipotency has been reported to be restored by mechanical stimuli in 3D cultures. A previous study demonstrated that shaking culture restored the multipotency of mouse 19 and human MSC spheroids after long-term expansion ex vivo 20 . In an iPSC study, mechanical stimulation using equiaxial stretching enhanced reprogramming efficiency, but did not enhance the transduction rate 21 .…”

Assessment of the Impacts of Centipeda minima (L.) on Cell Viability, and Osteogenic Differentiation of Mesenchymal Stem Cell Spheroids

“…In contrast to two-dimensional dedifferentiated fat cells, three-dimensional dedifferentiated fat spheroid promoted osteogenic differentiation and bone formation via canonical Smad 1/5 signaling pathways, according to research comparing the in vitro osteogenic potential of rat dedifferentiated fat cells cultured under osteogenic conditions in three-dimensional spheroids with that in two-dimensional monolayers [ 24 ]. The bone regeneration process was accelerated in vivo by neurosphere medium spheroids created under modified neurosphere culture conditions with constant shaking [ 25 ]. In contrast, constructions using two-dimensional and three-dimensional bone marrow mesenchymal stem cells behaved comparably in vivo despite a tendency for improved in vitro calcification [ 23 ].…”