Mesenchymal stromal/stem cells (MSCs) are promising candidates for regenerative medicine owing to their self-renewal properties, multilineage differentiation, immunomodulatory effects, and angiogenic potential. MSC spheroids fabricated by 3D culture have recently shown enhanced therapeutic potential. MSC spheroids create a specialized niche with tight cell-cell and cell-extracellular matrix interactions, optimizing their cellular function by mimicking the in vivo environment. Methods for 3D cultivation of MSCs can be classified into 2 main forms: static suspension culture and dynamic suspension culture. Numerous studies have reported the beneficial influence of these methods on MSCs, which is displayed by increased differentiation, angiogenic, immunomodulatory, and anti-apoptotic effects, and stemness of MSC spheroids. Particularly, recent studies highlighted the benefits of dynamic suspension cultures of the MSC spheroids in terms of faster and more compact spheroid formation and the long-term maintenance of stemness properties. However, only a few studies have compared the behavior of MSC spheroids formed using static and dynamic suspension cultures, considering the significant differences between their culture conditions. This review summarizes the differences between static and dynamic suspension culture methods and discusses the biological outcomes of MSC spheroids reported in the literature. In particular, we highlight the advantages of the dynamic suspension culture of MSC spheroids and contemplate its future applications for various diseases.

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.

Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) “shaking” to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85–95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable “undifferentiated MSC-pool” property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for cartilage regeneration as they can generate a wide spectrum of cell types. Here, we established a tetracycline (tet) controlled bone morphogenetic protein-4 ( BMP-4) expressing iPSC (iPSC- Tet/BMP-4) line in which transcriptional activation of BMP-4 was associated with enhanced chondrogenesis. Moreover, we developed an efficient and simple approach for directly guiding iPSC- Tet/BMP-4 differentiation into chondrocytes in scaffold-free cartilaginous pellets using a combination of transcriptional activation of BMP-4 and a 3D shaking suspension culture system. In chondrogenic induction medium, shaking culture alone significantly upregulated the chondrogenic markers Sox9, Col2a1, and Aggrecan in iPSCs- Tet/BMP-4 by day 21. Of note, transcriptional activation of BMP-4 by addition of tet (doxycycline) greatly enhanced the expression of these genes. The cartilaginous pellets derived from iPSCs- Tet/BMP-4 showed an oval morphology and white smooth appearance by day 21. After day 21, the cells presented a typical round morphology and the extracellular matrix was stained intensively with Safranin O, alcian blue, and type II collagen. In addition, the homogenous cartilaginous pellets derived from iPSCs- Tet/BMP-4 with 28 days of induction repaired joint osteochondral defects in immunosuppressed rats and integrated well with the adjacent host cartilage. The regenerated cartilage expressed the neomycin resistance gene, indicating that the newly formed cartilage was generated by the transplanted iPSCs- Tet/BMP-4. Thus, our culture system could be a useful tool for further investigation of the mechanism of BMP-4 in regulating iPSC differentiation toward the chondrogenic lineage, and should facilitate research in cartilage development, repair, and osteoarthritis.

The transcriptional regulation of induced pluripotent stem cells (iPSCs) holds promise for their directed differentiation into ameloblasts, which are usually lost after tooth eruption. Ameloblast differentiation is regulated by multiple signaling molecules, including bone morphogenetic proteins (BMPs). Epiprofin (Epfn), a transcription factor, is expressed in the dental epithelium, and epithelial Epfn overexpression results in ectopic ameloblast differentiation and enamel formation in mouse incisor, a striking phenotype resembling that of mice with deletion of follistatin (a BMP inhibitor). However, it remains unknown whether and how Epfn transcriptional activation promotes ameloblast induction from mouse iPSCs. Here, we generated doxycycline-inducible Epfn-expressing mouse iPSCs (Epfn-iPSCs). Ameloblasts, which are characterized by positive staining for keratin 14 and amelogenin and alizarin red S staining, were successfully derived from Epfn-iPSCs based on a stage-specific induction protocol, which involved the induction of the surface ectoderm, dental epithelial cells, and ameloblasts at stages 1, 2, and 3, respectively. Epfn activation by doxycycline at stages 2 and/or 3 decreased cell proliferation and promoted ameloblast differentiation, along with the upregulation of p-Smad1/5/8, a key regulator of the BMP-Smad signaling pathway. Gene analysis of the BMP-Smad signaling pathway-associated molecules revealed that Epfn activation decreased follistatin expression at stage 2, but increased BMP2/4/7 expression at stage 3. Perturbations in the ameloblast differentiation process were observed when the BMP-Smad signaling pathway was inhibited by a BMP receptor inhibitor (LDN-193189). Simultaneous LDN-193189 treatment and Epfn activation largely reversed the perturbations in ameloblast induction, with partial recovery of p-Smad1/5/8 expression, suggesting that Epfn activation promotes ameloblast induction from mouse iPSCs partially by upregulating BMP-Smad activity. These results reveal the potential regulatory networks between Epfn and the BMP-Smad pathway and suggest that Epfn is a promising target for inducing the differentiation of ameloblasts, which can be used in enamel and tooth regeneration.

Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx-expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor β1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.