Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair.

Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.

Selective vulnerability of human-induced pluripotent stem cells to dihydroorotate dehydrogenase inhibition during mesenchymal stem/stromal cell purification.

The use of induced mesenchymal stem/stromal cells (iMSCs) derived from human induced pluripotent stem cells (hiPSCs) in regenerative medicine involves the risk of teratoma formation due to hiPSCs contamination in iMSCs. Therefore, eradicating the remaining undifferentiated hiPSCs is crucial for the effectiveness of the strategy. The present study demonstrates the Brequinar (BRQ)-induced inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, selectively induces apoptosis, cell cycle arrest, and differentiation; furthermore, it promotes transcriptional changes and prevents the growth of 3-dimensional hiPSC aggregates. Contrastingly, BRQ-treated iMSCs showed no changes in survival, differentiation potential, or gene expression. The results suggest that BRQ is a potential agent for the effective purification of iMSCs from a mixed population of iMSCs and hiPSCs, which is a crucial step in successful iMSC-based therapy.

Hedgehog Activation Regulates Human Osteoblastogenesis.

Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling.

Stepwise strategy for generating osteoblasts from human pluripotent stem cells under fully defined xeno-free conditions with small-molecule inducers.

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

Three-dimensional system enabling the maintenance and directed differentiation of pluripotent stem cells under defined conditions.

The development of in vitro models for the maintenance and differentiation of pluripotent stem cells (PSCs) is an active area of stem cell research. The strategies used so far are based mainly on two-dimensional (2D) cultures, in which cellular phenotypes are regulated by soluble factors. We show that a 3D culture system with atelocollagen porous scaffolds can significantly improve the outcome of the current platforms intended for the maintenance and lineage specification of mouse PSCs (mPSCs). Unlike 2D conditions, the 3D conditions maintained the undifferentiated state of mouse embryonic stem cells (mESCs) without exogenous stimulation and also supported endoderm, mesoderm, and ectoderm differentiation of mESCs under serum-free conditions. Moreover, 3D mPSC-derived mesodermal cells showed accelerated osteogenic differentiation, giving rise to functional osteoblast-osteocyte populations within calcified structures. The present strategy offers a 3D platform suitable for the formation of organoids that mimic in vivo organs containing various cell types, and it may be adaptable to the generation of ectoderm-, mesoderm-, and endoderm-derived tissues when combined with appropriate differentiation treatments.

A novel photocrosslinkable and cytocompatible chitosan coating for Ti6Al4V surfaces.

BACKGROUND: In this work, chitosan (CH) was used to produce a novel coating for Ti6Al4V, the most widely used alloy in orthopedic implants, so as to improve the biological tissue response at the metallic surface. METHODS: The Ti6Al4V surface was sandblasted with alumina particles. CH was chemically modified, via carbodiimide chemistry, using lactobionic and 4-azidebenzoic acid to make it soluble at physiological pH and photocrosslinkable, respectively. The reaction was verified by FTIR, NMR and UV/vis spectroscopy. Ti6Al4V surfaces were coated with solutions of the modified CH and exposed to UV light, causing polymer crosslinking and formation of a hydrogel on the surface. The crosslinking reaction was monitored by FTIR at different exposure times. Coating morphology was observed by SEM. The coating's cytocompatibility was determined in vitro through the culture of rat bone marrow mesenchymal stem cells, using an MTT assay, with their morphology assessed by SEM. RESULTS: The developed coating behaved as a hydrogel on the Ti6Al4V and was stable on the surface. FTIR and NMR confirmed the crosslinking mechanism, based on an arile ring expansion, and subsequent reaction with the CH amine groups. Furthermore, the coating was able to support cell proliferation and osteogenic differentiation. CONCLUSIONS: UV crosslinking of CH is easy to apply and has potential for future metallic implant surface modifications. Due to its nature as a hydrogel, the coating could be used for further studies in the encapsulation of bioactive molecules to improve osteogenic potential at the tissue-implant interface.