Supplementary Materials7082679. lack of bone tissue pursuing trauma, tumor resection, or long-term teeth reduction in dentistry exceeds the organic healing capability, and bone tissue regeneration in huge defects is a substantial problem in the center [1]. Although bone tissue regeneration using autologous bone tissue grafts may be the yellow metal standard, harvesting from the graft needs an invasive MC-Val-Cit-PAB-rifabutin medical procedure and is connected with donor site morbidity [2]. Stem cell-based bone tissue tissue engineering can be an alternative method of eliminate the disadvantages of current medically utilized treatments, especially donor site morbidity and limited availability [3]. In addition, this approach has osteoinductive properties that MC-Val-Cit-PAB-rifabutin are crucial for efficient bone repair in clinical applications [4]. Stem cell-based therapy methods in bone regeneration have been under development for years and generally involve growing mesenchymal stem cells (MSCs) on biomaterial scaffolds enriched with growth factors. However, such approaches have MC-Val-Cit-PAB-rifabutin not been able to achieve complete bone healing in large defects due to fibrous tissue encapsulation, degradation of designed tissue, immune responses to the scaffold material [3, 5], and migration and death of the transplanted MSCs [6]. Therefore, there is an unmet need for effective protocols for efficient bone tissue engineering to achieve sufficient regeneration. Induced pluripotent stem cells (iPSCs) derived from reprogramming of somatic cells [7] have self-organizing potential that contributes to three-dimensional MC-Val-Cit-PAB-rifabutin (3D) tissue or organ construction without requiring the use of a scaffold; thus, iPSCs could be a encouraging source to generate tissue-engineered bone. Previous studies exhibited osteogenic differentiation of iPSCs in 2D adherent culture [8C10]. We previously fabricated iPSC-derived 3D-osteogenic constructs with high expression of osteocalcin, a crucial extracellular matrix (ECM) molecule for bone formation [11]. However, the inner region of the fabricated constructs showed central necrosis and thus might not be suitable for MC-Val-Cit-PAB-rifabutin clinical application. In addition, the osteogenic constructs showed teratoma formation, suggesting incomplete osteogenic differentiation. The microenvironment of stem cells, including aspects of the stem cell niche such as growth factors, cell-cell contact, and cell-matrix interactions, has been reported to govern stem cell fate and behavior. The translation of stem cell-based therapies to treat degenerated tissue relies on stem cell lineage commitment in the region of interest, in which the microenvironment precisely controls the commitment and success [12]. This microenvironment concept has been applied to promote stem cell commitment toward osteogenic lineages in coculture [13, 14] and 3D cell-scaffold culture systems [15, 16]. The formation of iPSC aggregates, i.e., the so-called embryoid body (EBs), prior to differentiation provides microenvironments for stem cells and influences multiple pathways that may control the differentiation trajectory [17, 18]. Several methods have been developed to form and culture iPSC aggregates. Among them, microspace culture, in which iPSCs in different microspaces accumulate and then form aggregates, could be a candidate for tissue engineering [19] to provide a large number of homogenous iPSC aggregates in a less time-consuming manner [20]. Recently, Takebe et al. attained reproducible and SQSTM1 substantial production of 3D liver bud organoids from iPSCs using microspace culture plates [21]. Therefore, microspace lifestyle may represent a appealing microplatform to facilitate self-organizing differentiation of iPSCs by giving a proper microenvironment for bone tissue tissue engineering. Furthermore, how big is the microspace continues to be reported to have an effect on the differentiation potential of pluripotent stem cells [22, 23]. Nevertheless, ramifications of microspace size on osteogenic differentiation of 3D-iPSC constructs never have yet been looked into. In this scholarly study, we utilized microspace well plates (Elplasia; Kuraray) to fabricate and lifestyle iPSC aggregates during osteogenic differentiation. We hypothesized a particular microspace size could facilitate self-organizing differentiation of iPSCs to create.