## 3 Undifferentiated Cells (Stem Cells)
### Induced Pluripot Stem Cells (iPSCs)
#### 3.2.2 Applications in BTE  
Compared to other cell types, the use of iPSCs for BTE applications opens a new horizon. In order to differentiate iPSCs toward osteogenic lineages, the use of protocols applied for ESCs was suggested since both cell types show similar properties [86]. One of the ways proposed for preparing iPSCs with a commitment towards osteogenic lineage is genetic manipulations. On this matter, [PERSON] et al. transduced an exogenous Runx2 gene into mouse iPSCs via adenoviral vectors [87]. This gene transduction resulted in a higher production of ALP and calcium levels in the transduced cells as compared to controls. This study is considered as one of the first experiments regarding the production of iPSC-derived osteoblasts. As a novel approach, the use of some specific anti-miRNA was reported as an effective way of improving osteogenic differentiation. For instance, [PERSON] et al. showed that the transfection of anti-miRNAs of 124a, 181a, 10a, 10b, 9-3p, and 19b into mouse iPSCs significantly promote the osteoblastic differentiation [88].  
Culturing iPSCs in the osteogenic medium is another proposed approach for obtaining osteogenic lineages. In this regard, [PERSON] et al. cultured iPS cells in an osteogenic medium containing low-glucose DMEM supplemented with 15% FBS, 50 ug/mL ascorbate-2-phosphate, 10 nmol/L dexamethasone, and 10 mmol/L \(\beta\)-glycerophophate for two weeks [89]. They used resveratrol to promote differentiation of iPSCs into osteocyte-like cells, protect the iPSC-derived osteocyte-like cells from glucocorticoid-induced oxidative damage, and reduce the risk of tumorigenicity of iPSCs.  
Even though the production of iPSC-derived osteoblast-like cells is now a common process, the in vitro and in vivo stability of the generated cell's phenotype is even more significant. This is a very important issue when iPSCs are used in in-vivo experiments. In this regard, [PERSON] et al. reported the expression of osteoblast markers (e.g., RUNX2) in the implanted mouse iPSC-derived osteoblasts together with Gelfoam sponges at 12 weeks of post-implantation [90]. Their results proved the effectiveness of appropriate scaffolds in improving the osteogenic differentiation of iPSCs and bone formation in vivo.  
It has been documented that BM-MSCs have a limited differentiation potential due to their senescence in vitro. To address this problem, the use of iPSCs-derived MSCs (iMSCs) was suggested since this source provides an unlimited source of MSCs. For example, [PERSON] et al. differentiated human iPSCs toward functional MSCs for use in bone repair and regeneration [91]. In order to direct differentiation of iPSCs towards MSCs, the authors exposed embryoid bodies to TGF-\(\beta\) for a short period. They could prepare two types of iMSCs including early and late iMSCs. The in vitro results revealed that, even though both cell types maintained their multi-differentiation potential, the early iMSCs showed higher osteogenic activity as compared to other cells (the late iMSCs and BM-MSCs). Moreover, the in vivo results indicated that the ectopic administration of the early iMSCs overexpressing BMP6 resulted in a substantial bone formation in comparison to the other groups. The authors concluded that iMSCs could exhibit a self-renewal property without tumorigenic activity and considered them as very good candidates for bone regeneration.