Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone in vivo has been variable. We hypothesized that given the multistage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time point of healing. Using a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome proliferator-activated receptor γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were coadministered with ECM, they remained at the injury, well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized bone defects in mice in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be used at optimal times to substantially and reproducibly improve bone repair.

It is widely accepted that canonical Wnt (cWnt) signaling is required for the differentiation of osteoprogenitors into osteoblasts. Furthermore, tumor-derived secretion of the cWnt-antagonist Dickkopf-1 (Dkk-1) is known to cause bone destruction, inhibition of repair and metastasis in many bone malignancies, but its role in osteosarcoma (OS) is still under debate. In this study, we examined the role of Dkk-1in OS by engineering its overexpression in the osteochondral sarcoma line MOS-J. Consistent with the known role of Dkk-1 in osteoblast differentiation, Dkk-1 inhibited osteogenesis by the MOSJ cells themselves and also in surrounding tissue when implanted in vivo. Surprisingly, Dkk-1 also had unexpected effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro and caused the formation of larger and more destructive tumors than controls upon orthotopic implantation. These effects were attributed in part to upregulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1). Direct inhibition of ALDH1 reduced viability under stressful culture conditions, whereas pharmacological inhibition of cWnt or overexpression of ALDH1 had a protective effect. Furthermore, we observed that ALDH1 was transcriptionally activated in a c-Jun-dependent manner through a pathway consisting of RhoA, MAP-kinase-kinase-4 and Jun N-terminal Kinase (JNK), indicating that noncanonical planar cell polarity-like Wnt signaling was the mechanism responsible. Together, our results therefore demonstrate that Dkk-1 enhances resistance of OS cells to stress by tipping the balance of Wnt signaling in favor of the non-canonical Jun-mediated Wnt pathways. In turn, this results in transcriptional activation of ALDH1 through Jun-responsive promoter elements. This is the first report linking Dkk-1 to tumor stress resistance, further supporting the targeting of Dkk-1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells.

Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25–40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary due to biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. We finally show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.