Effect of extracellular matrix proteins on the differentiation of human pluripotent stem cells into mesenchymal stem cells.

The transplantation of human mesenchymal stem cells (hMSCs), such as bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs), has shown beneficial effects in protecting transplanted tissues and cells against graft-versus-host disease (GVHD). Human pluripotent stem cell (hPSC)-derived mesenchymal stem cells (MSCs) can also be used to generate hMSCs with stable characteristics without limitations. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs, H-M5) and human embryonic stem cells (hESCs, H9) into hMSCs on dishes coated with different extracellular matrix (ECM) proteins to study the effect of cell culture biomaterials on hPSC differentiation into hMSCs. hPSC-derived MSCs cultured on Matrigel (MAT)-coated, collagen (COL)-coated and laminin-521 (LN-521)-coated tissue culture polystyrene (TCP) dishes showed excellent proliferation speed and reduced aging over 10 passages. High MSC surface marker (CD44, CD73, CD90 and CD105) expression was also observed on hPSC-derived MSCs cultured on MAT-coated, COL-coated and LN-521-coated TCP dishes as well as uncoated TCP dishes. Analysis of late osteogenic differentiation by evaluation of mineral deposition revealed that hPSC-derived MSCs cultured on fibronectin (FN)-coated and LN-521-coated TCP dishes showed high osteogenic differentiation. ECM proteins are effective as coating materials on cell culture biomaterials to regulate the proliferation and differentiation fate of hPSC-derived MSCs.

Quality control processes in allografting: A twenty-year retrospective review of a hospital-based bone bank in Taiwan.

Musculoskeletal allografts are now commonly used. To decrease the potential risks of transmission of pathogenic bacteria, fungi, or viruses to the transplant recipients, certain issues regarding the management of patients who receive contaminated allografts need to be addressed. We aimed to clarify the incidence and extent of disease transmission from allografts by analyzing the allografting procedures performed in the bone bank of our hospital over the past 20 years. We retrospectively reviewed the data from our allograft registry center on 3979 allografts that were implanted in 3193 recipients throughout a period of two decades, from July 1991 to June 2011. The source of the allografts, results of all screening tests, dates of harvesting and implantation, and recipients of all allografts were checked. With the help of the Center for Infection Control of our hospital, a strict prospective, hospital-wide, on-site surveillance was conducted, and every patient with healthcare-associated infection was identified. Fisher's exact test was used to compare the infection rate between recipients with sterile allografts and those with contaminated allografts. The overall discard and infection rates were, respectively, 23% and 1.3% in the first decade (1991-2001); and 18.4% and 1.25% in the second decade (2001-2011). The infection rate of contaminated allograft recipients was significantly higher than that of sterile allograft recipients (10% vs. 1.15%, P < 0.01) in the second decade. Both infection and discard rates of our bone bank are comparable with those of international bone banks. Strict allograft processing and adequate prophylactic use of antibiotics are critical to prevent infection and disease transmission in such cases.

Nimbolide Induces ROS-Regulated Apoptosis and Inhibits Cell Migration in Osteosarcoma.

Osteosarcoma (OS) is a primary malignant tumor of bone and is most prevalent in children and adolescents. OS is frequently associated with pulmonary metastasis, which is the main cause of OS-related mortality. OS has a poor prognosis and is often unresponsive to conventional chemotherapy. In this study, we determined that Nimbolide, a novel anti-cancer therapy, acts by modulating multiple mechanisms in osteosarcoma cells. Nimbolide induces apoptosis by increasing endoplasmic reticulum (ER) stress, mitochondrial dysfunction, accumulation of reactive oxygen species (ROS), and finally, caspase activation. We also determined that Nimbolide inhibits cell migration, which is crucial for metastasis, by reducing the expression of integrin αvß5. In addition, our results demonstrate that integrin αvß5 expression is modulated by the PI3K/Akt and NF-κB signaling cascade. Nimbolide has potential as an anti-tumor drug given its multifunctional effects in OS. Collectively, these results help us to understand the mechanisms of action of Nimbolide and will aid in the development of effective therapies for OS.

Cyr61 promotes epithelial-mesenchymal transition and tumor metastasis of osteosarcoma by Raf-1/MEK/ERK/Elk-1/TWIST-1 signaling pathway.

BACKGROUND: Osteosarcoma is the most common primary malignant tumor in children and young adults, and its treatment requires effective therapeutic approaches because of a high mortality rate for lung metastasis. Epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior during cancer progression. The cysteine-rich angiogenic inducer 61 (Cyr61) gene, a member of the CCN gene family, is responsible for the secretion of Cyr61, a matrix-associated protein that is involved in several cellular functions. A previous study showed that Cyr61 expression is related to osteosarcoma progression. In addition, Cyr61 could promote cell migration and metastasis in osteosarcoma. However, discussions on the molecular mechanism involved in Cyr61-regulated metastasis in osteosarcoma is poorly discussed. RESULTS: We determined that the expression level of Cyr61 induced cell migration ability in osteosarcoma cells. The Cyr61 protein promoted the mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers (TWIST-1 and N-cadherin) and inhibiting the epithelial marker (E-cadherin). Moreover, the Cyr61-induced cell migration was mediated by EMT. The Cyr61 protein elicited a signaling cascade that included αvß5 integrin, Raf-1, mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and Elk-1. The reagent or gene knockdown of these signaling proteins could inhibit Cyr61-promoted EMT in osteosarcoma. Finally, the knockdown of Cyr61 expression obviously inhibited cell migration and repressed mesenchymal phenotypes, reducing lung metastasis. CONCLUSION: Our results indicate that Cyr61 promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves αvß5 integrin, Raf-1, MEK, ERK, and Elk-1.

Hyperthermia induces apoptosis through endoplasmic reticulum and reactive oxygen species in human osteosarcoma cells.

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.

Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway.

Osteosarcoma is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Transforming growth factor alpha (TGF-α) is classified as the EGF (epidermal growth factor)-like family, which is involved in cancer cellular activities such as proliferation, motility, migration, adhesion and invasion abilities. However, the effect of TGF-α on human osteosarcoma is largely unknown. We found that TGF-α increased the cell migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced TGF-α-mediated cell migration. We also found that the phosphatidylinositol 3'-kinase (PI3K)/Akt/NF-κB pathway was activated after TGF-α treatment, and TGF-α-induced expression of ICAM-1 and cell migration was inhibited by the specific inhibitors and siRNAs of PI3K, Akt, and NF-κB cascades. In addition, knockdown of TGF-α expression markedly decreased cell metastasis in vitro and in vivo. Our results indicate that TGF-α/EGFR interaction elicits PI3K and Akt activation, which in turn activates NF-κB, resulting in the expression of ICAM-1 and contributing the migration of human osteosarcoma cells.