L-ascorbic acid 2-phosphate and fibroblast growth factor-2 treatment maintains differentiation potential in bone marrow-derived mesenchymal stem cells through expression of hepatocyte growth factor.

l-ascorbic acid 2-phosphate (Asc-2P) acts as an antioxidant and a stimulator of hepatocyte growth factor (HGF) production. Previously, we reported that depletion of growth factors such as fibroblast growth factor (FGF)-2, epidermal growth factor (EGF), FGF-4 and HGF during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF). In this study, we investigated the proliferation and differentiation potential of BMSCs by FGF-2 and Asc-2P. Co-treatment with FGF-2 and Asc-2P induced optimal proliferation of BMSCs and increased the accumulation rate of BMSC numbers during a 2-month culture period. Moreover, differentiation potential was maintained by co-treatment with FGF-2 and Asc-2P via HGF expression. Adipogenic differentiation potential by FGF-2 and Asc-2P was dramatically suppressed by c-Met inhibitors (SU11274). These data suggest that co-treatment with FGF-2 and Asc-2P would be beneficial in obtaining BMSCs that possess "stemness" during long-term culture.

The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.

Systemic administration of human mesenchymal stromal cells infected with polymer-coated oncolytic adenovirus induces efficient pancreatic tumor homing and infiltration.

Oncolytic adenovirus (oAd)-mediated gene therapy is a promising approach for cancer treatment because of its cancer cell-restricted replication and therapeutic gene expression. However, systemic administration of oAd is severely restricted by their immunogenic nature and poor tumor homing ability, thus oAd cannot be utilized to treat disseminated metastases. In this study, human bone marrow-derived mesenchymal stromal cell (hMSCs) was used as a viral replication-permissive carrier for oAd with an aim to improve the systemic delivery of the virus to tumor tissues. To overcome the poor delivery of oAd into hMSCs, a relaxin (RLX)-expressing oncolytic Ad (oAd/RLX), which degrades dense tumor extracellular matrix of highly desmoplastic pancreatic cancer, was complexed with biodegradable polymer (poly (ethyleneimine)-conjugated poly(CBA-DAH); PCDP), generating oAd/RLX-PCDP complex. oAd/RLX-PCDP complex enhanced the internalization of oAd into hMSC, leading to superior viral production and release from hMSCs, along with high RLX expression. Furthermore, systemic administration of oAd/RLX-PCDP-treated hMSCs elicited more potent antitumor effect compared to naked oAd/RLX or oAd/RLX-treated hMSC in pancreatic tumor model. This potent antitumor effect of systemically administered oAd/RLX-PCDP-treated hMSCs was achieved by superior viral replication in tumor tissues than any other treatment group. In conclusion, these results demonstrate that hMSCs are effective carriers for the systemic delivery of oAd to tumor sites and treatment of pancreatic cancer.

Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis.

The therapeutic effects of stem cell transplantation in ischemic disease are mediated by the production of paracrine bioactive factors. However, the bioactive factors secreted by human mesenchymal stem cells (hMSCs) and their angiogenic activity are not clearly identified or determined. We here found that hMSC-derived conditioned media (hMSC-CdM) stimulated in vitro angiogenic activity of endothelial cells and contained significant levels of various growth factors and cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-ß1). The angiogenic activity of hMSC-CdM was significantly inhibited by pretreatment with neutralizing antibodies against VEGF, MCP-1, and IL-6, but not against TGF-ß1 and HGF. A mixture of those inhibitory antibodies blocked CdM-mediated activation of angiogenic signals, as well as inhibited CdM-mediated in vivo angiogenesis. Moreover, local injection of CdM increased angiogenesis and promoted blood flow in mice with hindlimb ischemia, and these effects were inhibited by co-treatment with these inhibitory antibodies. These results indicate that hMSC-CdM represents a promising cell-free therapeutic strategy for neovascularization in ischemic diseases. These results suggest the combination of VEGF, MCP-1, and IL-6 as a commercial application for therapeutic angiogenesis.

Adipose tissue-derived mesenchymal stem cells cultured at high density express IFN-ß and suppress the growth of MCF-7 human breast cancer cells.

Although it has been reported that mesenchymal stem cells (MSCs) suppress tumor growth in vitro and in vivo, little is known about the underlying molecular mechanisms. We found that type I interferon is expressed in adipose tissue-derived stem cells (ASCs) cultured at high density, and ASCs and their conditioned medium (ASC-CM) suppress the growth of MCF-7 cells in vitro. Growth inhibition was amplified by glucose deprivation that resulted from high density culture of ASCs after 3days. The cytotoxic effect of the ASC-CM obtained from high density culture of ASCs was neutralized by anti-IFN-ß antibody. STAT1 was phosphorylated in MCF-7 cells treated with ASC-CM, and JAK1/JAK2 inhibitor treatment decreased STAT1 phosphorylation. The cytotoxic effect of ASC-CM was reduced especially by JAK1 inhibitors in MCF-7 cells. Our findings suggest that ASCs cultured at high density express type I interferons, which suppresses tumor growth via STAT1 activation resulting from IFN-ß secretion in MCF-7 breast cancer cells.

Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells.

The present study employed a combinatorial strategy using poly(D,L-lactide-co-glycolide) (PLGA) scaffolds seeded with human mesenchymal stem cells (hMSCs) to promote cell survival, differentiation, and neurological function in a completely transected spinal cord injury (SCI) model. The SCI model was prepared by complete removal of a 2-mm length of spinal cord in the eighth-to-ninth spinal vertebra, a procedure that resulted in bilateral hindlimb paralysis. PLGA scaffolds 2 mm in length without hMSCs (control) or with different numbers of hMSCs (1 × 10(5), 2 × 10(4), and 4 × 10(3)) were fitted into the completely transected spinal cord. Rats implanted with hMSCs received Basso-Beattie-Bresnahan scores for hindlimb locomotion of about 5, compared with ~2 for animals in the control group. The amplitude of motor-evoked potentials (MEPs) averaged 200-300 µV in all hMSC-implanted SCR model rats. In contrast, the amplitude of MEPs in control group animals averaged 135 µV at 4 weeks and then declined to 100 µV at 8 weeks. These results demonstrate functional recovery in a completely transected SCI model under conditions that exclude self-recovery. hMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating in vivo survival of implanted hMSCs. Immunohistochemical staining revealed differentiation of implanted hMSCs into nerve cells, and immunostained images showed clear evidence for axonal regeneration only in hMSC-seeded PLGA scaffolds. Collectively, our results indicate that hMSC-seeded PLGA scaffolds induced nerve regeneration in a completely transected SCI model, a finding that should have significant implications for the feasibility of therapeutic and clinical hMSC-delivery using three-dimensional scaffolds, especially in the context of complete spinal cord transection.

Pilot study of mass screening for Wilson's disease in Korea.

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism with copper accumulation in the liver as well as in the central nervous system. Treatment of WD includes oral chelating agents and diet and it is effective. However, once irreversible damage has occurred, the effect of treatment is diminished and the patient's quality of life is compromised. It is estimated that at least half of the patients with WD remain undiagnosed and die of untreated disease. Early detection of patients presymptomatically has been hampered by the lack of effective methods for mass screening. Recently, a sandwich ELISA method for ceruloplasmin measurement in blood spots was developed. We have used this method to analyze blood specimens collected on filter paper from 3667 children aged 3 months-15 years. The mean value of ceruloplasmin was 30.5+/-9.5 mg/dL. Among these children, we identified one WD case, a 32-month-old boy with markedly reduced ceruloplasmin concentration (2.3 mg/dL). Measurement of CP level in dried blood spot sample is proposed as a reliable method for population screening of WD.