A Novel Method for Human Adipose-Derived Stem Cell Isolation and Cryopreservation.

Adipose-derived stem cells (ADSCs) are isolated from abundant adipose tissue and have the capacity to differentiate into multiple cell lineages. ADSCs have raised big interest in therapeutic applications in regenerative medicine and demonstrated to fulfill the criteria for a successful cell therapy. There are several methods for isolation of ADSCs from adipose tissue and cryopreservation of ADSCs. Here, novel methods for the isolation and cryopreservation of ADSCs are presented and focused. Microscopic pieces of adipose tissue were placed on transwell inserts, and the ADSCs were induced to migrate to the lower wells for 1 week. We compared the properties of our ADSCs with those isolated by enzymatic digestion and enzyme-free method of culture plate, and our ADSCs were found to be more stable and healthier. In addition, we proposed a novel cryoprotectant solution (FNCP) containing pectin and L-alanine, which was compared with standard cryoprotectant solution. Overall, our methods proved more useful for ADSCs isolation than other methods and did not require consideration of "minimal manipulation" by the U.S. Food and Drug Administration (FDA). Furthermore, our FNCP did not contain dimethyl sulfoxide and fetal bovine serum, therefore stable storage is possible in xeno-free and animal-free cryopreservation solutions.

In vitro combinatorial anti-proliferative and immunosuppressive effects of Brucea javanica extract with CX-4945 and imatinib in human T-cell acute lymphoblastic leukemia cells.

Since 1970, the isolated and identified components of Brucea javanica (L.) Merr. have been known to contain anticancer effects, particularly antileukemic effect. In this study, the inhibitory effect of Brucea javanica (BJ) on cell growth and inflammation was confirmed in human T-cell acute lymphocytic leukemia (T-ALL) cells, and its efficacy as an antileukemic agent was verified. Our results showed that BJ extract induced caspase-dependent apoptosis of T-ALL Jurkat cells through inhibition of the CK2-mediated signaling pathway, while exerting no significant cytotoxicity in normal peripheral blood mononuclear cells. Moreover, BJ extract suppressed the NF-κB signaling pathway, thus, inhibiting the interleukin (IL)-2 expression induced by phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA). Notably, combined treatment with BJ extract plus CX-4945 or imatinib exerted enhanced inhibitory effects on T-ALL cell growth and IL-2 production. Overall, these results suggest that BJ extract can be a potent therapeutic herbal agent for T-ALL treatment and prevention of IL-2 mediated inflammatory immune responses.

Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells.

Vitamin C-linker-conjugated Ala-His-Lys tripeptide (Vit C-AHK) is a derivative of Vitamin C-conjugated tripeptides, which were originally developed as a component of a product for collagen synthesis enhancement or human dermal fibroblast growth. Here, we investigated the effect of Vit C-AHK on bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Vit C-AHK enhanced proliferation of C2C12 cells and induction of BMP-2-induced alkaline phosphatase, a typical marker of osteoblast differentiation. Vit C-AHK also stimulated the phosphorylation and translocation of Smad1/5/8 to the nucleus and phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38. In addition, Vit C-AHK enhanced the BMP-2-induced mRNA expression of osteoblast differentiation-related genes such as ALP, BMP-2, Osteocalcin, and Runx2. Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smad1/5/8 and MAPK ERK1/2 and p38 signaling and without significant cytotoxicity. These results provide important data for the development of peptide-based bone-regenerative agents and treatment of bone-related disorders.

Synergistic therapeutic effect of diethylstilbestrol and CX-4945 in human acute T-lymphocytic leukemia cells.

Human acute T-lymphocytic leukemia (T-ALL) is one of the most commonly diagnosed hematological disorders, and is characterized by poor prognosis and survival rate. Despite the development of new therapeutic approaches, leukemia treatment options remain limited. In this study, we investigated the immunosuppressive and anti-proliferative effects of the synthetic estrogen diethylstilbestrol (DES), both alone and combined with the casein kinase 2 (CK2) inhibitor CX-4945. Our results indicated that DES induced caspase-dependent apoptosis in a human T-ALL cell line (Jurkat cells), while exerting no significant cytotoxicity in normal peripheral blood mononuclear cells (PBMCs). Phytohaemagglutinin and phorbol 12-myristate 13-acetate induced interleukin (IL)-2 production and activation of NF-κB signaling pathways, which were both inhibited by DES. Moreover, DES exerted synergistic effects with CX-4945 on proliferation and IL-2 production in Jurkat cells. Our results demonstrated that DES exerts anti-proliferative and immunosuppressive effects through inhibition of CK2 and the NF-κB signaling pathway in human T-ALL Jurkat cells.

Synergistic inhibition effect of TNIK inhibitor KY-05009 and receptor tyrosine kinase inhibitor dovitinib on IL-6-induced proliferation and Wnt signaling pathway in human multiple myeloma cells.

Multiple myeloma is a fetal form of plasma cell malignancy characterized by abnormal clonal proliferation of plasma cells. Especially, the canonical Wnt signaling pathway mediated by ß-catenin is activated in multiple myeloma cells, stimulating their proliferation. Here, we investigated the relationship between interleukin-6-induced proliferation of multiple myeloma cells and Traf2- and Nck-interacting kinase (TNIK) expression in Wnt signaling. Interleukin-6 increased the proliferation of multiple myeloma cells and TNIK mRNA and protein expression. In addition, we examined the effect on TNIK of TNIK inhibitor KY-05009 and receptor tyrosine kinase inhibitor dovitinib and whether inhibition of TNIK suppresses the interleukin-6-induced proliferation of multiple myeloma cells. KY-05009 and dovitinib synergistically inhibited interleukin-6-stimulated proliferation and induced apoptosis through the inhibition of Wnt signaling in MM cells. Our results provide crucial information that TNIK is involved in the interleukin-6-dependent proliferation of multiple myeloma cells and inhibition of Wnt signaling involving TNIK could be a therapeutic strategy for the treatment of interleukin-6-dependent multiple myeloma.