CH02 Peptide Promotes CD34+ Umbilical Cord Blood Hematopoietic Stem Cells Ex Vivo Expansion

Background: Umbilical cord blood (UCB) has been clinically used for human hematopoietic stem cells (HSCs) transplantation. However, limited numbers of the functional UCB-HSCs from single cord blood restricts its application in adults, while most of the strategies for stem cells expansion in vitro are either inefficient or costly. To overcome these obstacles, we evaluated the potential role of our newly identified CH02 peptide in ex vivo culture expansion of CD34+ UCB-HSCs. Methods: Enriched human CD34+ progenitor/stem cells populations were cultured in serum-free medium supplemented with different cytokines combinations for 8 days. These cytokines combinations included various concentration of CH02 peptide or the FLT3 ligand, with a cocktail of several growth factors such as IL-6, SCF and TPO. In addition, the global gene expression profile of the CD34+ cells cultured under different conditions were monitored through RNA-seq experiments. Furthermore, the expanded CD34+ cells were topically transplanted into the dorsal wounds of diabetic mice, and the wound closure was observed to evaluate the pro-repair ability of CH02-cultured CD34+ cells.Results: We herein report that the combination of CH02 peptide and other cytokines under the serum-free medium can effectively expand the CD34+ HSCs into 12-fold within 7 days while maintaining their stem cell properties. Moreover, CH02 peptide increased the anti-inflammatory and growth-promoting capacity of CD34+ cells, and thus accelerating wound healing of diabetic mice via promoting the anti-inflammatory and inhibiting the inflammatory factors.Conclusions: Together, our CH02 peptide demonstrated promising potentials to improve HSCs expansion for clinical application.

Fatty Acid Modification of the Anticancer Peptide LVTX-9 to Enhance Its Cytotoxicity against Malignant Melanoma Cells

Spider venom is a valuable resource for the development of novel anticancer drugs. In this study, we focused on novel linear amphipathic α-helical anticancer peptide LVTX-9, which was derived from the cDNA library of the venom gland of the spider Lycosa vittata. The cytotoxicity of LVTX-9 against murine melanoma cells in the range of 1.56–200 μM was tested and found to be significantly lower than those of most anticancer peptides reported. Its IC50 was determined to be 59.2 ± 19.8 μM in a serum or 76.3 ± 12.7 μM in serum-free medium. Fatty acid modification is a promising strategy for improving peptide performance. Therefore, to enhance the cytotoxic activity of LVTX-9, fatty acid modification of this peptide was performed, and five different carbon chain length lipopeptides named LVTX-9-C12-C20 were produced. Among them, the lipopeptide LVTX-9-C18 showed the highest cytotoxic activity in relation to B16-F10 cells, whether in a serum or serum-free medium. Most importantly, the cytotoxic activity of LVTX-9-C18 was improved by about 12.9 times in a serum medium or 19.3 times in a serum-free medium compared to that of LVTX-9. Subsequently, assays including scanning electron microscopy, trypan blue staining, lactate dehydrogenase leakage assay, and hemolytic activity could indicate that the potential direct cell membrane disruption is the main mechanism of LVTX-9-C18 to induce cancer cell death. Furthermore, the LVTX-9-C18 also showed strong cytotoxicity in relation to 3D B16-F10 spheroids, which indicates it might be a promising lead for developing anticancer drugs.

Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

Serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of mesenchymal stem cells on experimental renal fibrosis

Background Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI). Methods SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Dual Effect Exhibited by Insulin in Myeloma and Lymphoblastoid Cells

Many reports have documented the role of INS (insulin) as growth factors in a variety of cancers. Epidemiological studies revealed that INS therapy causes increased mortality in multiple myeloma (MM) patients with pre-existing or steroid-induced type 2 diabetes. However, there is limited experimental evidence of this association. In the present study, the dual effect of INS on the viability of myeloma RPMI8226 and lymphoblastoid IM9 cells was revealed. In serum-containing medium exogenous INS serves as a growth factor, whereas INS decreases the number of cells under serum-free medium. In the last case, the main mechanism of decreasing the cell population is apoptosis through up-regulation of Cas-3 and downregulation of Bcl-2 expression. INS has also been shown to be involved in the regulation of necrotic cell death.