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Transplantation of neural progenitor cells (NPCs) derived from human-induced pluripotent stem cells (hiPSCs) is one of the promising treatment strategies for motor neuron diseases (MNDs). However, the inefficiency in committed differentiation of NPCs in vivo limits its application. Here, we tried to establish a potential therapeutic strategy for MNDs by in vivo directional differentiation of hiPSCs engineered with motor neuron (MN) specific transcription factors and Tet-On system.We engineered hiPSCs with three MN-specific transcription factors and Tet-On system. The engineered cells were directly transplanted into immunodeficient mice through subcutaneous, intra-spinal cord and intracerebroventricular injections. Following doxycycline (Dox) induction, teratoma formation, and motor MN differentiation were evaluated.We generated genetically engineered hiPSCs, in which the expression of Ngn2, Isl1, and Lhx3 was controlled by a drug-inducible transgenic system. These cells showed normal pluripotency and proliferative capacity, and were able to directionally differentiate into mature motor neurons (MNs) and NPCs with high efficiency in spinal cords and cerebral lateral ventricles under the induction of Dox. The grafts showed long-term survival in the recipient mice without formation of teratoma.The induced mature MNs and NPCs were expected to replace the damaged endogenous MNs directly, and play a role of de novo stem cell stock for long-term neuron damage repair, respectively. Therefore, in vivo directional differentiation of the hiPSCs engineered with MN-specific transcription factors and Tet-On system via Dox induction could be a potential therapeutic strategy for MNDs with high efficacy and safety.

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene

The application of saponins has been restricted by problems such as hemolysis, low bioavailability, and poor solubility. So it is imperative to find a strategy to deliver saponins safely and efficiently. Here, through bottom-up technique, we design and prepare two saponin-cholesterol (Cho) nano-complex: dioscin (Dio, steroid saponin)-Cho nanofibers (NFs) and escin Ia (EIa, triterpene saponin)-Cho nanoparticles (NPs). It is found that the hydrophobic force and hydrogen bonding drive the two pairs of molecules to bind in different directions (the 3β-OH of Cho face the sugar chain of EIa and the 22α-O of Dio, respectively) and finally show spherical NPs (EIa-Cho) and fibrous NFs (Dio-Cho). The equimolar saponin-Cho complex, Dio NFs and EIa NPs, reveal potent cytotoxicities against mouse breast cancer cells (4T1) in vitro. In vivo results confirm the antitumor (4T1 mice model) efficacy of PEGylation Dio NFs (10 mg/kg, i.v.) with a tumor inhibition rate of 61%, meanwhile, it does not cause extreme irritation and pain as free Dio does to mice. Moreover, compared with the free drug, the prepared nano-complex can significantly reduce hemolysis and organ toxicity. Our research reduces the toxicity of saponins while retaining their antitumor activity, providing a new strategy for the delivery of saponins.

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency—naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten−/− mouse ESCs with the CRISPR-Cas9 system and verified that Pten−/− ESCs maintained naïve pluripotency by blocking Gsk3β activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3β modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.

The potential in vivo application of liposome for polycationic colistin has been hindered by the poor entrapment efficiency (EE) due to their phospholipid membrane permeability. The objective of this study is to investigate the loading mechanism and validity of applying electrostatic attraction for the colistin entrapment and delivery in liposomes. Anionic lipids with various structures were used for colistin entrapment, and the properties of resulting liposomes (i.e. zeta-potential, EE and release rate) were highly dependent on the structure of anionic lipids. Based on consideration of intermolecular interactions, the retention of electrostatically entrapped colistin is essentially determined by the balance of interfacial hydrophobic attraction and electrostatic repulsion. The liposomal colistin showed the reduced bacterial killing rate, but did not compromise the in vitro antibacterial activity. Specially, the PEGylated liposomal colistin of sodium cholesteryl sulfate (Chol-SO4-) showed the best drug retention, resulting in the significantly increased maximum-tolerated dose, prolonged blood circulation and decreased colistin distribution in kidney after intravenous administration in mice. These results highlight the potential utility of electrostatically entrapped liposome for polycationic colistin delivery.

The potential use of liposomes for the pulmonary delivery of colistin has been hindered by their phospholipid membrane permeability resulting in a very low entrapment of colistin in the liposomes. To increase the entrapment capacity of colistin in liposomes, the anionic lipid sodium cholesteryl sulfate (Chol-SO4

Subcutaneous liraglutide-loaded multivesicular liposomes (Lrg-MVLs) were developed as a sustained drug-delivery system for treating diabetes and their properties were characterized. The Lrg-MVLs prepared using a two-step water-in-oil-in-water double emulsification process had a spherical appearance with a mean diameter of 6.69 μm and an encapsulation efficiency of 82.23 ± 4.78% without any initial burst release. Their pharmacodynamics (PD) and pharmacokinetics (PK) were also studied after a single subcutaneous administration to Sprague-Dawley (SD) rats with diabetes. The PD results demonstrated that Lrg-MVLs presented sustained glucose-lowering effects for nearly a week, while the pharmacokinetic parameters showed that the plasma liraglutide concentration of the designed preparation produced Cmax of 81.979 ± 12.140 pg/ml and an MRT0-t of 88.224 ± 3.893 h. Furthermore, retention of Lrg-MVLs at the injection site was studied semiquantitatively by an in vivo imaging system, which can be used to evaluate the drug release from MVLs in vivo. In conclusion, MVLs are a promising carrier for liraglutide and Lrg-MVLs deserve further study for the treatment of diabetes.

Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1−/− ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.

Potential use of liposome for polycationic colistin is hindered by their phospholipid membrane permeability. In this study, liposomes were modified with sodium cholesteryl sulphate (Chol-SO4(-)) for improving the colistin loading by enhancing the colistin-bilayer electrostatic attraction. We have evaluated two liposomes: colistin-entrapped liposome of Chol-SO4(-) (CCL) and coated Chol-SO4(-)/colistin complex liposome (CCCL). In comparison with CCL which formed large aggregates at Chol-SO4(-)/colistin charge ratio below 2:1, CCCL showed a smaller size less dependent on the charge ratio, probably arising from more colistin entrapped on the inner leaflet of bilayer. Both liposomes exhibited significantly increased entrapment efficiency as compared with the liposome without Chol-SO4(-). But colistin released upon dilution, implying free transfer of colistin through bilayers. Pharmacokinetics results showed the approximately four-fold increase in the plasma AUC0-8 h for CCCL and CCL as compared with colistin solution, showing potential benefit for infectious target localisation by prolonging the systemic circulation of colistin.

The BRE gene, alias BRCC45, produces a 44 kDa protein that is normally distributed in both cytoplasm and nucleus. In this study, we used adult fibroblasts isolated from wild-type (WT) and BRE knockout (BRE−/−) mice to investigate the functional role of BRE in DNA repair and cellular senescence. We compared WT with BRE−/− fibroblasts at different cell passages and observed that the mutant fibroblasts entered replicative senescence earlier than the WT fibroblasts. With the use of gamma irradiation to induce DNA damage in fibroblasts, the percentage of SA-β-Gal+ cells was significantly higher in BRE−/− fibroblasts compared with WT cells, suggesting that BRE is also associated with DNA damage-induced premature senescence. We also demonstrated that the gamma irradiation induced γ-H2AX foci, a DNA damage marker, persisted significantly longer in BRE−/− fibroblasts than in WT fibroblasts, confirming that the DNA repair process is impaired in the absence of BRE. In addition, the BRCA1-A complex recruitment and homologous recombination (HR)-dependent DNA repair process upon DNA damage were impaired in BRE−/− fibroblasts. Taken together, our results demonstrate a role for BRE in both replicative senescence and DNA damage-induced premature senescence. This can be attributed to BRE being required for BRCA1-A complex-driven HR DNA repair.

The lack of suitable animal model for HIV-1 infection has become a bottleneck for the development of AIDS vaccines and drugs. Wild-type rabbits can be infected by HIV-1 persistently and HIV-1 can be efficiently replicated resulting in syncytia in rabbit cell line co-expressing human CD4 and CCR5.Therefore, a rabbit highly expressing human CD4 and CCR5 may be an ideal animal model for AIDS disease study. In the present report, by using the efficient gene targeting technology, transcription activator-like effector nuclease (TALEN), we explored the feasibility of generating a HIV-1 model by knocking in human CD4 and CCR5 into rabbit genome. First we constructed two TALEN vectors targeting rabbit CCR5 gene and a vector with homologous arms. TALEN mRNAs and donor DNA were then co-injected into fertilized oocytes. After 3?5 days, 24 embryos were collected and used to conduct mutation analysis with PCR and sequencing. All the 24 embryos were detected with CCR5 knockouts and 5 were human CD4 and CCR5 knockins. Our results laid a foundation for establishing a new animal model for the study of AIDS.

Rosa26 -targeted swine models for stable gene over-expression and Cre-mediated lineage tracing