Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus.

BACKGROUND/AIMS: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. METHODS: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. RESULTS: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. CONCLUSION: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications.

A bi-functional anti-thrombosis protein containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities.

UNLABELLED: Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in 'Compendium of Materia Medica', the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. OBJECTIVES: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. METHODS AND RESULTS: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. CONCLUSIONS: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo.