Behaviour of human induced pluripotent stem cell-derived neural progenitors on collagen scaffolds varied in freezing temperature and laminin concentration

Biomaterial technology, when combined with emerging human induced pluripotent stem cell [hiPSC] technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors [hiPSC-NPs]. In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 Micro m for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. The results have suggested that the pore structure and laminin coating of collagen scaffolds significantly impact cell behavior. These biocompatible three-dimensional laminin-coated collagen scaffolds are good candidates for future hiPSC-NPs biomedical nerve tissue engineering applications

Cloning, expression, and functional characterization of in-house prepared human leukemia inhibitory factor

Leukemia inhibitory factor [LIF] plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF [hLIF] is an essential growth factor for the maintenance of mouse embryonic stem cells [ESCs] and induced pluripotent stem cells [iPSCs] in a pluripotent, undifferentiated state. In this experimental study, we cloned hLIF into the pENTR-D/TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami[TM] 2[DE3] pLacI competent cells to produce the His6-hLIF fusion protein. This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. Our results showed no significant differences in function between laboratory produced and commercialized hLIF