Study On Neural Differentiation Of Human Embryonic Stem Cells In Combination With Silk Fibroin Biomaterials

Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and retain the developmental potency of embryonic founder cells, being able to differentiate into cells and tissues of all three germ layers in vitro and in vivo. They are valuable not only for the study of early human development but also for regenerative medicine. Silk fibroin in various formats (films, nets, membranes, sponges and so on) has been shown to support stem cell adhesion, proliferation and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using three-dimensional silk fibroin scaffolds has expanded the use of silk-based biomaterials as one of the most promising scaffolds.The feasibility of hESCs to combine with silk fibroin for neural differentiation was invaluable and unknown. In this study, we first explored the biocompatibility of silk fibroin materials with hESCs and the possible effect of silk fibroin on the differentiation of hESCs. The HUES-17 cells were cultured on silk fibroin and characterized by morphology, colony efficiency and population doubling time analysis, alkaline phosphatase and Oct-4 detection, and in vitro differentiation. The results showed that:(1)HUES-17 cells can form into typical colony with sharpness of border and high nucleo-cytoplasmic ratio(;2)the colony efficiency (0.2967±0.0272%)and population doubling time (47.25±1.40 hrs)were similar to the conventional culture system; (3)Alkaline phosphatase and Oct-4 detection were positive; (4)EBs derived from hESCs can differentiate into cells of all three germ layers. Together, these results demonste the good biocompatibility of silk fibroin materials with hESCs.Subsequently, we studied the neural differentiation of HUES-17 cells combination with silk fibroin biomaterials. It is critical to choose a suitable induced method for the success of this experiment. The method was as followes: hESC colonies were detached by collagenaseⅣand transferred onto bacterial culture dishes and incubated in EB medium for EB formation. Seven days after formation of the EBs, they were transferred onto gelatin-coated culture dishes. For the selection of neural progenitor cells (NPs), the attached EBs were cultured for 10-14 days in N2 medium. The foci cells were manually picked with a glass pipette under a phase-contrast microscope and cultured again in bacterial culture dishes to form floating neurospheres. After that, the neurospheres were dissociated into single cells and induced them into neurons on Tussah silk films in N2/B27 medium. The results showed that cells were diferentiated into neural cells and the the proportion ofβ-Ⅲ-tubulin positive cells was up to 70%. This study may provide important experimental information for tissue engineering, in which ES cells-derived neuron cells and silk fibroin materials are scaffolds, and also offer a source for cell therapy research of neurodegenerative disease.