Differentiation Of Schwann-like Cells Derived From Human Neural Stem Cells And Effect Of Microvesicles Of Schwann-like Cells On Axon Regeneration

Background In peripheral nervous system(PNS)injury,axonal regeneration is the basic process of nerve function recovery.Neural stem cells(NSCs)are capable of differentiating into various neural cells in vitro,such as neurons and oligodendrocytes.Currently,these abilities have been widely used to generate a variety of nerve cells for clinical application and treat neurological diseases as potential means.In peripheral nerve regeneration,Schwann cells(SCs)are closely related to the regeneration of nerves.They are distributed along the neurites and wrapped around nerve fibers to form myelin.SCs can promote the survival of damaged neurons and the regeneration of axons by secreting neurotrophic factors,proteins and other active substances,and participate in the formation of nerve fibers in PNS.After peripheral nerve injury,SCs are activated to dedifferentiate and participate in the removal of myelin sheath fragments,providing a pathway for axon regeneration.Transplanted SCs have been shown to promote nerve regeneration in animal studies.However,the clinical therapeutic effect of SCs is often limited by its source.Previous research of our research group has obtained embryonic stem cell derived neural stem cells(h ESC-NSCs).Theoretically,schwann-like cells differentiated from human embryonic stem cell derived-neural stem cell(hereinafter referred to as SC-like cells)can continuously induce differentiation from h ESC-NSCs.Meanwhile,we hypothesized that SC-like cells could promote axonal growth and nerve regeneration by secreting various active substances just like traditional SCs.Microvesicles(MVs)are tiny membranous organelle structures derived from parental cells that transfer cytoplasmic contents,including proteins,lipids and RNA between cells and can be isolated from conditioned cell culture media.This kind of intercellular MVs transmission is a new intercellular communication mechanism in recent years.Based on our previous studies,h ESC-NSCs released MVs(h ESC-NSC-MVs)to promote functional recovery after sciatic nerve injury in rats.Therefore,microvesicles of schwan-like cells derived from human neural stem cells(hereinafter referred to as SC-like cell MVs)may better promote axonal regeneration and repair of neurons than h ESC-NSC-MVs,and preliminarily explored the possible mechanism of SC-like cell MVs in promoting axonal growth.Objectives We aimed to investigate the induced SC-like cells,the repaired effect of SC-like cell MVs on 5mm sciatic nerve transection in rats,and the mechanism of promoting the growth of neuronal axons.Methods(1)h ESC-NSCs were induced and differentiated into SC-like cells,which were identified by means of cell morphology,immunofluorescence staining and RT-q PCR.(2)SC-like cell MVs were extracted from the collected SC-like cells conditioned medium by ultracentrifugation,and then the characterization of SC-like cell MVs was detected.(3)Establishment of a model of transverse sciatic nerve injury in SD rats,and the feasibility of using high-resolution X-ray imaging system to detect the injury model.Sciatic nerve functional index(SFI),HE staining and immunostaining were used to evaluate the repair effect of SC-like cell MVs on sciatic nerve injury.(4)Primary culture of dorsal root ganglion was used to establish the model of neurons in vitro.(5)SC-like cell MVs were co-cultured with dorsal root ganglion to observe the growth of axon.(6)SC-like cell MVs were co-cultured with neuron-like N2 a cell lines to detect differentiation,number of neurites and length,and RT-q PCR was used to detect gene expression related to axon of N2 a cells and glycogen staining of N2 a cells.Western Blot was used to detect the protein expression of SC-like cell MVs-stimulated N2 a and PC12 cell lines.Results(1)SC-like cells induced by h ESC-NSCs have similar morphology to traditional schwann cells,and highly express S100β,GFAP,HNK-1 and other genes related to SCs.(2)SC-like cell MVs significantly promoted the growth of neuronal axons in dorsal root ganglion.(3)The model of transverse sciatic nerve injury in rats was successfully constructed and feasible.Six weeks after sciatic nerve injury repair,the morphological and functional recovery and axon regeneration length of the injured hind limbs in the SC-like cell MVs group were excelled than those in the PBS group.(4)SC-like cell MVs can be engulfed by N2 a cells and promote the expression of GAP43 m RNA,neurite growth and glycogen generation.SC-like cell MVs promote the axon growth of N2 a and PC12 cells through the PTEN/PI3K/Akt cascade signaling pathway with a certain concentration dependence.Conclusions(1)SC-like cells can be differentiated by h ESC-NSCs and express Glial SC markers.(2)SC-like cell MVs promote axonal growth and glycogen generation,which can promote the expression of GAP43 protein in neurons through the PTEN/PI3K/Akt cascade signaling pathway,and the expression of GAP43 protein is dependent on the concentration of SC-like cell MVs.(3)SC-like cell MVs have a repairing effect on sciatic nerve transection injury.