Transplantation Of Human Neural Precursor Cells For Mice Following Traumatic Brain Injury

BackgroundTraumatic brain injury(TBI)often occurs in traffic accidents,violent conflicts and natural disasters,leading to a high mortality and disability rate.Although the current medical technology has significantly reduced the mortality rate of traumatic brain injury,due to the complexity and heterogeneity of TBI,irreversible neuronal degeneration or apoptosis in the lesion area leads to long-term motor and cognitive deficits in patients.This will seriously affect the health and quality of life of the patients,and the need to strengthen the rehabilitation training for a long time will bring a heavy burden to the individual family and the national economy.Nerve regeneration is an urgent problem after traumatic brain injury.Stem cell transplantation has great potential in nerve repair.As a new method for the treatment of craniocerebral injury,it has a very broad application prospect.Neural stem cells possess the potential of self-renewal and multi-directional differentiation:into neurons,astrocytes and oligodendrocytes,providing a good cure prospect for traumatic brain injury.ObjectiveTo investigate the efficacy and nerve repair mechanism of human embryonic stem cells derived neural progenitor cells transplantation in the treatment of neurological deficits in mice following traumatic brain injury.MethodHuman embryonic stem cells transduced with DsRed gene were induced to differentiate into neural progenitor cells(Hl-DsRed-NPCs)in vitro.Fluorescent staining,Q-PCR and flow cytometry were performed to identify the properties of stem cells.Compression injury was used to establish a model of traumatic brain injury with stable behavior.Healthy adult male C57BL/6 mice were randomly divided into sham operation group,TBI + hNPCs group and TBI + vehicle group(n=11/group).One day after injury,intracranial injection of human precursor stem cells(hNPCs)or control medium was performed.The rotarod task was used to evaluate movement capacity at 0,1,3,7,14,28 and 35 days post-injury,and Morris water maze was used to test learning and memory ability at day28 to day33.Two-photon Ca2 + imaging and neural tracing were performed to observe the neural integration ability of exogenous stem cells after transplantation.Thirty-five days after transplantation,mice were sacrificed for collecting brain tissue.Fluorescent staining was used to observe the survival,differentiation and migration of hNPC.Results1.The DsRed gene was successfully knocked into hESCs(H1-DsRed).They could be seen directly under the fluorescence microscope from day 0 of neural induction to day 16 of neural harvest,and in the subsequent passages of hNPC.A large number of neural precursor cells can be induced from human embryonic stem cells in vitro.A homogenous neural differentiation of H1-DsRed was confirmed by fluorescent staining of nestin.Q-PCR analysis after neural differentiation shown a high expression of neural ectoderm transcription factors PAX6 and SOX1 as well as the neural stem cell markers nestin,but without pluripotency markers such as OCT4 and NANOG.And FACS analyses on PAX6 was expressed highly.These experiments help to confirm the identity of neural precursor cells.2.Motor impairment(P<0.05)and cognitive dysfunction(P<0.05)occurred in traumatic brain injury mice.Compared with vehicle transplantation group,the latency of rotarod task was increased as well as the latency in water maze acquired training was decreased,and the number of crossing platform was increased in hNPC treatment group.All the differences were statistically significant(P<0.05).The calcium activity of exogenous cells was detectable by Ca2+ imaging,and the synaptic connections between the exogenous cells and the host was observed by neural tracing.Histological analysis revealed less lesion volume in hNPC treatment group as compared to vehicle transplantation group.Immunofluorescence staining showed that hNPC continued to survive in the TBI mouse brain,differentiated into DCX positive and Tuj1 positive neurons,and migrated to the surrounding area including the hippocampus at 5 weeks after transplantation.Conclusion1.Human embryonic stem cells can produce neural precursor cells with self-renewal and three lineage differentiation ability,which provides a good source of cells for stem cell transplantation.2.The present findings demonstrated that early transplanted hNPC could differentiate into neuronal lineage and migrated extensively in TBI mice,form synaptic connections with the host and integrate into the host circuit,so as to alleviate lesions and improve injury-related cognitive and motor neurological deficits,which provided reliable evidences for further hNPC transplantation in clinical practice.