Abstract: BACKGROUND: Cell transplantation is one of the effective approaches for repairing spinal cord injury. Our research team previously found that transplanted cerebrospinal fluid-contacting neurons can survive and promote motor function recovery in mice with spinal cord injury. However, whether these transplanted cerebrospinal fluid-contacting neurons differentiate into functional neurons and thereby facilitate motor function recovery remains unclear.
OBJECTIVE: To investigate whether transplanted cerebrospinal fluid-contacting neurons differentiate into functional neurons in vivo and contribute to motor function recovery after spinal cord injury.
METHODS: Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical spinal cord of C57BL/6 mice within 24 hours after birth and cultured in adherent conditions. The cells were transduced with a lentivirus carrying a multimodal imaging fusion gene and selected with puromycin to purify cerebrospinal fluid-contacting neurons. Differentiation was induced using serum-containing medium. Immunofluorescence staining was performed to detect the expression of neuronal marker NeuN and motor neuron marker ChAT after differentiation. Thirty C57BL/6 mice were randomly divided into three groups. The T10 segment spinal cord injury model was established in the transplantation group and PBS group by clamping method, and the sham operation group only opened the lamina. One week after spinal cord injury, the transplantation group received cell transplantation, and the PBS group was injected with an equivalent volume of PBS. At weeks 1, 4, and 8 post-transplantation, spinal cord tissues were collected for immunofluorescence to assess ChAT expression. At week 8, the expression of synaptic marker SYN, inhibitory neurotransmitter marker GAD65/67, and excitatory neurotransmitter marker vGLUT1 was further evaluated. Hematoxylin and eosin staining was used to observe spinal cord morphology. Motor function recovery was assessed using Basso Mouse Scale scoring and footprint analysis.
RESULTS AND CONCLUSION: (1) Cerebrospinal fluid-contacting neurons exhibited neural stem cell characteristics in vitro and could differentiate into motor neurons. (2) Transplanted cerebrospinal fluid-contacting neurons survived long-term and differentiated into motor neurons in vivo. (3) The proportion of cerebrospinal fluid-contacting neurons differentiating into motor neurons reached the highest level at 8 weeks post-transplantation (P < 0.000 1). (4) At 8 weeks post-transplantation, cerebrospinal fluid-contacting neurons co-expressed SYN, GAD65/67, and vGLUT1, indicating synaptic formation between transplanted cerebrospinal fluid-contacting neurons and host neurons. (5) The Basso Mouse Scale scores of the PBS group remained significantly lower than those of the transplantation group (P < 0.001). Transplanted mice exhibited more coordinated gait patterns with only minor toe dragging, whereas PBS-treated mice displayed severe hindlimb dragging. (6) Hematoxylin and eosin staining revealed large cavitation areas in the injury site of PBS-treated mice, whereas the cavitation area was significantly reduced in the transplantation group. These findings confirm that transplanted cerebrospinal fluid-contacting neurons can differentiate into motor neurons both in vitro and in vivo, establish synaptic connections, and ultimately improve motor function in spinal cord injury mice. 

Key words: spinal cord injury, cerebrospinal fluid-contacting neuron, neural stem cell, motor neuron, transplantation, synaptic reconstruction, motor function recovery, multimodal imaging gene lentivirus