Cerebrospinal fluid-contacting neurons differentiating into motor neurons promote functional recovery in spinal cord-injured mice

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 neonatal mice within 24 hours of birth and cultured adherently. Cells were transduced with a lentivirus carrying a multimodal imaging gene, and cerebrospinal fluid-contacting neurons were selected and purified using puromycin. Differentiation was induced with serum-containing differentiation medium, and expression of neuronal marker NeuN and motor neuron marker ChAT was detected by immunofluorescence. Thirty C57BL/6 mice were randomly divided into three groups: transplantation group and PBS group underwent T10 spinal cord injury by clip compression, while sham group only had laminectomy. One week after injury, cerebrospinal fluid-contacting neurons were transplanted in situ in the transplantation group, and an equal volume of PBS was injected in the PBS group. At 1, 4, and 8 weeks after transplantation, immunofluorescence was used to detect expression of motor neuron marker ChAT in spinal cord tissue. At 8 weeks, immunofluorescence was used to detect synaptic marker SYN, inhibitory transmitter marker GAD65/67, and excitatory transmitter marker vGLUT1; hematoxylin-eosin staining was used to observe spinal cord morphology; BMS motor function score and footprint analysis were used to assess motor function recovery. RESULTS AND CONCLUSION: (1) Cerebrospinal fluid-contacting neurons expressed neural stem cell characteristics in vitro and could differentiate into motor neurons. (2) Transplanted cerebrospinal fluid-contacting neurons could survive long-term in vivo and differentiate into motor neurons. (3) The proportion of cerebrospinal fluid-contacting neurons differentiating into motor neurons was highest at 8 weeks (P < 0.0001). (4) At 8 weeks after transplantation, cerebrospinal fluid-contacting neurons co-expressed SYN, GAD65/67, and vGLUT1, indicating synaptic connections with host neurons. (5) BMS scores of PBS group were consistently lower than those of transplantation group (P < 0.001); footprint analysis showed more coordinated gait in transplantation group with only toe dragging, while PBS group showed obvious hindlimb dragging. (6) Hematoxylin-eosin staining showed large cavities in the injured area of PBS group, while cavities were reduced in transplantation group. These results indicate that transplanted cerebrospinal fluid-contacting neurons can differentiate into motor neurons both in vitro and in vivo, form synaptic connections, and thereby improve motor function in spinal cord-injured mice.

1. Introduction

Spinal cord injury (SCI) leads to severe motor, sensory, and autonomic dysfunction, and currently there is no effective treatment. Existing strategies, including surgical intervention and systematic rehabilitation, have limited efficacy in promoting neural regeneration and functional recovery. Over the past three decades, cell replacement therapy has shown promise in treating various neurological disorders. Neural stem cells, due to their potential for directed differentiation into neural tissues and good biocompatibility, are considered a promising avenue for SCI treatment. However, simple transplantation of neural stem cells faces issues such as insufficient integration, low survival rate, and limited differentiation efficiency. Therefore, selecting the appropriate type of neural stem cells for transplantation is crucial.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are a type of multifunctional, multimodal cells located in the central nervous system, directly contacting the cerebrospinal fluid. They reside in neural stem cell niches in the spinal cord and possess mechanosensory functions, playing important roles in regulating posture and fine motor control in mammals. Recent studies have shown that CSF-cNs exhibit neural stem cell potential both in vivo and in vitro, and are a special neuronal population with endogenous neural stem cell properties, playing a key role in recovery after SCI. Using flow cytometry, adult endogenous CSF-cNs can form neurospheres in vitro and differentiate into neurons and glial cells. In previous research, we used lentivirus to label GFP-positive CSF-cNs and transplanted them into SCI mice; these cells survived under SCI conditions.