Preclinical Exploring Research Of Human Pluripotent Stem Cell Differentiated Spinal Cord Gabaergic Neural Progenitors For Treating Spinal Cord Injury

Part 1 Long term safety of intraspinal graft of human pluripotent stem cell differentiated spinal GABAergic neural progenitorsObjective: To determine the safety of intraspinal graft of spinal GABAergic progenitors differentiated from human pluripotent stem cell over prolonged time.Methods: Human pluripotent stem cells were gene edited to express hM3 Dq and KORD simultaneously,then patterned caudally and dorsally into spinal d I4 neural progenitors with small molecules,and grafted into cervical spinal cord of na(?)ve immunodeficient Nude rats through stereotaxic injection.Motor and sensory function of both fore and hind limbs were evaluated every 3 month,and by 12 months after transplantation,they were analyzed before and after activation and deactivation of grafted neurons by application of CNO and Sal B,respectively;rats were sacrificed after another 6 months observation,spinal cords were harvested for immunofluorescence and laser scanning confocal microscopy.Results: High purity spinal d I4 neural progenitors with Bi-DREADDs expression were generated from human pluripotent stem cells,when grafted into Nude rat spinal cord,they survived,migrated,differentiated and matured into neurons,extending large numbers of axons rostrally and caudally,forming synapses with host neural circuits,however,the survival,body growth,motor and sensory function of Nude rats were not altered by grafted human spinal GABA neuron,even after activation or deactivation of grafted neurons with CNO or Sal B.Conclusion: No significant neural dysfunction was caused by intraspinal graft of human spinal GABAergic neurons,which makes it a safe strategy for spinal cord repair.Part 2 Preliminary study of grafting human pluripotent stem cell derived spinal GABAergic neural progenitors for treating spinal cord injury in nonhuman primate Macaca MulattaObjective: To determine whether spinal GABAergic neural progenitors derived from human pluripotent stem cell survive after grafted into injured spinal cord of nonhuman primate Macaca Mulatta,and whether they promote locomotion,reduce neuropathic pain and alleviate muscle spasm.Methods: Human pluripotent stem cells were gene edited to express h M3 Dq and KORD simultaneously,then patterned caudally and dorsally into spinal cord d I4 neural progenitors with small molecules,and grafted into rostral part of left side hemisected thoracic spinal cord of Macaca Mulatta through stereotaxic injection.Locomotion,mechanical allodynia threshold,and muscle tone were assessed every month until 7 month,after then,monkey was sacrificed,spinal cord was harvested for immunofluorescence and laser scanning confocal microscopy.Results: Spinal GABAergic neural progenitors derived from human pluripotent stem cell survived up to 7 months after grafted into injured monkey spinal cord,they differentiated and matured into spinal GABAergic neurons,grew numerous axons,and synapsed with host neural circuits.Although the mechanical allodynia threshold continued to decline,locomotion ability gradually recovered,and muscle tone didn't increase.Conclusion: Transplanting spinal GABAergic neural progenitors from human pluripotent stem cell is practical and safe for treating spinal cord injury in nonhuman primate.