DOI: 10.25442/hku.31238890.v1
title: Supporting data for ‘Directed differentiation of functional dorsal spinal GABAergic neurons by defined transcriptional factors for treating spinal cord injury and central neuropathic pain’

description: Spinal cord injury (SCI) results in significant loss of γ-aminobutyric acid (GABA) interneurons in the dorsal horn, leading to increased neuronal excitability—a key feature of central neuropathic pain (CNP). This condition manifests as spontaneous hyperalgesia and allodynia, severely impacting quality of life. Following SCI, excessive glutamate release causes excitotoxicity, further damaging neurons and oligodendrocytes. Conversely, GABA exhibits neuroprotective effects, reducing secondary injury and supporting nerve regeneration during development and after CNS injury. Despite ongoing research, there are no effective treatments for CNP. Human neural stem cell (NSC) therapies show promise in replacing lost neurons. However, the hostile injury environment and the default pathways of differentiation hinder their ability to generate mature GABAergic neurons with the appropriate dorsal spinal identity, limiting their therapeutic potential.<br>In this study, we employed a small molecule differentiation strategy using human induced pluripotent stem cells (hiPSCs) to identify five key transcription factors including BRN2 (B), PTF1A (P), LBX1 (L), PAX2 (p), and ASCL1 (A) that are crucial for specifying dorsal spinal GABAergic neurons. We demonstrated that these transcription factors are sufficient to directly convert hiPSCs into dorsal spinal GABAergic progenitors. These induced GABAergic cells, characterized by their dorsal spinal identity, effectively counteract the harmful effects of the injured microenvironment, showing improved survival, resilience, and an inherent ability to mature into functional GABAergic neurons. Notably, these cells also exert non-cell autonomous effects by reducing apoptosis and promoting the generation of new neurons in the host tissue after SCI. Integration of these transplanted cells into the host circuitry resulted in significant improvements in neuropathic pain and locomotor function, highlighting their potential for future clinical applications. To monitor reprogramming efficiency and analyze gene expression profiles, we developed an adeno-associated virus (AAV)-based GABA-reporter system, validating its effectiveness both in vitro and in vivo.<br>Despite these promising findings, challenges such as the risk of tumor formation from pluripotent stem cells, heterogeneity of grafted cell populations, and mismatched integration with the host tissue may limit clinical translation. Therefore, producing homogeneous dorsal spinal GABAergic neurons directly from patient somatic cells via lineage reprogramming is crucial. Based on data from small molecule differentiation and transcription factor reprogramming of hiPSCs, we identified a specific combination of transcription factors-PSLA that effectively guides the development and maturation of GABAergic neurons in the dorsal spinal cord. These factors enable the direct reprogramming of human cells into dorsal spinal GABAergic neurons. Future efforts will focus on optimizing transcription factor combinations for direct reprogramming from human urine cells and evaluating their therapeutic potential through transplantation in SCI models. The ability to generate GABAergic neurons via direct reprogramming offers significant promise for advancing research and developing regenerative therapies for neurological diseases.