Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice

ARTICLE https://doi.org/10.1038/s42003-023-04893-0 OPEN Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice 1234567890():,; Miriam Aceves 1,2, Ashley Tucker1,2,5, Joseph Chen1,5, Katie Vo1, Joshua Moses1, Prakruthi Amar Kumar Hannah Thomas1, Diego Miranda1, Gabrielle Dampf1, Valerie Dietz1, Matthew Chang1, Aleena Lukose1, Julius Jang1, Sneha Nadella1, Tucker Gillespie1, Christian Trevino1, Andrew Buxton3, Anna L. Pritchard3, Peyton Green4, Dylan A. McCreedy1,2,3 & Jennifer N. Dulin 1,2 ✉ 1, Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentallyrestricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI. 1 Department of Biology, Texas A&M University, College Station, TX 77843, USA. 2 Texas A&M Institute for Neuroscience, Texas A&M University, College Station, TX 77843, USA. 3 Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA. 4 Ganado High School, Ganado, TX 77962, USA. 5These authors contributed equally: Ashley Tucker, Joseph Chen. ✉email: COMMUNICATIONS BIOLOGY | (2023)6:544 | https://doi.org/10.1038/s42003-023-04893-0 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04893-0 S pinal cord injury results in immediate and permanent loss of spinal cord neurons, frequently causing lifelong neurological dysfunction including but not limited to paralysis, loss of sensation, autonomic dysfunction, and chronic neuropathic pain1–3. Transplantation of neural progenitor cells is viewed as a promising neuronal replacement strategy with potential to regenerate neural circuits and improve functional outcomes following SCI4,5. Indeed, in the past few decades, there have been several clinical trials evaluating the therapeutic potential of neural stem- and progenitor cell transplantation for treatment of SCI in humans6–11. Despite advancement to clinical trials, further characterization of graft biology and therapeutic mechanism is sorely needed. For example, although it has been shown that spinal cord NPC grafts are populated with diverse neuronal subtypes12–19, it is still poorly understood how graft cellular composition influences regeneration of host axons and functional outcomes following SCI. Over the past four decades, a great deal of knowledge has been gained from experimental rodent studies transplanting fetal rodent spinal cord NPCs19. These cells are exposed to normal developmental patterning cues and differentiate into multiple endogenous spinal cord neuronal subtypes following transplantation12–18,20–23, making them a gold standard cell source to characterize graft/host biology in cellular transplantation studies. A 1983 study by Reier, Perlow, and Guth was the first to demonstrate the survival and neurogenic potential of rat fetal spinal cord solid tissue grafts, derived from embryonic days 12 to 17 (E12–E17), following transplantation into the injured adult central nervous system (CNS)24. Due to their long-term survival and neural differentiation, E14–E15 embryos were concluded to be the “most optimal source” for spinal cord transplants compared to later-stage grafts24. Based on a literature survey of 70 fetal rodent spinal cord NPC transplantation studies, E14 rat spinal cord (developmentally equivalent to E12.5 in mouse25) remains the most commonly used source of NPC donor tissue, with 86.6% of these studies utilizing cells derived from this embryonic stage (Table 1). Despite this widespread use of a single developmental age of donor tissue, spinal cord neurogenesis occurs over a five-day period in the rodent26,27. Distinct populations of spinal cord neurons are born at different intervals within the period of neurogenesis, with varying abundances of the 11 progenitor populations over time26. This raises the possibility that transplantation of NPCs obtained from distinct days within the neurogenic period might produce grafts with varying neuronal subtype composition. We and others have previously shown that dorsal- or ventralrestricted populations of spinal cord NPCs impart distinct effects on host axon regeneration12 and respiratory function28 after SCI. Here, we explore how the developmental restriction of donor NPCs influences graft neuronal subtype composition, host axon regeneration into grafts, and recovery of sensorimotor function following SCI. Following the isolation of spinal cord NPC populations from E11.5, E12.5, and E13.5 mouse embryos, we assessed the abundances of distinct progenitor populations and spinal cord neuronal subtypes in vitro and in vivo. We also analyzed the effects of graft type on gliogenesis, graft axon outgrowth, and host axon regeneration into grafts. Finally, we determined the effects of NPC graft type on recovery of locomotor function and sensory function following transplantation into sites of thoracic SCI. Results Developmental stage of spinal cord neural progenitor cells affects the abundance of distinct progenitor populations and postmitotic cell populations in vitro. Mouse spinal cord neurogenesis occurs from embryonic days E9.5 to E13.5. Relative abundances of the 11 cardinal spinal cord neural progenitor populations (dp1-dp6, p0-p3, pMN) shift over time during this neurogenic period26,27,29. Recent work has demonstrated that ventral progenitor populations are most abundant during early neurogenesis, and dorsal progenitors dominate in the later stages of neurogenesis26. The overall goal of our study is to determine how the transplantation of developmentally restricted NPC populations into sites of spinal cord injury affects graft cellular composition and integration with the injured host spinal cord. We first sought to characterize the cellular makeup of spinal cord isolates obtained from different days within the neurogenic period. We dissociated whole spinal cords from E11.5, E12.5, or E13.5 mouse embryos, then cultured the cell suspensi (...truncated)