Exploring the importance of axial identity in transplantation studies using neural progenitors of specific anteroposterior identity
Creators: name: Alonso Torrens, Aaron
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: anteroposterior
spinal cord
neural precursor cells
Hox genes
neuromesodermal progenitors
posterior CNS
axial identity

description: After an injury, axons in the mammalian spinal cord fail to regenerate, and the spinal cord cannot repair itself. A promising approach to improving recovery after spinal cord injury (SCI) is the transplantation of neural stem or progenitor cells (NPCs) derived from pluripotent cells into the injury site. However, there are significant challenges to this potential therapeutic approach. Grafted NPCs must survive, differentiate, grow neurites, and integrate appropriately into the host. One reported cause of the failure of integration is a mismatch between the anteroposterior identity of the grafted cells and the host site. The differentiation of pluripotent cells in vitro to posterior NPCs corresponding to a thoracic/lumbosacral spinal cord identity has been achieved by using neuromesodermal progenitors (NMP) as an intermediate. Cells of the most caudal/sacral identity have not been produced. I first tested several conditions for generating NPC of defined identities and characterised the cells produced. I confirmed that the generation of NMPs is an essential step to generate NPCs of a posterior identity and that the addition of RA in NMP cultures is not sufficient to induce the expression of the most posterior Hox genes. GDF11 is reported to posteriorize differentiating human ESC towards a sacral identity. I tested the effects of this protein in an established NMP culture protocol. I showed that timing of GDF11 treatment during NMP differentiation is critical for the optimal expression of sacral/caudal Hox genes and the overall cellular phenotype acquired. The addition of GDF11 early during NMP differentiation factors results in cells acquiring an endoderm fate. In contrast, GDF11 added to established NMP populations in culture results in cells developing a neural identity capable of expressing the late paralog groups (PGs), including PG13. To determine functional integration, survival, neurite outgrowth and pathfinding of cells after grafting, I differentiated Epiblast stem cells (EpiSCs) towards NPCs of anterior (NA), hindbrain (NH) and posterior thoracic spinal cord identity (NS) and grafted them into E10.5 mouse embryos at homotopic and heterotopic locations. Since the culture period of whole embryos is limited, I also grafted cells of defined axial identities into dissected spinal cord slices and allowed these to grow in culture for five days. Grafted NPCs survive and integrate into the host while retaining their neural and anteroposterior axial identity when grafted into the neural tube. In addition, we show that cells grafted in isotopic sites show higher rates of proliferation and integration into the tissue. Overall, this suggests that cells' axial identity is fixed in the grafted NPCs and contributes to successful integration, highlighting the importance of axial identity in cellular therapies.