Anti-mitotic signaling regulation to enhance cell grafting outcomes at CNS injuries

Introduction: Central Nervous System (CNS) injuries such as stroke and spinal cord injury (SCI) are leading causes of death with limited treatment options1. CNS injuries form lesions that are devoid of neural cells but contain fibrotic scars and immune cells that do not support repair2. Grafting neural progenitor cells or immature astrocytes are promising strategies to improve neural repair outcomes and we recently demonstrated that cell grafts direct repair in small ischemic strokes but have limited survival and are incapable of directing repair in more severe lesions such as hemorrhagic stroke injuries and SCI3-4. Augmenting cell graft-directed repair at larger CNS lesions, will require regulating the effects of lesion derived cues, including mitogen and anti-mitotic factors, on grafted cells to sustain their proliferation, post transplantation. Transforming Growth Factor beta (TGF-β) superfamily proteins are canonical anti-mitotic cues upregulated at CNS injuries, that control neural cell differentiation and maintenance of quiescence as well as counteracting the effects of pro-proliferative growth factors5-6. Small molecule drugs including SB43125, and DMH-1 have been identified as effective TGF-β inhibitors7. The goal of this study is to evaluate the cell and molecular differences in CNS lesion environments that direct good and poor grafting outcomes and investigate the use of small molecule regulators to augment proliferation of grafted cells. We hypothesize that SB43125 and DMH-1 through dual SMAD inhibition of TGF- β family will promote proliferation and survival of cell graft in high serum in vitro environments as well a severe CNS injuries.

Materials and

Methods: Mouse embryonic stem cells (mESC) that constitutively express the ribosomal protein Rp122 with a hemagglutinin (HA) tag (RiboTag) were used to generate passagable Neural Progenitor Cells (NPC) that were differentiated into quiescent astrocyte (qAs). NPCs and qAs were exposed to high serum environments with and without the TGF- β inhibitors for 72 hours. Cells were fixed on coverslips and using immunocytochemistry (ICC) were stained for proliferation markers; Top2a and Ki67. Image analysis was conducted to obtain the percentage of proliferative cells. Cells were also manually counted to obtain the growth rate. For in vivo studies, NPCs were injected into L-NIO induced stroke lesions at 1-, 2-, 3-, 4-, 5- and 7 days, collected at 2 weeks for immunohistochemistry (IHC) and stained with astrocyte, neuronal and inflammatory cells markers. Images were quantified for cell survival and preferential spatial distribution of cells.

Results, Conclusions, and Discussions: Pro-proliferative growth factors and serum had opposing effects on the proliferation state of both NPC and qA and treatment with TGF-β inhibitors SB43125 and DMH-1 had individual and synergistic effects that rescued proliferation capacity in these cells during serum exposure. Grafted NPC showed increased survival and more effective modulation of lesion derived inflammation when grafted at 2 days compared to 7days after L-NIO stroke demonstrating that there is temporal regulation of grafting outcome at CNS injury lesion environments. NPCs grafted at 1-2-,3-,4- and 5- days showed differences in spatial distribution and minor differences in survival.
SB43125 and DMH-1 inhibit anti-mitotic activity of TGF-β superfamily in NPCs and qA and rescue proliferation in high serum environment. We identified that there is a temporal factor that contributes to NPC graft survival and inflammatory cell density when grafts are made at 2 versus 7 days after stroke. Future studies will identify transcriptomic differences of lesion environments of mild and severe stroke lesions at key grafting timepoints to identify key lesion regulatory cues that influence grafting outcomes.

Acknowledgements (Optional): Boston University Core Facilities and Staff. This work was supported by funding from Boston University (T.M.O), Craig H. Neilsen Foundation (T.M.O.) and Schlumberger Foundation (H.A).