Ischemic stroke, predominantly affecting the white matter, leads to significant disability and mortality. Current treatments are ineffective. White matter infarcts cause sensorimotor and cognitive dysfunction, driven in part by neuroinflammation. Microglia and macrophages release pro-inflammatory cytokines and matrix metalloproteinases, disrupting the blood-brain barrier and promoting T lymphocyte infiltration, amplifying inflammation, demyelination, and infarct exacerbation. This persistent pro-inflammatory environment hinders remyelination and recovery. Previous research demonstrated that transplanting rat brain microvascular endothelial cells and human iPSC-derived vascular endothelial cells (iVECs) improves locomotor function, reduces infarct size, promotes remyelination, and suppresses neuroinflammation. iVEC transplantation also increases oligodendrocyte precursor cells (OPCs) and that extracellular vesicles (EVs) from endothelial cells promote OPC survival, proliferation, and migration. However, the mechanism of iVECs' anti-inflammatory effects remained unclear. This study aimed to clarify the contribution of the immune system, particularly Tregs, to iVECs' therapeutic effects on white matter infarcts.

The literature extensively documents the role of T cells in stroke responses. Studies have shown that Tregs play a crucial role in suppressing neuroinflammation after ischemic stroke and in neurodegenerative diseases such as multiple sclerosis. Treg deficiency worsens injury in these conditions. The infiltration of CD4+ T cells, including Tregs, into the ischemic brain is well-established. However, the interaction between transplanted iVECs and the immune system, specifically the role of Tregs in mediating the beneficial effects of iVECs, needed further investigation.

Eight-week-old male Sprague-Dawley rats underwent white matter infarct induction via endothelin-1 (ET-1) injection into the internal capsule. Human iPSCs were differentiated into iVECs. Human Tregs were isolated from healthy donors' peripheral blood mononuclear cells (PBMCs) using magnetic-activated cell sorting (MACS) and expanded in vitro. Seven days post-ET-1 injection, iVECs, Tregs, or both were transplanted into the ischemic area. A group received daily intraperitoneal fingolimod (FTY720) from day 7 to 14 to sequester lymphocytes in lymph nodes. Infarct size was assessed using magnetic resonance imaging (MRI). Immunohistochemistry was used to quantify ED-1+ (activated microglia/macrophages), CD4+ T cells, Foxp3+ Tregs, and Olig2+ (oligodendrocyte lineage cells). Myelin regeneration was evaluated using Luxol fast blue staining. Statistical analysis included two-tailed Student's t-tests and one-way ANOVAs with post-hoc Tukey-Kramer tests.

iVEC transplantation significantly reduced ED-1+ inflammatory cells and CD4+ T cells while increasing Foxp3+ Tregs in the ischemic area over time. FTY720 treatment significantly decreased both CD4+ T cells and Tregs, highlighting their peripheral origin. FTY720 strongly suppressed neuroinflammation but did not enhance remyelination. iVEC transplantation increased Tregs from the periphery, and this recruitment was essential for the observed remyelination. In FTY720-treated rats, co-transplantation of iVECs and Tregs significantly promoted remyelination without altering Olig2+ cell numbers, suggesting that Tregs directly influenced myelin regeneration rather than increasing oligodendrocyte progenitor cells. Transplanted human iVECs and Tregs survived at least one week post-transplantation.

This study demonstrates that iVEC transplantation suppresses neuroinflammation in ischemic white matter, but this suppression alone is insufficient for remyelination. The recruitment of Tregs by iVECs is crucial for promoting remyelination, likely through influencing the maturation and differentiation of oligodendrocyte precursor cells rather than increasing their number. This finding highlights a novel mechanism by which iVEC transplantation exerts its therapeutic effects. The specific mechanisms by which iVECs recruit Tregs remain to be elucidated, although factors such as chemokine ligands are potential candidates.

iVEC transplantation promotes remyelination in ischemic white matter by increasing oligodendrocyte lineage cells and recruiting Tregs. Tregs significantly contribute to remyelination, possibly by promoting the maturation and differentiation of oligodendrocyte precursor cells. Future research should focus on identifying the specific factors mediating Treg recruitment by iVECs and further characterizing the role of Tregs in the remyelination process.

The study used a rat model of ischemic stroke, which may not fully recapitulate the complexities of human stroke. The sample size was relatively small. The mechanisms by which iVECs recruit Tregs and the precise roles of Tregs in remyelination require further investigation.