Engineered human CD34+ hematopoietic stem cells as an innovative approach to modulate the immunoinflammatory response during atherosclerosis

Aim The increase in plasma cholesterol levels drives atherosclerosis via the accumulation of lipids in the arterial wall and the activation of an immune-inflammatory response. The growing recognition of the role of immune cells, coupled with the availability of biologics targeting specific pathways, highlight the need for experimental models that allow to translate molecular mechanisms and therapeutic strategies for atherosclerosis. Here we will present the immune-metabolic characterization of an innovative immunodeficient mouse – on an atheroprone genetic background -, that is humanized with human hematopoietic cells. Methods Humanized TKO-LDLr KO mice (by crossing LDLr-KO with immunodeficient Rag2-KO/IL2rg-KO/CD47-KO mice, HuTKOL) were generated by hepatic injection of commercial or iPSCs-derived hCD34+ cells (250.000-300.000 cells/mouse), following low-dose irradiation (250 cGy) of 2-3 days old pups. Human cells engraftment was checked through tail blood and flow cytometry after 12 weeks. HuTKOL were then fed 12-week high-cholesterol diet to investigate immunometabolic phenotype and atherosclerosis. Results HuTKOL presented human leukocytes (%hCD45+ cells/total number of live leukocytes: 34,55%, SE7,02%) in the circulation after engraftment with commercial hCD34+ cells, where CD19+ B lymphocytes were the most abundant population (%hCD19+/hCD45+: 79,15%, SE±1,76%), but they decreased over time (%hCD19+/hCD45+: 4,95%, SE±1,88%). Instead, CD4+ and CD8+ T lymphocytes increased (%hCD4+/hCD45+: 3,02%, SE±0,58%; %hCD8+/hCD45+: 5,51%, SE±1,26%), becoming the predominant population (%hCD4+/hCD45+: 37,44%, SE±6,79%; %hCD8+/hCD45+: 22,84%, SE±2,58%) at 24 weeks, similar to human lymphocyte profile. HuTKOL developed dyslipidemia (plasma cholesterol levels: 1211,96 mg/dl, SE± 63,72) and atherosclerosis (% aortic sinus plaque occlusion: 18,35%, SE±1,90%), when fed a high-cholesterol diet. Experiments are ongoing on iPSCs-derived CD34+, which showed a differentiation of 88.51% (SE±0,55%) in CD34+CD45- and 5.94% (SE±0.22%) in CD34-CD45- cells over all live cells by day 12 of the protocol. Conclusions hCD34+ humanized TKO-LDLR mice represent a valuable platform for studying the immune-metabolic response during atherosclerosis and could represent a valuable tool to test immunotherapies for CVD.