We profiled human central nervous system (CNS)-associated macrophages (CAMs) in anatomically dissected CNS interface tissue from typical, fetal and glioblastoma-affected brains using single-cell multi-omics and spatially resolved transcriptomic techniques. Analyses of CAM (and microglia) turnover rates in stem-cell-transplanted glioblastoma and prenatal tissues highlighted the developmental phenotypes of these cells in patients, which lays the groundwork for potential replacement therapies.