Graphical AbstractHighlights d Myeloid scRNA-seq atlas across brain regions and developmental stages d Limited transcriptomic heterogeneity of homeostatic microglia in the adult brain d Phase-specific gene sets of proliferating microglia along cell cycle pseudotime d Phagocytic PAM subset uncovered by scRNA-seq sharing a DAM gene signature SUMMARY Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous.We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

Cancer is often viewed as a caricature of normal developmental processes, but the extent by which its cellular heterogeneity truly recapitulates multi-lineage differentiation processes of normal tissues remains unknown. Here, we implement “single-cell PCR gene-expression analysis” (SINCE-PCR) to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single-cell (n = 1), we show that transcriptional diversity of cancer tissues is largely explained by in vivo multi-lineage differentiation, not only by clonal genetic heterogeneity. Finally, we show that perturbations in gene-expression programs linked to multi-lineage differentiation strongly associate with patient survival. Guided by SINCE-PCR data, we develop two-gene classifier systems (KRT20 vs CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard-ratios superior to pathological grade and comparable to microarray-derived multi-gene expression signatures.

Summary There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with a post-transplant therapy that combines immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients), and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment, and offer a potential application of the virome state to predict immunocompetence.