Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.

The identity and degree of heterogeneity of glial progenitors and their contributions to brain tumor malignancy remain elusive. By applying lineage-targeted single-cell transcriptomics, we uncover an unanticipated diversity of glial progenitor pools with unique molecular identities in developing brain. Our analysis identifies distinct transitional intermediate states and their divergent developmental trajectories in astroglial and oligodendroglial lineages. Moreover, intersectional analysis uncovers analogous intermediate progenitors during brain tumorigenesis, wherein oligodendrocyte-progenitor intermediates are abundant, hyper-proliferative, and progressively reprogrammed toward a stemlike state susceptible to further malignant transformation. Similar actively cycling intermediate progenitors are prominent components in human gliomas with distinct driver mutations. We further unveil lineage-driving networks underlying glial fate specification and identify Zfp36l1 as necessary for oligodendrocyte-astrocyte lineage transition and glioma growth. Together, our results resolve the dynamic repertoire of common and divergent glial progenitors during development and tumorigenesis and highlight Zfp36l1 as a molecular nexus for balancing glial cell-fate decision and controlling gliomagenesis. (A) Immunolabeling for GFAP and GS in the cortex of P5 hGFAP-GFP mice. (B) The percentage of indicated cells among hGFAP-GFP+ cells in P5 mouse cortices (n = 4 for GFAP; n = 3 for GS and PDGFRa). (C) Immunolabeling for GFAP, Olig2, and Slc1a3 from P5 hGFAP-GFP mice. (D) Zoom on boxed area in (C). (E) The percentage of Olig2+ and Olig2À cells among hGFAP-GFP+GFAP+ (left) or hGFAP-GFP+Slc1a3+ (right) cells in P5 mouse cortices (n = 3). (F) Immunolabeling of Blbp in the cortices from hGFAP-GFP mice at P3. (G) Expression of PDGFRa in the cortices of P5 hGFAP-GFP mice. (H) Immunolabeling for Ppp1r14b and Olig2 in the cortices of hGFAP-GFP mice at P3. (I) Immunolabeling for Olig2 and Ki67 from P5 hGFAP-GFP mice. (J) (Left) Enlarged images of (I) show cells co-labeled with Ki67 (arrows) and cells without Ki67 (arrowheads). (Right) Percentage of Olig2+ and Olig2À cells among Ki67+ hGFAP-GFP+ double-positive cells is shown (>300 cell counts from 3 cortices).

BackgroundCurrent management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment.MethodsWe performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments.ResultsOur findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors—including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)—and of several novel genes (Ahnak, Sh3bgrl3, and Col18a1) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a. Moreover, AKI caused the formation of “mixed-identity cells” (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis.ConclusionsThe scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.

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