Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels--random sequences that label individual molecules--can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

SummaryThe murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis.

Graphical Abstract Highlights d Deconstruction of full-thickness skin by single-cell RNA-seq and in situ RNA staining d Basal ORS, suprabasal ORS, companion layer, and LPC cells constitute outer HF layers d Inner HF layers form from unfated progenitors and mature via intermediate states d Fibroblasts spatiotemporally separate into three major subtypes and one cell state SUMMARYSkin homeostasis is orchestrated by dozens of cell types that together direct stem cell renewal, lineage commitment, and differentiation. Here, we use single-cell RNA sequencing and single-molecule RNA FISH to provide a systematic molecular atlas of fullthickness skin, determining gene expression profiles and spatial locations that define 56 cell types and states during hair growth and rest. These findings reveal how the outer root sheath (ORS) and inner hair follicle layers coordinate hair production. We found that the ORS is composed of two intermingling but transcriptionally distinct cell types with differing capacities for interactions with stromal cell types. Inner layer cells branch from transcriptionally uncommitted progenitors, and each lineage differentiation passes through an intermediate state. We also provide an online tool to explore this comprehensive skin cell atlas, including epithelial and stromal cells such as fibroblasts, vascular, and immune cells, to spur further discoveries in skin biology. IFE C -IFE basal, cycling (1 pop.) IFE B -IFE basal (1 pop.) IFE SB -IFE suprabasal (2 pop.) uHF B -Upper HF basal (1 pop.) uHF SB -Upper HF suprabasal (1 pop.) SG -Sebaceous gland (1 pop.) OB -Outer bulge (1 pop.) HG -Hair germ (1 pop.) FIB -Skin fibroblasts (4 pop.) DS -Dermal sheath (2 pop.) DP -Dermal papilla (2 pop.) EC -Endothelial cells (2 pop.) vSM -Vascular smooth muscle (1 pop.) LV -Lymph vessel cells (1 pop.) TC -T-cells (1 pop.) MC -Macrophages (3 pop.) DC -Dendritic cells (1 pop.) LC -Langerhans cells (1 pop.) SM -Skeletal muscle (1 pop.) RBC -Red blood cells (1 pop.) MEL -Melanocytes (2 pop.) SC -Schwann cells (1 pop.) ORS -Outer root sheath (3 pop.) LPC -Lower proximal cup (1 pop.) CP -Companion layer (2 pop.) GL -Germinative layer (4 pop.) IRS -Inner root sheath (6 pop.) CX -Cortex / cuticle (5 pop.) MED -Medulla (3 pop.) H Krt5 hi (sbOL) and Krt5 dim cells (bOL) Krt5+ / Krt6+ cells (mCP) KRT5 + Krt5 + Krt6a + DAPI + BF Summary A Heterogeneity and RNA velocity of outer layer (OL) cells ORS SB uCP mCP ORS B1 ORS B2 LPC mCP -mid-part companion layer (36 cells) ORS B1 -basal ORS (101 cells) ORS SB -suprabasal ORS (51 cells) ORS B2 -basal ORS (102 cells) LPC -lower proximal cup (17 cells) uCP -upper companion layer (28 cells)

Highlights d Single-cell RNA-seq identifies distinct states of Lgr5 and Lgr6 progeny upon wounding d Wound healing activates IFE-like signature and downregulates bulge genes in Lgr5 progeny d Adaptation in Lgr5 SCs occurs within 1 day, enabling them to respond to wound signals d Lgr6 SCs are already primed to respond to wound signals in unwounded healthy skin

A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.

SummaryThe dynamics and interactions between stem cell pools in the hair follicle (HF), sebaceous gland (SG), and interfollicular epidermis (IFE) of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE. We show that these Lgr6+ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6+ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6+ and Lgr6− cells did not reveal a distinct Lgr6-associated gene expression signature, raising the question of whether Lgr6 expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6+ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.

Regulation of adipose tissue stem cells (ASCs) and adipogenesis impact the development of excess body fat-related metabolic complications. Animal studies have suggested the presence of distinct subtypes of ASCs with different differentiation properties. In addition, ASCs are becoming the biggest source of mesenchymal stem cells used in therapies, which requires deep characterization. Using unbiased single cell transcriptomics we aimed to characterize ASC populations in human subcutaneous white adipose tissue (scWAT). The transcriptomes of 574 single cells from the WAT total stroma vascular fraction (SVF) of four healthy women were analyzed by clustering and t-distributed stochastic neighbor embedding visualization. The identified cell populations were then mapped to cell types present in WAT using data from gene expression microarray profiling of flow cytometry-sorted SVF. Cells clustered into four distinct populations: three adipose tissue-resident macrophage subtypes and one large, homogeneous population of ASCs. While pseudotemporal ordering analysis indicated that the ASCs were in slightly different differentiation stages, the differences in gene expression were small and could not distinguish distinct ASC subtypes. Altogether, in healthy individuals, ASCs seem to constitute a single homogeneous cell population that cannot be subdivided by single cell transcriptomics, suggesting a common origin for human adipocytes in scWAT.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0701-4) contains supplementary material, which is available to authorized users.

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.