How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

The transition of epithelial cells into a mesenchymal state (epithelial-to-mesenchymal transition or EMT) is a highly dynamic process implicated in various biological processes. During EMT, cells do not necessarily exist in ‘pure’ epithelial or mesenchymal states. There are cells with mixed (or hybrid) features of the two, which are termed as the intermediate cell states (ICSs). While the exact functions of ICS remain elusive, together with EMT it appears to play important roles in embryogenesis, tissue development, and pathological processes such as cancer metastasis. Recent single cell experiments and advanced mathematical modeling have improved our capability in identifying ICS and provided a better understanding of ICS in development and disease. Here, we review the recent findings related to the ICS in/or EMT and highlight the challenges in the identification and functional characterization of ICS.

Highlights d Generate combined scATAC-seq and scRNA-seq libraries of adult mouse MECs d Uncover luminal cell heterogeneity and molecular underpinning driving cell type/state d Identify the pre-committed lactation progenitor within the luminal progenitor compartment d Regulatory the grammar of MEC types and putative enhancer identification

Highlights d Maintenance of mammary basal cell fate and stem cell quiescence requires Zeb1 d Zeb1 promotes basal cell fate in part through EMTassociated gene regulation d Zeb1 acts in quiescent basal cells to promote self-renewal and suppress proliferation d Zeb1 suppression of Wnt signaling is important for basal stem cell maintenance

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<b><i>Introduction and Objective:</i></b> The weight gained during pregnancy could determine the immediate and future health of the mother-child dyad. Excessive gestational weight gain (EGWG) due to abnormal adipose tissue (AT) accumulation is strongly associated with adverse perinatal outcomes as gestational diabetes, macrosomia, obesity, and hypertension further in life. Dysregulation of adipokine, AT dysfunction, and an imbalance in the prooxidant-antioxidant systems are critical features in altered AT accumulation. This study was aimed to investigate the association between adipokines and oxidative stress markers in pregnant women and the influence of the GWG on this association. <b><i>Methods:</i></b> Maternal blood samples were obtained in the third trimester of pregnancy (<i>n</i> = 74) and serum adipokines (adiponectin, leptin, and resistin), oxidative damage markers: 8-oxo-2′-deoxyguanosine (8-oxodG), lipohydroperoxides (LOOH), malondialdehyde (MDA), and carbonylated proteins (CP), and glucose a metabolic marker were measured. <b><i>Results:</i></b> Women with EGWG had low adiponectin levels than women with adequate weight gain (AWG) or insufficient weight gain (IWG). Multiple linear regression models revealed a positive association between adiponectin and 8-oxodG in women with AWG (<i>B</i> = 1.09, 95% CI: 164–222, <i>p</i> = 0.027) and IWG (<i>B</i> = 0.860, 95% CI: 0.199–1.52, <i>p</i> = 0.013) but not in women with EGWG. In women with EGWG, leptin was positively associated with LOOH (<i>p</i> = 0.018), MDA (<i>p</i> = 0.005), and CP (<i>p</i> = 0.010) oxidative markers. <b><i>Conclusion:</i></b> Our findings suggest that concurrent mechanisms regulate adipokine production and oxidative stress in pregnant women and that this regulation is influenced by GWG, probably due to an excessive AT accumulation.

How stem cells give rise to human interfollicular epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest the "transitional" basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed "transitional" basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity. Joost et al., 2018). Despite these studies, epidermal stem cell heterogeneity of human IFE remains unresolved. To address this issue, we interrogated epidermal cell heterogeneity within human neonatal foreskin epidermis using droplet-enabled scRNA-seq and identify four spatially distinct basal stem cell subpopulations. Interrogation of the "transitional" basal subpopulations that spatially occupy both the basal and suprabasal layers indicate their essential role in epidermal homeostasis. Our findings argue against a single population of progenitor cells and suggest a more complex model of multiple epidermal stem cell transitions that maintain epidermal homeostasis. RESULTS Single cell transcriptome analysis reveals robust cellular heterogeneity in human neonatal epidermis.To define the cellular heterogeneity of human IFE, we isolated viable, single cells from discarded and de-identified human neonatal foreskin epidermis and subjected them to droplet-enabled scRNA-seq to resolve their individual transcriptomes ( Figure 1A; Supplementary Figure 1; Supplementary Table 1; n = 5). We chose foreskin epidermis because it is composed of mostly IFE and contains few rudimentary skin appendages, such as hair follicles and sweat glands (Tuncali et al., 2005). We processed a total of 17,553 cells and performed quality control analysis on individual libraries using Seurat (Supplementary Figure 2) (Macosko et al., 2015). We used Similarity matrix-based OPtimization for Single Cell (SoptSC) to bioinformatically parse and analyze our data . The SoptSC algorithm is based on a cell-cell similarity matrix that can coherently perform many inference tasks -including unsupervised clustering, pseudotemporal ordering, cell lineage inference, cell-cell communication, and network inference.We used library three as a repr...

Breast cancer is a heterogenous disease that can be classified into multiple subtypes including the most aggressive basal-like and triple-negative subtypes. Understanding the heterogeneity within the normal mammary basal epithelial cells holds the key to inform us about basal-like cancer cell differentiation dynamics as well as potential cells of origin. Although it is known that the mammary basal compartment contains small pools of stem cells that fuel normal tissue morphogenesis and regeneration, a comprehensive yet focused analysis of the transcriptional makeup of the basal cells is lacking. We used single-cell RNA-sequencing and multiplexed RNA in-situ hybridization to characterize mammary basal cell heterogeneity. We used bioinformatic and computational pipelines to characterize the molecular features as well as predict differentiation dynamics and cell–cell communications of the newly identified basal cell states. We used genetic cell labeling to map the in vivo fates of cells in one of these states. We identified four major distinct transcriptional states within the mammary basal cells that exhibit gene expression signatures suggestive of different functional activity and metabolic preference. Our in vivo labeling and ex vivo organoid culture data suggest that one of these states, marked by Egr2 expression, represents a dynamic transcriptional state that all basal cells transit through during pubertal mammary morphogenesis. Our study provides a systematic approach to understanding the molecular heterogeneity of mammary basal cells and identifies previously unknown dynamics of basal cell transcriptional states.