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Cited by 221Hair Growth and DisordersSingle-cell and spatial transcriptomicsSkin and Cellular Biology Research

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Single cell transcriptomics of human epidermis identifies basal stem cell transition states

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Wang, Shuxiong, Drummond, Michael L., Guerrero-Juarez, Christian F., Tarapore, Eric, MacLean, Adam L., Stabell, Adam R., Wu, Stephanie C., Gutierrez, Guadalupe, That, Bao T., Benavente, Claudia A., Nie, Qing, Atwood, Scott X.·2020·Nature Communications
DOI 10.1038/s41467-020-18075-7PMC PMC7447770

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Abstract

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.

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Figures

Fig. 1 Defining human neonatal epidermal cell populations using scRNA-seq. a Schematic of epidermal cell isolation from human neonatal foreskin. b Clustering of 16,360 single cells isolated from Libraries 1-5 that passed quality control metrics using SoptSC and displayed using Graph embedding. Cell proportions from putative cellular communities are quantified on the right. c Batch IDs for each library are superimposed onto the Graph embedded clusters and show no substantial batch effects. d Violin plots of relative gene expression of known epidermal marker genes split by cell cohorts using SoptSC. e Feature plots showing expression of keratinocyte markers from basal, spinous, and granular layers, including melanocytes.
Fig. 1 Defining human neonatal epidermal cell populations using scRNA-seq. a Schematic of epidermal cell isolation from human neonatal foreskin. b Clustering of 16,360 single cells isolated from Libraries 1-5 that passed quality control metrics using SoptSC and displayed using Graph embedding. Cell proportions from putative cellular communities are quantified on the right. c Batch IDs for each library are superimposed onto the Graph embedded clusters and show no substantial batch effects. d Violin plots of relative gene expression of known epidermal marker genes split by cell cohorts using SoptSC. e Feature plots showing expression of keratinocyte markers from basal, spinous, and granular layers, including melanocytes.CC BY · PubMed Central
Fig. 2 Differential gene expression highlight basal stem cell heterogeneity. a Heatmap showing top 300 differentially expressed genes per cluster. Dotted lines outline differentially expressed genes. b Violin plots of relative expression of marker genes split by cell cohorts and color-coded by cell community as in A. c Library 3 violin plots of relative expression of GJB2 and RHCG. d Feature plots showing expression of select basal and granular cell marker genes. e Immunostaining of KRT14 (red), KRT10 (green), and DAPI (blue) in human neonatal skin. Scale bar 100 µm. n = 3. Immunostaining of differentially expressed proteins (red) from the (f) BAS-III cluster (ASS1 [n = 6] and COL17A1 [n = 4]), (g) BAS-III/IV clusters (KRT19 [n = 3] and GJB2 [n = 4]), (h) BAS-I cluster (PTTG1 [n = 6] and CDC20 [n = 6]), (i) BAS-II cluster (RRM2 [n = 6] and PCLAF [n = 2]), (j) GRN cluster (SPINK5 [n = 3] and CALML5 [n = 2]), and (k) the MEL (MLANA [n = 2]) and LAN (CD74 [n = 2]) clusters in human neonatal skin. COL7A1 (green) and DAPI (blue) are costained. Dotted lines denote position of basement membrane where COL7A1 staining is absent. Scale bar 100 µm. l Quantification of ASS1 (n = 14 ridges) and KRT19 (n = 10 ridges) staining intensity at the top and bottom of Rete ridges. ***p < 0.0001. m Quantification of PTTG1 (n = 102 cells) and RRM2 (n = 104 cells) distance from the basement membrane, with KRT14 + basal cell (n = 167 cells) or KRT10+ suprabasal cell (n = 142 cells) distance shown as controls. Black stars represent significance compared with basal cell position whereas green stars represent significance compared with suprabasal position. ***p < 0.0001. Box represents 25th to 75th percentiles. Whiskers represent minimum and maximum data points. Bar represents mean. Significance was determined by unpaired two-tailed t test.
Fig. 2 Differential gene expression highlight basal stem cell heterogeneity. a Heatmap showing top 300 differentially expressed genes per cluster. Dotted lines outline differentially expressed genes. b Violin plots of relative expression of marker genes split by cell cohorts and color-coded by cell community as in A. c Library 3 violin plots of relative expression of GJB2 and RHCG. d Feature plots showing expression of select basal and granular cell marker genes. e Immunostaining of KRT14 (red), KRT10 (green), and DAPI (blue) in human neonatal skin. Scale bar 100 µm. n = 3. Immunostaining of differentially expressed proteins (red) from the (f) BAS-III cluster (ASS1 [n = 6] and COL17A1 [n = 4]), (g) BAS-III/IV clusters (KRT19 [n = 3] and GJB2 [n = 4]), (h) BAS-I cluster (PTTG1 [n = 6] and CDC20 [n = 6]), (i) BAS-II cluster (RRM2 [n = 6] and PCLAF [n = 2]), (j) GRN cluster (SPINK5 [n = 3] and CALML5 [n = 2]), and (k) the MEL (MLANA [n = 2]) and LAN (CD74 [n = 2]) clusters in human neonatal skin. COL7A1 (green) and DAPI (blue) are costained. Dotted lines denote position of basement membrane where COL7A1 staining is absent. Scale bar 100 µm. l Quantification of ASS1 (n = 14 ridges) and KRT19 (n = 10 ridges) staining intensity at the top and bottom of Rete ridges. ***p < 0.0001. m Quantification of PTTG1 (n = 102 cells) and RRM2 (n = 104 cells) distance from the basement membrane, with KRT14 + basal cell (n = 167 cells) or KRT10+ suprabasal cell (n = 142 cells) distance shown as controls. Black stars represent significance compared with basal cell position whereas green stars represent significance compared with suprabasal position. ***p < 0.0001. Box represents 25th to 75th percentiles. Whiskers represent minimum and maximum data points. Bar represents mean. Significance was determined by unpaired two-tailed t test.CC BY · PubMed Central
Fig. 3 Cell-cell interaction modeling of the WNT signaling pathway. a Hierarchical clustering of similar cell-cell signaling probability scores from library 3 and visualized on a tSNE plot. b Visualization of signaling probability scores of Ligand-Receptor pairs and their downstream signaling components. Dot size represents number of averaged cells with probability scores between clusters. c Cell-cell communication networks predicted for the entire WNT pathway or for (d) individual Cluster networks from B. Edge weights represent the probability of signaling between cell clusters.
Fig. 3 Cell-cell interaction modeling of the WNT signaling pathway. a Hierarchical clustering of similar cell-cell signaling probability scores from library 3 and visualized on a tSNE plot. b Visualization of signaling probability scores of Ligand-Receptor pairs and their downstream signaling components. Dot size represents number of averaged cells with probability scores between clusters. c Cell-cell communication networks predicted for the entire WNT pathway or for (d) individual Cluster networks from B. Edge weights represent the probability of signaling between cell clusters.CC BY · PubMed Central
Fig. 4 Keratinocyte differentiation trajectories highlight epigenetic modifiers in epidermal homeostasis. a Epidermal keratinocyte subclustering using SoptSC. b Pseudotime inference of epidermal keratinocytes. Cell lineage inference displayed on the right. Edge weights denote probability of transition to each cluster. Dot size denotes number of cells. c Cell ID vs. pseudotime. Dot size denotes number of cells in pseudotime score. d Cellular Entropy (ξ) of epidermal keratinocytes plotted in a Waddington landscape. Clusters color-coded as in A. Blue and black arrows show predicted low energy paths along differentiation. e RNA velocity of epidermal keratinocytes using 2500 vectors. Quantification of Cellular Entropy displayed on the right. Cells or clusters color-coded as in A. f Rolling wave plot showing pseudotime-dependent gene expression dynamics. Average gene expression dynamics of each cluster pattern shown on the right and independently color-coded. g Rolling wave plots showing pseudotime-dependent gene expression dynamics of transcription factors (TF) and epigenetic modifiers (EpiGen). h Feature plots showing expression of UHFR1 and HELLS and their expression along pseudotime. Expression along epidermal cell lineage displayed at the bottom. i Knock-down (KD) of UHFR1 and HELLS, with a scramble control, in human skin equivalent organoids grown for 16 days. Hematoxylin and eosin (H&E) staining shown at top. Scale bar 100 µm. KI67 (red) and DAPI staining shown at the bottom. Scale bar 50 µm. Dotted lines denote the position of the basement membrane. n = 6 experiments each condition. j Quantification of the total cells per squared area (***p < 0.0001; n = 12 areas [HELLS] or 13 areas [UHRF1]), (k) KI67 + cells per 200 µm area along the basement membrane (**p = 0.0012; ***p < 0.0001; n = 12 areas [HELLS] or 19 areas [UHRF1]), and (l) qPCR of the respective transcripts for each KD condition (*p < 0.0292; n = 2–4 experiments each condition). For whisker and box plots: box represents 25th to 75th percentiles, whiskers represent minimum and maximum data points, and bar represents mean. For scatter plot: data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed t test.
Fig. 4 Keratinocyte differentiation trajectories highlight epigenetic modifiers in epidermal homeostasis. a Epidermal keratinocyte subclustering using SoptSC. b Pseudotime inference of epidermal keratinocytes. Cell lineage inference displayed on the right. Edge weights denote probability of transition to each cluster. Dot size denotes number of cells. c Cell ID vs. pseudotime. Dot size denotes number of cells in pseudotime score. d Cellular Entropy (ξ) of epidermal keratinocytes plotted in a Waddington landscape. Clusters color-coded as in A. Blue and black arrows show predicted low energy paths along differentiation. e RNA velocity of epidermal keratinocytes using 2500 vectors. Quantification of Cellular Entropy displayed on the right. Cells or clusters color-coded as in A. f Rolling wave plot showing pseudotime-dependent gene expression dynamics. Average gene expression dynamics of each cluster pattern shown on the right and independently color-coded. g Rolling wave plots showing pseudotime-dependent gene expression dynamics of transcription factors (TF) and epigenetic modifiers (EpiGen). h Feature plots showing expression of UHFR1 and HELLS and their expression along pseudotime. Expression along epidermal cell lineage displayed at the bottom. i Knock-down (KD) of UHFR1 and HELLS, with a scramble control, in human skin equivalent organoids grown for 16 days. Hematoxylin and eosin (H&E) staining shown at top. Scale bar 100 µm. KI67 (red) and DAPI staining shown at the bottom. Scale bar 50 µm. Dotted lines denote the position of the basement membrane. n = 6 experiments each condition. j Quantification of the total cells per squared area (***p < 0.0001; n = 12 areas [HELLS] or 13 areas [UHRF1]), (k) KI67 + cells per 200 µm area along the basement membrane (**p = 0.0012; ***p < 0.0001; n = 12 areas [HELLS] or 19 areas [UHRF1]), and (l) qPCR of the respective transcripts for each KD condition (*p < 0.0292; n = 2–4 experiments each condition). For whisker and box plots: box represents 25th to 75th percentiles, whiskers represent minimum and maximum data points, and bar represents mean. For scatter plot: data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed t test.CC BY · PubMed Central
Fig. 5 Subclustering basal stem cells point to the BAS-I cluster as essential to epidermal homeostasis. a Subclustering basal clusters (BAS-I – BAS-IV) using SoptSC. b Heatmap showing top 100 differentially expressed genes per cluster. c Correlation between overlapping differentially expressed genes from basal clusters and subclustering of basal clusters (kBAS) in A. d Pseudotime inference of kBAS keratinocytes. e RNA velocity of kBAS keratinocytes using 1000 vectors. Cells color-coded as in A. f Relative gene expression of KRT14 and KRT10 across the kBAS subclusters. g Differentiation score of kBAS subclusters using average gene expression of GRN genes. h Violin plot of relative gene expression of PTTG1 and RRM2 across kBAS subclusters. i Relative mRNA expression of indicated genes along Ca2+-induced differentiation of primary human keratinocytes. n = 3 experiments each time point (n = 2 for PCNA). j Coimmunostaining of PTTG1 (red) and RRM2 (green) in human neonatal skin. Green arrowheads highlight RRM2-only expressing cells; yellow arrowheads point to PTTG1-RRM2 double-positive cells. White dotted line denotes basement membrane. Scale bar 50 µm. n = 3. k Knock-down (KD) of PTTG1 and RRM2, with a scramble control, in human skin equivalent organoids grown for 16 days. Hematoxylin and eosin (H&E) staining shown. Dotted lines denote basement membrane. Scale bar 100 µm. n = 4 experiments each condition. l Quantification of total cells per squared area (*p = 0.0104; ***p < 0.0001; n = 12 areas each [PTTG1 and RRM2 KD]), (m) KI67 + cells per 200 µm area along the basement membrane (*p = 0.0123; ***p < 0.0001; n = 26 areas [PTTG1] or n = 25 areas [RRM2]), and (n) qPCR of the respective transcripts for each KD condition (PTTG1: p = 0.0056, 0.0018; RRM2: p = 0.0485, 0.025; ASS1: p = 0.0025; GJB2: p = 0.0037; PCNA: p = 0.0339, 0.0295; n = 3 experiments each [PTTG1 and RRM2 KD]). For whisker and box plots: box represents 25th to 75th percentiles, whiskers represent minimum and maximum data points, and bar represents mean. For bar graph: error bar represents SEM. For scatter plot: data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed t test.
Fig. 5 Subclustering basal stem cells point to the BAS-I cluster as essential to epidermal homeostasis. a Subclustering basal clusters (BAS-I – BAS-IV) using SoptSC. b Heatmap showing top 100 differentially expressed genes per cluster. c Correlation between overlapping differentially expressed genes from basal clusters and subclustering of basal clusters (kBAS) in A. d Pseudotime inference of kBAS keratinocytes. e RNA velocity of kBAS keratinocytes using 1000 vectors. Cells color-coded as in A. f Relative gene expression of KRT14 and KRT10 across the kBAS subclusters. g Differentiation score of kBAS subclusters using average gene expression of GRN genes. h Violin plot of relative gene expression of PTTG1 and RRM2 across kBAS subclusters. i Relative mRNA expression of indicated genes along Ca2+-induced differentiation of primary human keratinocytes. n = 3 experiments each time point (n = 2 for PCNA). j Coimmunostaining of PTTG1 (red) and RRM2 (green) in human neonatal skin. Green arrowheads highlight RRM2-only expressing cells; yellow arrowheads point to PTTG1-RRM2 double-positive cells. White dotted line denotes basement membrane. Scale bar 50 µm. n = 3. k Knock-down (KD) of PTTG1 and RRM2, with a scramble control, in human skin equivalent organoids grown for 16 days. Hematoxylin and eosin (H&E) staining shown. Dotted lines denote basement membrane. Scale bar 100 µm. n = 4 experiments each condition. l Quantification of total cells per squared area (*p = 0.0104; ***p < 0.0001; n = 12 areas each [PTTG1 and RRM2 KD]), (m) KI67 + cells per 200 µm area along the basement membrane (*p = 0.0123; ***p < 0.0001; n = 26 areas [PTTG1] or n = 25 areas [RRM2]), and (n) qPCR of the respective transcripts for each KD condition (PTTG1: p = 0.0056, 0.0018; RRM2: p = 0.0485, 0.025; ASS1: p = 0.0025; GJB2: p = 0.0037; PCNA: p = 0.0339, 0.0295; n = 3 experiments each [PTTG1 and RRM2 KD]). For whisker and box plots: box represents 25th to 75th percentiles, whiskers represent minimum and maximum data points, and bar represents mean. For bar graph: error bar represents SEM. For scatter plot: data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed t test.CC BY · PubMed Central

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Authors
Wang, S., Drummond, M. L., Guerrero-Juarez, C. F., Tarapore, E., MacLean, A. L., Stabell, A. R., Wu, S. C., Gutierrez, G., That, B. T., Benavente, C. A., Nie, Q., & Atwood, S. X.
Affiliations
University of California, Irvine (US)
Corresponding author
  • Nie, Qing
  • Atwood, Scott X.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-08-25
Volume
11
Issue
1
Pages
4239-4239
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-18075-7
PMC
PMC7447770
OpenAlex
W3081219157
Cited by
218
References
131
Citations by year
2026: 32 · 2025: 40 · 2024: 47 · 2023: 38 · 2022: 37 · 2021: 18 · 2020: 6
FWCI
28.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
  • Concern Foundation (CF204525)
  • U.S. Department of Health & Human Services | National Institutes of Health (P30AR075047)
Topics
  • Health Sciences › Medicine › Dermatology › Hair Growth and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
Keywords
basal stem cells, interfollicular epidermis, epidermal stratification, epidermal homeostasis, intercellular communication, pseudotime, RNA velocity, skin equivalents, rete ridges
MeSH terms
Epidermal Cells, Cell Communication, Cell Differentiation, Epidermis, Gene Expression Regulation, Homeostasis, Humans, Infant, Newborn, Male, Models, Biological, Stem Cells, Signal Transduction, Keratinocytes, Cell Lineage, Gene Expression Profiling, Foreskin

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