Science - USA (2022-04-15)

Capolupoet al.,Science 376 , eabh1623 (2022) 15 April 2022 6 of 12

AB

Cycle G1/S

Cycle G1

Cycle G2

Cycle M

Cycle M/G1

Middle

Basal

Inflammatory1

Vascular Fibrogenic1

Fibrogenic2

Contractile

Inflammatory2

Vascular

fibrogenic

Smooth Muscle

Fibrolytic2

Fibrolytic1

UMAP1

UMAP2

BasalCycle G1Cycle G1/SCycle G2Cycle MCycle M/G1MiddleContractileFibrogenic1Fibrogenic2Smooth MuscleVascularfibrogenicInflammatory1Inflammatory2Fibrolytic1Fibrolytic2Vascular

Basal

Cycle G1/SCycle G1

Cycle G2Cycle M

Cycle M/G1Middle

Contractile

Fibrogenic1Fibrogenic2

V. fibrogenicSm. Muscle

Immuno1Immuno2

Fibrolytic1

Fibrolytic2Vascular

Basal Proliferative Fibrogenic InflammatoryFibrolyticVascular

CAV1

S100A10

ADIRFMCM4MCM3HELLS

HIST1H4CHIST1H1B

RRM2KPNA2PLK1AURKACCNB1PTTG1CDC20TUBA1B

HNRNPA1

HNRN

PA2B1GTSE1UBE2CTOP2AGAPDHMYL9PFN1COL1A1

LBH

PS

AT1

CALD1CHRM2

COL12A1RSPO4OLFM2COL12A1

THBS2

COL12A1 P

PAP2BMXRA5SOD2CCL2TNFAIP2ISG15

MX1IFIT1

NEAT1CTSKMMP14MMP1SOD2STC1PTGIS

FN1FGF7

% of c

ells

expression

in

group

PROLIFERATIVE

BASAL

INFLAMMATORY FIBROGENIC

FIBROLYTIC

VASCULAR

Diffusion

map

PAGA

graph

DC1

DC3

DC2

C

D

H

ACTA2

LMNA

0 40

UMI counts

0 60

UMI counts

C

h

Tx

B

A

CT

A2

ACTA2

ShTxB

1 a

LM

N

A

LM

N

A

C

hT

xB

Sh

TxB2

e

IJ

E F

G

Triple+ ShTxB1a+/2e+

ChTxB+ ShTxB2e+

A

vg

. Z
-s
core

0.75

Signatures -0.75

IGF2

ACANINMT

HAPLN3ACVR2A

IGFBP7SFRP1

VCAM1MEG3

SERPINB2

KRTAP1-5NEAT1

ADAM33APP

MMP14TWIST2

CPS1

EFNA1DSG2

CCAT1KRT17

SLC7A2NPTX1

FAM83A

CELF4HAS2

CAPZA1GREB1

FAM76APEG3

B3GALT1CHIT1

C

hTxB/ShTxB1a

+

ChTxB

+

ShTxB2e

+

ShTxB1a

+/2e

+

Top Genes

RNA-seq

condition

Av

g. R

PM

0

Max

Avg. Z-score

% cells

| Z-score | > 0.2

Fibrogenic 1

V. fibrogenic

Fibrogenic 2

Sm. Muscle

Fibrolytic 1

Inflammatory 2

Fibrolytic 2

Contractile

Inflammatory 1

Basal

Vascular

Cycle M/G1

Middle

Cycle G1

Cycle G1/S

Cycle G2

Cycle M

-0.25

0

0.25

15%

30%

60%

Triple

+

ChTxB

+

ShTxB2e

+

ShTxB1a

+/2e

+

Signatures

States

C

hT

xB

+

Triple

Sh

T

xB2e

+

S

h

T

xB1a

+/2e

+

(^10080)
(^6040)
20
Fig. 4. Lipotype mapping to transcriptional cell states.(A) UMAP embedding
analysis of scRNA-seq of 5652 individual dHFs colored by the assigned cluster.
(B) Gene expression dot plot of cluster marker genes. Genes for each cluster were
identified using the Wilcoxon rank-sum test. (C) Diffusion map visualization of single
dHF cells from (A) highlighting the axes of transcriptional variation among the different
cell states. (D) PAGA applied to scRNA-seq data of control dHFs. Nodes indicate
cell type states, and the length of edges indicates the degree of similarity between
states, with shorter edges corresponding to greater state similarity. (E) Heatmap
reporting the average gene expression of enriched genes for each of the FACS-sorted
lipotype populations (bulk RNA-seq data). For each lipotype, the top eight genes,
ranked by fold change, are shown. (F) UMAP embedding colored by the different
lipotype gene signature scores. The 250 top differentially expressed genes were
used to calculate the signature score. (G) Dot plot colored by the average lipotype
z-score of cells of the different clusters. Size of the dots represents the number
of cells with magnitude of the score >0.35. (H) PAGA applied to scRNA-seq data of
control dHFs and based on the ShTxB2e+, ShTxB1a/2e+, ChTxB+, and triple+
lipotype signatures. Nodes are positioned corresponding to cell states in (D).
Color of nodes corresponds toz-score signatures, with a positivez-score (red)
indicating a greater correspondence of the particular cell type to the particular
lipotype state. Color bar is the same as in (G). (I) UMAP embedding of dHFs
colored by the expression of the two canonical markers for fibrogenic (ACTA2) and
basal (LMNA) cell states. (J) Confocal micrographs of cells stained with ShTxB1a
(green), ShTxB2e (red), and ChTxB (blue) and counterstained by antibodies
against ACTA2 and LMNA (magenta). Scale bar, 100mm.
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