Identification of a mesenchymal progenitor cell hierarchy ...€¦ · distinct mesenchymal cell...

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RESEARCH ARTICLE SUMMARY DEVELOPMENTAL BIOLOGY Identification of a mesenchymal progenitor cell hierarchy in adipose tissue David Merrick*, Alexander Sakers*, Zhazira Irgebay, Chihiro Okada, Catherine Calvert, Michael P. Morley, Ivona Percec, Patrick SealeINTRODUCTION: Adipose tissue is a highly plastic organ that plays a central role in regulating whole-body energy metabolism. The capacity for adipocytes to store chemical energy in lipid droplets protects other organs against the toxic effects of ectopic lipid dep- osition. In the setting of chronic nutritional excess, such as obesity, adipose tissue expands through increases in fat cell size (hypertrophy) and/or fat cell number (hyperplasia). Adipocyte hypertrophy causes fibrosis and inflammation, thereby promoting metabolic disease. Con- versely, hyperplastic growth, mediated by the differentiation of progenitor cells into new adipocytes, is critical for preserving adipose tissue function and protecting against meta- bolic disease. However, the precise cell types, lineage dynamics, and mechanisms governing the development of adipocytes are incompletely understood. RATIONALE: Adipose mesenchymal pro- genitors constitute a heterogeneous pool of diverse cell types. Previous attempts to char- acterize these cells have relied on nonspecific mesenchymal markers and candidate lineage marker genes. We used single-cell RNA se- quencing to identify distinct types of progen- itor cells in murine and human adipose tissues and to predict lineage relationships in an un- biased manner. Functional assessments of these cell types in vitro and in vivo define a me- senchymal cell hierarchy involved in adipocyte formation. RESULTS: Single-cell RNA sequencing and cell trajectory analyses identified a lineage hi- erarchy consisting of several distinct mesen- chymal cell types in murine and human adipose. Dipeptidyl peptidase4expressing (DPP4+) cells are highly proliferative and multipotent progenitors that are relatively resistant to dif- ferentiation into adipocytes. Intercellular adhe- sion molecule1expressing (ICAM1+) cells are committed preadipocytes that express Pparg and are poised to differentiate into mature adipocytes with minimal stimulation. CD142+/ Clec11a+ cells represent a distinct adipogenic population in murine subcutaneous adipose that shares many properties with ICAM1+ pre- adipocytes. In vivo cell transplantation studies showed that DPP4+ progenitors give rise to both ICAM1+ and CD142+ preadipocytes before differentiation into mature adipocytes. DPP4+ cells depend on transforming growth factorb (TGFb) signaling to maintain their progenitor identity, whereas ICAM1+ preadipocytes are re- fractory to the proliferative and anti-adipogenic actions of the TGFb pathway. Obesity and in- sulin resistance lead to a depletion of DPP4+ mesenchymal progenitors and a reduction in the adipogenic differentiation competency of preadipocytes, specifically in visceral white adi- pose tissue. Single-cell analysis of human sub- cutaneous adipose tissue revealed distinct DPP4+ and ICAM1+ populations that displayed func- tional properties similar to those of the analogous mouse populations. Histological examination of murine subcutaneous adipose tissue showed that ICAM1+ preadipocytes are intercalated between ma- ture adipocytes, occupying a perivascular niche. The DPP4+ progenitor cells are localized in an anatomically distinct niche surrounding the adipose depot, which we term the reticular interstitium. CONCLUSION: Our studies define a develop- mental hierarchy of adipose progenitors con- sisting of DPP4+ interstitial progenitors that give rise to committed ICAM1+ and CD142+ preadipocytes, which are poised to differen- tiate into mature adipocytes. Targeting one or more of these cell populations may be beneficial for promoting adaptive hyperplas- tic adipose growth to ameliorate metabolic disease. A key finding from this work is that adipose progenitor cells reside in the reticular interstitium, a recently appreciated, but not well-studied, fluid-filled network of collagen and elastin fibers that encases many organs, including adipose depots. Our results raise the possibility that DPP4+ cells, in addition to serving as progenitor cells for adipocytes in fat depots, may play important roles in the de- velopment and regeneration of other tissues. RESEARCH Merrick et al., Science 364, 353 (2019) 26 April 2019 1 of 1 *These authors contributed equally to this work. The list of author affiliations is available in the full article online. Corresponding author. Email: sealep@pennmedicine. upenn.edu Cite this article as D. Merrick et al., Science 364, eaav2501 (2019). DOI: 10.1126/science.aav2501 Adipocytes (Adipoq, Plin1) Insulin Adipocytes Reticular Interstitum (RI) RI DPP4 PREF1 50 µM Group 3 Cells (CD142, Clec11a) Interstitial Progenitor Cells (DPP4, Anxa3, Wnt2) Committed Preadipocytes (ICAM1, PREF1, Pparg) 50 µM DPP4+ progenitors reside in the reticular interstitium and give rise to committed preadipocytes. DPP4+ multipotent progenitor cells give rise to ICAM1+ and CD142+ committed preadipocytes, which are poised to differentiate into mature adipocytes (top). Committed preadipocytes (green) are intercalated between mature adipocytes,whereas DPP4+ progenitors (red) reside in the reticular interstitium, an anatomically distinct fluid-filled network of collagen and elastin fibers that encases many organs, including adipose depots. ON OUR WEBSITE Read the full article at http://dx.doi. org/10.1126/ science.aav2501 .................................................. on December 12, 2020 http://science.sciencemag.org/ Downloaded from

Transcript of Identification of a mesenchymal progenitor cell hierarchy ...€¦ · distinct mesenchymal cell...

Page 1: Identification of a mesenchymal progenitor cell hierarchy ...€¦ · distinct mesenchymal cell types that are present in both mouse and human adipose tissues. Cells marked by dipeptidyl

RESEARCH ARTICLE SUMMARY◥

DEVELOPMENTAL BIOLOGY

Identification of a mesenchymalprogenitor cell hierarchy inadipose tissueDavid Merrick*, Alexander Sakers*, Zhazira Irgebay, Chihiro Okada, Catherine Calvert,Michael P. Morley, Ivona Percec, Patrick Seale†

INTRODUCTION: Adipose tissue is a highlyplastic organ that plays a central role inregulating whole-body energy metabolism.The capacity for adipocytes to store chemicalenergy in lipid droplets protects other organsagainst the toxic effects of ectopic lipid dep-osition. In the setting of chronic nutritionalexcess, such as obesity, adipose tissue expandsthrough increases in fat cell size (hypertrophy)and/or fat cell number (hyperplasia). Adipocytehypertrophy causes fibrosis and inflammation,thereby promoting metabolic disease. Con-versely, hyperplastic growth, mediated by thedifferentiation of progenitor cells into newadipocytes, is critical for preserving adiposetissue function and protecting against meta-bolic disease. However, the precise cell types,lineage dynamics, and mechanisms governingthe development of adipocytes are incompletelyunderstood.

RATIONALE: Adipose mesenchymal pro-genitors constitute a heterogeneous pool ofdiverse cell types. Previous attempts to char-acterize these cells have relied on nonspecificmesenchymal markers and candidate lineagemarker genes. We used single-cell RNA se-quencing to identify distinct types of progen-itor cells inmurine and human adipose tissues

and to predict lineage relationships in an un-biasedmanner. Functional assessments of thesecell types in vitro and in vivo define a me-senchymal cell hierarchy involved in adipocyteformation.

RESULTS: Single-cell RNA sequencing andcell trajectory analyses identified a lineage hi-erarchy consisting of several distinct mesen-chymal cell types inmurine andhumanadipose.Dipeptidyl peptidase–4–expressing (DPP4+)cells are highly proliferative andmultipotentprogenitors that are relatively resistant to dif-ferentiation into adipocytes. Intercellular adhe-sion molecule–1–expressing (ICAM1+) cells arecommitted preadipocytes that express Ppargand are poised to differentiate into matureadipocytes withminimal stimulation. CD142+/Clec11a+ cells represent a distinct adipogenicpopulation in murine subcutaneous adiposethat sharesmany properties with ICAM1+ pre-adipocytes. In vivo cell transplantation studiesshowed that DPP4+ progenitors give rise toboth ICAM1+andCD142+preadipocytes beforedifferentiation into mature adipocytes. DPP4+cells depend on transforming growth factor–b(TGFb) signaling to maintain their progenitoridentity, whereas ICAM1+ preadipocytes are re-fractory to the proliferative and anti-adipogenic

actions of the TGFb pathway. Obesity and in-sulin resistance lead to a depletion of DPP4+mesenchymal progenitors and a reduction inthe adipogenic differentiation competency ofpreadipocytes, specifically in visceral white adi-pose tissue. Single-cell analysis of human sub-cutaneous adipose tissue revealed distinctDPP4+and ICAM1+ populations that displayed func-tional properties similar to thoseof the analogousmouse populations. Histological examination

of murine subcutaneousadipose tissue showed thatICAM1+preadipocytes areintercalated betweenma-tureadipocytes, occupyinga perivascular niche. TheDPP4+ progenitor cells

are localized in an anatomically distinct nichesurrounding the adipose depot, whichwe termthe reticular interstitium.

CONCLUSION: Our studies define a develop-mental hierarchy of adipose progenitors con-sisting of DPP4+ interstitial progenitors thatgive rise to committed ICAM1+ and CD142+preadipocytes, which are poised to differen-tiate into mature adipocytes. Targeting oneor more of these cell populations may bebeneficial for promoting adaptive hyperplas-tic adipose growth to ameliorate metabolicdisease. A key finding from this work is thatadipose progenitor cells reside in the reticularinterstitium, a recently appreciated, but notwell-studied, fluid-filled network of collagenand elastin fibers that encases many organs,including adipose depots. Our results raisethe possibility that DPP4+ cells, in addition toserving as progenitor cells for adipocytes in fatdepots, may play important roles in the de-velopment and regeneration of other tissues.▪

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Merrick et al., Science 364, 353 (2019) 26 April 2019 1 of 1

*These authors contributed equally to this work. The list ofauthor affiliations is available in the full article online.†Corresponding author. Email: [email protected] this article as D. Merrick et al., Science 364, eaav2501(2019). DOI: 10.1126/science.aav2501

Adipocytes(Adipoq, Plin1)

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RIDPP4 PREF1 50 µMGroup 3 Cells

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DPP4+ progenitors reside in the reticular interstitium and give rise to committed preadipocytes. DPP4+ multipotent progenitor cellsgive rise to ICAM1+ and CD142+ committed preadipocytes, which are poised to differentiate into mature adipocytes (top). Committedpreadipocytes (green) are intercalated between mature adipocytes, whereas DPP4+ progenitors (red) reside in the reticular interstitium, ananatomically distinct fluid-filled network of collagen and elastin fibers that encases many organs, including adipose depots.

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RESEARCH ARTICLE◥

DEVELOPMENTAL BIOLOGY

Identification of a mesenchymalprogenitor cell hierarchy inadipose tissueDavid Merrick1,2*, Alexander Sakers1,2*, Zhazira Irgebay1,2, Chihiro Okada1,2,Catherine Calvert3, Michael P. Morley4, Ivona Percec3, Patrick Seale1,2†

Metabolic health depends on the capacity of adipose tissue progenitor cells to undergode novo adipogenesis. The cellular hierarchy and mechanisms governing adipocyteprogenitor differentiation are incompletely understood. Through single-cell RNA sequenceanalyses, we show that the lineage hierarchy of adipocyte progenitors consists ofdistinct mesenchymal cell types that are present in both mouse and human adiposetissues. Cells marked by dipeptidyl peptidase–4 (DPP4)/CD26 expression are highlyproliferative, multipotent progenitors. During the development of subcutaneous adiposetissue in mice, these progenitor cells give rise to intercellular adhesion molecule–1(ICAM1)/CD54–expressing (CD54+) committed preadipocytes and a related adipogeniccell population marked by Clec11a and F3/CD142 expression. Transforming growthfactor–b maintains DPP4+ cell identity and inhibits adipogenic commitment of DPP4+ andCD142+ cells. Notably, DPP4+ progenitors reside in the reticular interstitium, a recentlyappreciated fluid-filled space within and between tissues, including adipose depots.

White adipose tissue (WAT) stores ex-cess nutritional energy in the form oftriglycerides, which can be released foruse in other tissues during periods ofenergy demand, such as fasting and

physical activity. The capacity for adipocytes tostore chemical energy in lipid droplets protectsother organs against the toxic effects of ectopiclipid deposition. Adipose tissue expands by in-creases in fat cell size (hypertrophy) and/or num-ber (hyperplasia). Hyperplastic growth, mediatedby the differentiation of progenitor cells (adipo-genesis), is critical for proper adipose tissue func-tion (1–3). Defects in this process cause adiposefibrosis and inflammation, contributing to sys-temic insulin resistance (4). Conversely, enhanc-ing adipogenesis preventsmaladaptive adipocytehypertrophy, reduces ectopic lipid accumulation,and ameliorates metabolic disease (5–7).Adipose progenitor cells have been isolated on

the basis of their expression of common progen-itor or mesenchymal cell surface markers, includ-ing platelet-derived growth factor receptor–a

(PDGFRa), CD29, CD34, stemcell antigen–1 (SCA1)/LY6A, and CD24 (8–20). In particular, CD24-expressing (CD24+) progenitor cells, which residein the vasculature, efficiently form functionaladipocytes when transplanted into lipodystrophicmice (21). Preadipocyte factor–1 (PREF1) [alsocalled delta-like homolog–1 (DLK1)] and the adi-pose lineage marker peroxisome proliferator–activated receptor–g (PPARg) are also reported tomark adipogenic cells that are closely associatedwith blood vessels in adipose tissue (18, 19). Lastly,mural and smooth muscle–related cells thatexpress Acta2 (encoding smooth muscle actin),Myh11, or Pdgfrb contribute to adipocyte forma-tion under certain conditions (5, 10, 16, 22, 23).However, the overlap, heterogeneity, and devel-opmental interrelationships among these andother described populations are incompletelyunderstood.

Profiling and trajectory analysis ofadipose progenitors

We applied single-cell RNA sequencing (RNA-seq) to identify and profile progenitor cells inan unbiased manner from the developing sub-cutaneous inguinal WAT (iWAT) of 12-day-old(p12) mice. At this stage, adipocytes are rapidlydeveloping from progenitor cells, allowing us tocapture the continuum of cell states spanningdifferentiation. Mature lipid-laden adipocytes,which are incompatible with the downstreamanalysis, were separated from stromal vascularcells (SVCs) by centrifugation. SVCs were thendepleted of CD45+ leukocytes and subjectedto single-cell RNA-seq (fig. S1). Unsupervised

clustering of the gene expression profiles iden-tified 10 cell types (Fig. 1A).Canonical mesenchymal progenitor markers

Cd34, Pdgfra, Ly6a (Sca1), and Thy1 (Cd90) wereexpressed predominantly in groups 1 to 3 (figs.S2 and S3). Group 1 cells (blue), which we named“interstitial progenitors,” were marked by the ex-pression ofDpp4 [encoding dipeptidyl peptidase–4(DPP4)], Wnt2, Bmp7, and Pi16 but did not ex-press adipocyte markers (Fig. 1B and figs. S2 andS3). Group 2 cells expressed Icam1 [encodingintercellular adhesion molecule–1 (ICAM1)] andDlk1 (Pref1), along with several adipocyte iden-tity genes, including Pparg, Fabp4, and Cd36,suggesting that these cells could be “committedpreadipocytes” (Fig. 1B and figs. S2 and S3).Group 3 cells expressed Clec11a and Fmo2, aswell as tissue factor (F3/CD142), which is amarker of “adipogenesis-regulatory cells” (Aregs)that were recently reported to inhibit adipo-genesis (24). A related cell cluster (group 4) wasmarked by Wnt6 and Sfrp5 expression but didnot showdetectable expression ofmesenchymalmarker genes, such as Pdgfra or Thy1. Group 7cells (yellow) were classified as “adipocytes”because of their high expression of matureadipocyte–specific genes (e.g., Adipoq, Plin1, andCar3) and lack of expression of progenitor mark-ers. These cells likely represent newly formedadipocytes without substantial lipid stores thatcopurified with the stromal cell fraction. TheSVC compartment of adult adipose tissue dis-plays similar cellular diversity, including prom-inent populations of mesenchymal cell groups 1to 3 (fig. S4).We examined potential precursor-product rela-

tionships between the identified mesenchymalpopulations by using in silico cell trajectoryanalyses. Pseudotemporal analysis predictedthat group 1 interstitial progenitors have twodevelopmental trajectories: group 2 preadipo-cytes terminating as group 7 adipocytes (cellfate A) and group 3 and 4 cells (cell fate B)(Fig. 1C and fig. S5). Group 3 cells appear torepresent an intermediary cell type betweengroup 1 interstitial progenitors and group 4 cells.Altogether, these analyses suggest that group1 progenitors (Dpp4+) provide a source of bothgroup 2 committed preadipocytes and group 3Cd142+ cells.

DPP4+ cells are multipotentmesenchymal progenitors

To investigate the function of these cell popula-tions, we developed fluorescence-activated cellsorting (FACS) strategies for the isolation ofgroup 1 [DPP4+, CD142(−)] cells (hereafter referredto as DPP4+ cells), group 2 [ICAM1+, CD142(−)]cells (hereafter referred to as ICAM1+ cells), andgroup 3 (CD142+) cells frommurine subcutaneousadipose tissue (see fig. S14 for the detailed sortingstrategy). The iWATs from p12 and adult (8- to10-week-old) mice contained similar proportionsof DPP4+, ICAM1+, and CD142+ cells (fig. S6).RNA-seqprofiling of freshly sortedDPP4+, ICAM1+,and CD142+ cells from p12 pups showed that thesecells expressed the same marker gene signatures as

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1Institute for Diabetes, Obesity and Metabolism, PerelmanSchool of Medicine at the University of Pennsylvania,Philadelphia, PA 19104, USA. 2Department of Cell andDevelopmental Biology, Perelman School of Medicine at theUniversity of Pennsylvania, Philadelphia, PA 19104, USA.3Division of Plastic Surgery, Perelman School of Medicine atthe University of Pennsylvania, Philadelphia, PA 19104, USA.4Penn Center for Pulmonary Biology, Perelman School ofMedicine at the University of Pennsylvania, Philadelphia, PA19104, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

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their respective single-cell groups (fig. S7), val-idating the cell purification methods. We nextassayed the differentiation and proliferativeactivities of these populations from adult mice,with treatments initiated within 48 hours ofcell isolation. All three populations underwentrobust adipocyte differentiation and activatedadipocyte-specific genes to comparably high

levels when treated with the standard completecocktail of adipogenic inducers (Fig. 2, A and C).However, under minimal adipogenic conditions,where the cocktail contained only a low concen-tration of insulin, ICAM1+ and CD142+ cellsdifferentiated efficiently into adipocytes (a resultsimilar to that seen with the complete cocktail)whereas DPP4+ cells displayed very low adipo-

genic capacity (Fig. 2B). ICAM1+ and CD142+cells expressed adipocyte-specific genes at ornear the levels observed in primary adipocytesisolated from adipose tissue, whereas DPP4+cultures displayed little to no induction of thesegenes (Fig. 2C).Classical features of mesenchymal progenitor

cells includeacapacity formultilineagedifferentiation

Merrick et al., Science 364, eaav2501 (2019) 26 April 2019 2 of 11

Fig. 1. Single-cell RNA-seq and cell trajectory analysis delineatethe lineage hierarchy of adipocyte progenitors. (A) Unsupervisedclustering of 11,423 cells (mean number of genes per cell = 1977)from the subcutaneous WAT of p12 pooled male and female C57BL/6Jmouse pups reveals 10 distinct cell groups represented on a tSNE map(relevant marker genes are listed in parentheses). (B) Individual gene tSNE

and violin plots showing the expression levels and distribution ofrepresentative marker genes. The y axis is the log-scale normalizedread count. (C) Pseudotemporal cell ordering of groups 1 to 4 andadipocytes along differentiation trajectories by using Monocle.Pseudotime (arbitrary units) is depicted from dark to light blue (left).Group identities were overlaid on the pseudotime trajectory map (right).

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and high proliferative activity. We found thatDPP4+ cells proliferated at a higher rate thanICAM1+ or CD142+ cells (Fig. 2D). Furthermore,the DPP4+ cell population displayed enhancedcompetence for differentiation into osteocytes,with higher induction of osteocyte-specific markergenes (Fig. 2E). Together, these data identifyDPP4+cells as highly proliferative multipotent progenitorspossessing many properties attributed to mesen-chymal stemcells. By contrast, ICAM1+andCD142+cells are relatively restricted to the adipocytelineage.

TGFb signaling maintains DPP4+progenitor cell identity

To identify signaling pathways regulating the di-vergent activities of DPP4+ and ICAM1+ cells, wecompared the bulk transcriptomes of freshlysorted DPP4+ cells and ICAM1+ cells by RNA-seq. Gene ontology (GO) analysis identified en-richment of the anti-adipogenic transforminggrowth factor–b (TGFb) andWNT signaling path-ways in DPP4+ cells (Fig. 3A) (8, 25). To assessthe importance of TGFb signaling for DPP4+cell activity, we treated freshly isolated DPP4+

cells with either recombinant TGFb or SB431542,a potent and specific TGFb receptor inhibitor.TGFb treatment induced the expression of manygroup 1 marker genes, including Dact2, Wnt10b,and Ptgs2 (Cox2), while also suppressing group 2and adipogenic marker genes (Fig. 3B). TGFb re-ceptor inhibition had the opposite effect, includ-ing strongly inducing group 2 and adipocytegenes in DPP4+ progenitor cells (Fig. 3B).At a functional level, TGFb treatment aug-

mented the proliferation of DPP4+ cells, whereasTGFb receptor antagonism suppressed prolifera-tion down to the levels of ICAM1+ cells (Fig. 3C).TGFb treatment inhibited adipocyte differentia-tion and suppressed the induction of adipocyte-specific genes in DPP4+ cells exposed to thecomplete induction cocktail (Fig. 3, D and E).By contrast, TGFb only mildly suppressed theadipogenic conversion of ICAM1+ cells and ex-erted an intermediate inhibitory effect on CD142+cells (Fig. 3, D and E). Reciprocally, the inhibitionof TGFb signaling enhanced adipocyte differentia-tion of DPP4+ cells exposed to minimal adipo-genic conditions but had no observable effect onICAM1+ or CD142+ cells (Fig. 3, F and G). Thus,

TGBb signaling regulates the identity and func-tional character of DPP4+ cells. Moreover, theseresults show that ICAM1+ cells are especially re-fractory to the anti-adipogenic effects of TGFb,reinforcing the notion that, among the mesen-chymal cell populations, ICAM1+ cells are themost highly committed to becoming adipocytes.Adipose tissue is organized into many discrete

depots that are distributed throughout the body.Flow cytometry analysis showed that the threemajor mesenchymal cell populations (DPP4+,ICAM+, andCD142+) identified in iWATwere alsopresent in subcutaneous axillaryWAT (axWAT),interscapular brown adipose tissue (iBAT), andvisceral epididymal WAT (eWAT) of adult mice(figs. S8 to S10). Of note, the relative proportionof DPP4+ cells was much lower in visceral WATthan in subcutaneous depots (iWAT or axWAT)(figs. S8 to S10). The cell populations isolatedfrom axWAT, eWAT, and iBAT displayed thesame pattern of adipogenic activity as those fromiWAT (fig. S8 to S10). Regardless of their depotof origin, ICAM1+ cells had greater adipo-genic competence and were more resistantto the inhibitory effects of TGFb than DPP4+

Merrick et al., Science 364, eaav2501 (2019) 26 April 2019 3 of 11

Fig. 2. DPP4+ progenitors display enhanced proliferative and multilineagedifferentiation capacity.Cells were isolated by using the following FACSstrategy: Lin− (CD45−, CD31−) cells were stained with anti-DPP4, anti-ICAM1,and anti-CD142. Groups were gated as follows: CD142+ cells were selectedfirst, followed by DPP4+ (CD142−, DPP4+) or ICAM1+ (CD142−, ICAM1+) cells.(A and B) Staining of adipocytes (with Bodipy lipid stain) (green) andquantification of adipogenesis in cell cultures from adult CD1 mice afterexposure to the complete adipogenic differentiation cocktail (A) or insulin

only (min) (B) [n=3biological replicates (BRs) per condition]. (C)mRNA levelsof adipocyte-specific genes in cultures from (A) and (B). Primary adipocytes(adipo) purified directly from adipose tissue were included for reference.(D) Quantification of cellular growth (representative of 3 BRs). (E)mRNA levelsof osteocyte-specific genes in cultures exposed to osteogenic differentia-tion inducers (n = 5 BRs). Statistical testing: not significant, P > 0.05;**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Dots represent BRs, and errorbars indicate SEM. Scale bars, 50 mM.

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cells (Fig. 3 and figs. S8 to S10). These resultssuggest thatDPP4+, ICAM1+, andCD142+mesen-chymal cells have conserved roles in all the majoradipose depots.

Reduced activity of visceral progenitorsin obese mice

We next examined the responses of these cellpopulations to adverse metabolic conditions.

DPP4+, ICAM1+, and CD142+ cells were isolatedfrom mice with diet-induced obesity that wereglucose intolerant (fig. S11A). The visceral eWATfrom obese animals had a lower proportion ofDPP4+ cells and a higher proportion of CD142+cells than that from lean controls (fig. S11B).ICAM1+ and CD142+ cells from the visceral de-pots of obese mice were less proliferative andhad lower adipogenic differentiation capacity than

corresponding cells from lean control animals (fig.S11, C andD).By contrast, ICAM1+andCD142+cellsfrom the subcutaneous iWAT of obese mice re-tained high adipocyte differentiation competency(fig. S11, E to G). These results imply that high-fat feeding and/or obesity depletes the DPP4+mesenchymal progenitor pool and impairs theadipogenic differentiation competency of pre-adipocytes, specifically in visceral WAT. Thus,

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Fig. 3. TGFb regulates the proliferation and adipogenic differentiationcompetency of multipotent DPP4+ progenitors. (A) RNA-seq andGO analysis of signaling pathways enriched in DPP4+ versus ICAM1+ cellsfrom pooled p12 pups (n = 3 BRs). Combined score = log P valuemultiplied by the z-score of deviation from the expected ranking.(B) mRNA levels of group 1, group 2, and adipocyte (adipo) marker genesin DPP4+ cells treated with vehicle control, TGFb, or the TGFb receptorinhibitor SB431542 (n = 4 BRs). (C) Quantification of cell growth incultures treated with TGFb or SB431542 (representative of 3 BRs).(D) Bodipy staining of adipocytes (green) differentiated with the completeinduction cocktail with or without TGFb treatment. Relative adipogenesis

is the adipogenic index of TGFb-treated cells relative to that of control cells(right) (n = 3 BRs). (E) Fold changes in mRNA levels of adipocyte-specificgenes in cultures from (D). (F) Bodipy staining of adipocytes (green)and quantification of differentiation in the indicated cultures (right) (n = 3 BRs).Relative adipogenesis is the adipogenic index of SB431542-treated cells(SB cpd) relative to that of control cells. Min, minimal cocktail(insulin only). (G) Adipocyte-specific mRNA levels in cultures from (F),displayed as fold change in treated cells relative to control cells. Scalebars, 50 mM. Statistical testing: not significant (ns), P > 0.05; *P ≤ 0.05;**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Dots represent BRs, anderror bars indicate SEM.

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A B C

D E

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Fig. 4. Human adipose contains analogous populations of DPP4+progenitors and ICAM1+ preadipocytes. (A) FACS isolation of cellpopulations from human adipose tissue (representative of eight individualdonors). Populations were purified as follows: Lin− (CD45−, CD31−) cells werestained with anti-DPP4, anti-ICAM1, and anti-CD142. CD142+ cells wereselected first, followed by DPP4+ (CD142−, DPP4+) and ICAM1+ (CD142−,ICAM1+) cells. DP, double positive (DPP4+, ICAM1+); DN, double negative(DPP4−, ICAM1−); FSC, forward scatter; k, thousand. (B) Quantification ofrelative progenitor abundance from n = 8 human tissue donors. (C) Relativeproliferation rates of human cells (n = 5 donors). (D) Bodipy staining ofadipocytes (green) in cultures treated with complete (top) or minimal (insulin

plus rosiglitazone) (bottom) induction cocktail. (E) Quantification of adipo-genic differentiation from cells shown in (D) (n = 6 donors). (F) Unsupervisedclustering of 11,338 cells (mean number of genes per cell = 1253) fromthe abdominal subcutaneous adipose tissue of a 31-year-old female donor(body mass index, 31.6) reveals five distinct cell groups represented ona tSNE map (relevant marker genes are in parentheses). (G) Individual genetSNE and violin plots showing the expression levels and distribution ofrepresentative marker genes.The y axis is the log-scale normalizedread count. Statistical testing: ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01;***P ≤ 0.001; ****P ≤ 0.0001. Dots represent BRs, and error bars indicateSEM. Scale bars, 50 mM.

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progenitor cell exhaustion and reduced precursordifferentiation may contribute to the pathologic re-modeling of visceral WAT, which is linked tometabolic disease progression.

Human adipose tissue containsanalogous stromal populations

An important question is whether analogous pop-ulations of adipose stromal cells are present inhumans. To address this, we used FACS to iden-tify and purify DPP4+, ICAM1+, and CD142+ cellsfrom fresh subcutaneous adipose tissue of humansubjects undergoing cosmetic plastic surgery(Fig. 4A). Human adipose tissue contained pro-portionally fewer DPP4+ cells than mouse adi-pose tissue (Fig. 4B).HumanDPP4+ cells, like theirmouse counterparts, displayed higher proliferativeactivity (Fig. 4C) and lower potential to differen-tiate into adipocytes than both ICAM1+ andCD142+ cells, especially under conditions of min-imal adipogenic stimulation (Fig. 4, D and E).Activation of TGFb signaling enhanced pro-liferation and profoundly inhibited adipocytedifferentiation in human DPP4+ cells, while

having less effect on ICAM1+ and CD142+ cells(fig. S12).To investigate the heterogeneity of human

adipose stromal cells in an unbiased manner,we performed single-cell expression profiling ofCD45-depleted SVCs from human abdominalsubcutaneousWAT. Clustering analysis of geneexpression profiles identified five cell populations,including twomain groups of cells (groups 1 and2) that expressedmesenchymalmarkersPDGFRa,PDGFRb, and SCA1. Group 1 cells selectively ex-pressed DPP4, CD55, andWNT2, comparable tomurine group 1 cells (Fig. 4, F and G, and fig. S13).Group 2 cells expressed ICAM1,PPARg, andGGT5,comparable to murine group 2 cells. However,murine group 3 markers CD142, CLEC11A, andFMO2 were broadly expressed across humangroups 1 and 2 (Fig. 4, F and G, and fig. S13).This suggests that human subcutaneous adiposetissue does not contain a distinct population ofCD142+ cells. In support of this, we did not de-tect any functional differences between sortedhuman ICAM1+ and CD142+ cells in our assays.We conclude that human and murine WATs

contain comparable DPP4+ and ICAM1+ cellpopulations with similar functional attributes.

DPP4+ progenitors produce ICAM1+ andCD142+ preadipocytes

To determine the in vivo fate of DPP4+, ICAM1+,and CD142+ cells in adipose tissue, we trans-planted mTomato-expressing DPP4+, ICAM1+,or CD142+ cells into the developing fat pads ofwild-type (WT) animals (Fig. 5 and fig. S14). Flowcytometry analyses of stromal cells isolated fromrecipient animals showed that DPP4+ cells ac-quired ICAM1 and CD142 expression as early asday 7 posttransplant and that a subset of theimplanted cells down-regulated DPP4 expressionby day 14 (Fig. 5A and fig. S15A). TransplantedICAM1+ cells acquired expression of CD142 butdid not produce substantial numbers of DPP4+cells (Fig. 5B and fig S15B). Transplanted CD142+cells did not acquire DPP4 expression, but asubset of these cells down-regulated CD142 andretained ICAM1 expression (Fig. 5C). Histologicexamination of the transplants after 50 days re-vealed robust development of mTomato+ mature

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Fig. 5. DPP4+ progenitors give rise to ICAM1+, CD142+ cells and matureadipocytes in vivo. (A) Sort-purified CD142(−), DPP4(+) and (B) CD142(−),ICAM1(+) cells from mTomato(+) donor mice were analyzed before trans-plantation (day 0) (left) into the subcutaneous adipose of 10-day-oldmTomato(−) recipientmice.Sevenand 14days after transplantation,mTomato(+)cells were recovered and analyzed for the expression of DPP4, ICAM1, andCD142 (shown is one representative transplant from n=4BRs). Fifty days after

transplantation, mTomato+ cells were visualized by immunofluorescence inrecipient iWAT (right) (representative image; n = 3 to 4 BRs). AF, auto-fluorescence (525/50 nM) showing host adipocytes. (C) Sort-purifiedCD142(−) cells were analyzed 14 days posttransplant for the expression ofDPP4, ICAM1, and CD142 (shown is one representative transplant fromn = 4 BRs).The left FACS plot depicts the CD142-positive gate, and the rightFACS plot shows analysis of cells from the CD142-negative gate.

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adipocytes from DPP4+, ICAM1+, and CD142+cells (Fig. 5, right panels). These results demon-strate that DPP4+ cells act as progenitors forboth ICAM1+ and CD142+ cells, with these pop-ulations further differentiating into mature adi-pocytes.Moreover, the results suggest that CD142+and ICAM1+/CD142− cells interconvert in vivo.

DPP4+ progenitors reside in thereticular interstitium

Lastly, given the divergent genetic and func-tional properties of DPP4+ and ICAM1+ cells, weexamined the anatomic relationship of thesepopulations by immunofluorescence staining forDPP4 and PREF1. PREF1 is another marker ofgroup 2 (ICAM1+) cells in developing adiposetissue that was also previously defined as a pre-

adipocyte marker (Fig. 1) (26). Histologicalexamination of transverse sections of iWATfrom2-day-old pups revealed amarked anatomicpartitioning of DPP4+ (group 1) and PREF1+(group 2) cells (Fig. 6A). PREF1+ cells wereintercalated between mature adipocytes, dis-tributed throughout the central body of theiWAT. By contrast, DPP4+ cells were localizedin the reticular interstitium (RI) (Fig. 6A, inset 2),a recently appreciated fluid-filled network ofcollagen and elastin fibers that encases manyorgans, including adipose depots (27). Many cellscoexpressing DPP4 and PREF1 were found at theleading edge of p2 iWAT, potentially represent-ing DPP4+ cells in the process of transiting toICAM1+ cells during adipogenesis (Fig. 6A,inset 1). At even earlier stages of development,

doubly positive cells were more prominentthroughout the body of the nascent iWAT depot(fig. S16). Staining in adult murine adipose tissuefor DPP4 along with the other group 1 markersANXA3 and CD55 shows a similar localizationof these cells in the RI (fig. S17). Previous reportsshow that preadipocytes and CD142+ cells occupya perivascular niche, an anatomically distinctstructure, characterized by a collagen-rich extra-cellular matrix, similar to the RI (16, 18, 24). No-tably, DPP4+ interstitial progenitors are excludedfrom the perivascular compartment and lack theexpression of classic pericyte markers (fig. S18).

Discussion

In this study, we identified and functionallyassessed threemajormesenchymal cell groups in

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Fig. 6. DPP4+ progenitorcells reside in the RI.(A) Full-thickness flanktissue from a 2-day-oldmouse pup was cross-sectioned. Murine skin andadipose were stained withhematoxylin and eosin(H&E) with labels localizingthe dermis (D), dermalfibroblasts (DF), thepanniculus carnosus (PC),the RI, and adipocytes(Ad). Anti-DPP4 (red),anti-PREF1 (green), andDAPI (blue) were used forimmunofluorescence.(Inset 1) Magnification ofthe leading edge of thedeveloping iWAT. (Inset 2)Cross-sectional magnifica-tion of the body of theiWAT. Arrows showexamples of DP (DPP4+,PREF1+) cells. The arrow-heads point to DPP4+,PREF1(−) cells. (B) Modeldepicting the lineagehierarchy relationships ofthe indicated cell types.(C) Model depicting theanatomical relationships ofthe indicated cell typesat the leading edge ofdeveloping adipose tissue.

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murine adipose: group 1 DPP4+ interstitial pro-genitor cells, group 2 ICAM1+ preadipocytes, andgroup 3 CD142+/Clec11a+ cells. Recent reports byother investigators using similar methods iden-tified analogous cell groups (24, 28, 29), demon-strating that these cell populations are presentacross a wide variety of adipose sources fromanimals of different ages and metabolic statuses.The high overall concordance between studieshighlights the robustness of single-cell RNA-seqtechnology and strengthens the notion that theidentified cell groups represent a general pa-radigm for adipose progenitor biology.Burl et al. performed single-cell RNA-seq on

SVCs from adult mouse iWAT and eWAT andidentified populations similar to those we foundin p12 pups (28). Specifically, the group-defininggenes for ASC2 (DPP4+/Pi16+) cells overlap exten-sivelywith the genes defining our group 1 (DPP4+)population, and the ASC1 (ICAM1+/Col4a2+)group is similar to our group 2 (ICAM1+) cells(fig. S19). Hepler et al. defined twomain subtypesof Pdgfrb-expressing (mural) cells inmurine eWAT:fibroinflammatory precursors (FIPs), whichexpress many markers overlapping with those ofour group 1DPP4+ cells, and adipocyte precursorcells, which are similar to our group 2 ICAM1+cells (fig. S20) (29). FIPs from visceral adiposehave low adipogenic differentiation capacity.However, the authors noted that theFACS strategyused to isolate FIPs from eWAT (with PDGFRb+,LY6a+, and CD9+ markers) did not identify ananalogous population in subcutaneous adipose.The three markers used to purify FIPs are notspecifically enriched in group 1 DPP4+ cells (fig.S20, top three genes), although many other FIP-definingmarkers are highly concordant betweenthe populations. Therefore, FIPs may represent asubset of DPP4+ cells that are present only in thevisceral eWAT depot.Schwalie et al. identify three cell clusters (24)

that largely coincide with the groups that westudied (fig. S21). Schwalie population 1 (P1) ex-presses a gene signature similar to that of ourgroup 1 (DPP4+) cells, P2 corresponds to ourgroup 2 (ICAM1+) cells, and P3 corresponds toour group 3 (CD142+/Clec11a+) cells. In agree-ment with Schwalie et al., we found that therewas a higher proportion of CD142+ cells in eWATthan in iWAT and in obese versus lean adiposetissue (fig. S11). However, a notable discrepancyconcerns the functional properties of CD142+group 3 and P3 cells. Schwalie et al. show thatmouse and human P3 (CD142+) cells, which theynamed Aregs, have low adipogenic potential andare also capable of inhibiting the differentiationof preadipocytes. By contrast, our study indicatesthat these cells are highly adipogenic both in vitroand upon transplantation in vivo. This divergencelikely stems from key differences in our respectiveFACS strategies for cell purification. For many oftheirmouse studies, Schwalie et al. used ABCG1 inaddition to CD142 to enrich for the P3 population.Examination of the expression pattern of Abcg1in our single-cell data shows almost no mRNAdetectable in groups 1 to 3 (fig. S21). We per-formed extensive validation of our flow sorting

strategy by bulk RNA-seq analyses, showing thatthe CD142+ cells we isolated were highly en-riched for expression of the corresponding group3–defining genes (fig. S7). Moreover, our analysesof human adipose reveal that CD142 and ICAM1are largely coexpressed in the same population ofadipogenic cells (Fig. 4). Together, these resultsdemonstrate that group 3 cells are an adipogenicpopulation, related to ICAM1+ group 2 cells, inthe adipose lineage. We speculate that the anti-adipogenic activity attributed to P3 CD142+ cellsby Schwalie et al. may reside in a different celltype captured in their flow sorting strategy.In conclusion, we define a developmental hi-

erarchy of adipose progenitors that are activeduring early murine adipogenesis, and we sug-gest that this may be a general paradigm foradipogenesis in bothmice and humans. In younganimals, DPP4+ interstitial progenitors produceCD142+ cells and ICAM1+ preadipocytes that arepoised to differentiate into adipocytes (Fig. 6B).We speculate that, in adults, DPP4+ cells under-go adipose lineage commitment in response tovarious stimuli, providing a renewable source ofpreadipocytes. It is also conceivable that othercell populations in adult adipose depots, includ-ing ICAM1+ or CD142+ cells residing within theperivascular niche, mediate tissue turnover andremodeling without further contribution fromDPP4+ cells. Future genetic lineage–tracingstudies will ascertain whether DPP4+ cells arethe major or exclusive progenitor source of adi-pocytes during development and in adult animals.A particularly notable finding is that adiposeprogenitor cells reside in a recently discoveredanatomic niche (Fig. 6C). The RI is a large tissuethat surrounds many organs but has not beenwell studied. Our results raise the possibility thatin addition to serving as a reservoir for adipocyteprogenitor cells in fat depots, the RI plays im-portant roles in the development and regenera-tion of other tissues. Lastly, the identification offunctionally distinct precursor populations couldpotentially inform the development of more tar-geted approaches to promote metabolically ben-eficial adipose growth.

Materials and MethodsHuman adipose tissue samples

All human adipose tissue samples were obtainedfrom living donors undergoing cosmetic surgery.Tissue samples consisted of 1- to 10-kg intactblocks of discarded tissue, including skin andsubcutaneous adipose, from procedures to re-move excess skin and adipose tissue from theabdomen and flanks. Collection of tissue andprocessing were initiated within 1 to 3 hours ofremoval from the patient. This work was per-formed under the approval of InstitutionalReview Board protocols 812150 and 824825.Human subject demographics are outlined

in fig. S22.

Isolation of SVCs from humansubcutaneous adipose

The skin and cauterized edges of human tissuesamples were removed to yield an intact block of

subcutaneous fat that was unmanipulated by thesurgical procedure. Three hundred to five hun-dred grams of tissue was manually minced anddigested with collagenase D (0.75 unit/ml; Roche)and dispase II (1.2 units/ml; Roche) in Dulbecco’smodified Eagle’s medium (DMEM) with Ham’sF12 medium (DMEM/F12) containing 0.4% fattyacid–free bovine serum albumin (Sigma) at 37°Cwith agitation for 45 min and shaking for 10 s at15-min intervals. Digestion was performed at aratio of 1 g of tissue to 1 ml of digestion medium.The digestion was quenched with DMEM con-taining 10% fetal bovine serum (FBS), and thedissociated cells were filtered twice through asingle layer of gauze to remove large undigestedparticles and then subjected to centrifugation at400 × g for 5 min at room temperature (RT). Theresulting supernatant containing mature adipo-cytes was aspirated, and the pellet, consisting ofSVCs, was resuspended, passed through a 100-mMfilter, and subjected to centrifugation at 400 × gfor 5 min at RT. Cells were resuspended in redblood cell lysis buffer (Biolegend) for 5 min at RTand then quenched in DMEM containing 10%FBS. Cells were then passed through a 40-mMfilter and collected by centrifugation at 400 × gfor 5 min. Cells were recovered in FACS buffer[Hanks’ balanced salt solution (HBSS) contain-ing 3% FBS; Fisher] and kept on ice for the dura-tion of processing.

Animals

C57BL/6J WT [stock number (no.) 000664] andC57BL/6J:ROSAmT/mG (stock no. 007676) mouselines were obtained from Jackson Laboratories.CD1WTmice (stock no. 022) were obtained fromCharles River. 129SVE mice (stock no. 129S6/SvEvTac) were obtained fromTaconic. Mice withdiet-induced obesity (stock no. 380050) and con-trol animals (stock no. 380056) were obtainedfrom Jackson Laboratories and maintained ontheir respective diets until harvest. All animalprocedures were performed under the guidanceof the University of Pennsylvania InstitutionalAnimal Care and Use Committee.

Isolation of SVCs from mouse adipose

Inguinal and axillary subcutaneous white adi-pose depots were surgically removed from WTmice and processed for SVC enrichment. Briefly,adipose tissues were manually minced and di-gested with collagenase D (1.5 units/ml; Roche)and dispase II (2.4 units/ml; Roche) in DMEM/F12 containing 0.8% fatty acid–free bovine serumalbumin (Sigma) at 37°Cwith agitation for 45minand vortexing for 10 s at 15-min intervals. Thedigestionwas quenchedwithDMEM/F12 contain-ing 10% FBS, and the dissociated cells were passedthrough a 100-mM filter and then subjected tocentrifugation at 400 × g for 5 min. The result-ing supernatant containing mature adipocyteswas aspirated, and the pellet, consisting of SVCs,was resuspended in red blood cell lysis buffer(Biolegend) for 5 min at RT and then quenchedin DMEM/F12 containing 10% FBS. Cells werethen passed through a 40-mM filter and collectedby centrifugation at 400 × g for 5 min. Cells were

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recovered in FACS buffer (HBSS containing 3%FBS; Fisher).

FACSMouse studies

SVCs from the subcutaneous adipose of ~20 to30 mice (p10 to p20) or 10 to 20 mice (8 weeksold or older) were pooled and resuspended inFACS buffer for incubation with the followingantibodies for 30 min at 4°C: CD26 (DPP4)–fluorescein isothiocyanate (FITC) (Biolegend,San Diego, CA; catalog no. 137806; 1:200), anti–mouse ICAM1–phycoerythrin (PE)/Cy7 (Biolegendcatalog no. 116122; 1:100), anti–mouse CD45–allophycocyanin (APC)/Cy7 (Biolegend catalogno. 103116; 1:1000), anti–mouse CD31–APC-Fire(Biolegend catalog no. 102528; 1:1000), and anti–mouse CD142 (Sino Biological catalog no. 50413-R001; 1:100). Antibodies were preconjugatedwith Biotium Mix-n-Stain CF647 (Sigma catalogno. MX647S100). 4′,6-Diamidino-2-phenylindole(DAPI) (Roche catalog no. 10236276001; 1:10,000)was added for the final 5 min. The cells werewashed three times with FACS buffer to removeunbound antibodies. The cells were sorted witha BD FACS Aria cell sorter (BD Biosciences)equippedwith a 100-mmnozzle and the followinglasers and filters: DAPI, 405 and 450/50 nm;FITC, 488 and 515/20 nm; mTomato, 532 and610/20 nm; PE/Cy7, 532 and 780/60 nm; CF647,640 and 660/20 nm; and APC/Cy7 and APC-Fire,640 and 780/60 nm. All compensation was per-formed at the time of acquisition in Diva softwareby using compensation beads (BioLegend catalogno. A10497) for single-color staining and SVCs fornegative staining and fluorescence (DAPI andmTomato).

Human studies

SVCs were resuspended in FACS buffer for in-cubation with the following antibodies for 60minat 4°C: CD26 (DPP4)–FITC (Biolegend catalogno. 302704; 1:100), anti–mouse ICAM1–PE/Cy7(Biolegend catalog no. 353116; 1:100), anti–mouseCD45–APC/Cy7 (Biolegend catalog no. 368516;1:100), anti–mouse CD31–APC/Cy7 (Biolegendcatalog no. 303120; 1:100), and anti–mouse CD142–APC (Biolegend catalog no. 365206; 1:100). DAPI(Roche catalog no. 10236276001; 1:10,000) wasadded for the final 5 min. The cells were washedthree times with FACS buffer to remove un-bound antibodies. The cells were sorted witha BD FACS Aria cell sorter (BD Biosciences)equippedwith a 100-mmnozzle and the followinglasers and filters: DAPI, 405 and 450/50 nm;FITC, 488 and 515/20 nm; PE/Cy7, 532 and 780/40 nm; APC, 640 and 660/20 nm; and APC/Cy7,640 and 780/60 nm. All compensation was per-formed at the time of acquisition in Diva soft-ware by using compensation beads (BioLegendcatalog no. A10497) for single-color staining andSVCs for negative staining and fluorescence (DAPI).

Single-cell RNA-seq with 10× Genomicschromium platform

For both mouse and human studies, SVCs wereisolated and flow sorted with gating to isolate

single cells away from debris, doublets, and deadcells. For the p12 pups (pooled male and femaleC57BL/6 mice) and the human single-cell study,the cells were further gated against CD45 to ex-clude leukocytes. For the adult mouse thermo-neutral (pooled male 129S6/SvEvTac) sample,CD45 cells were segregated and remixed withthe SVCs at a ratio of ~20% of the total cells.The sorted cells were loaded onto a GemCodeinstrument (10× Genomics, Pleasanton, CA) togenerate single-cell barcoded droplets (GEMs)according to the manufacturer’s protocol withthe 10× single-cell 3′ v2 chemistry. The resultinglibraries were sequenced on an Illumina HiSeq2500 instrument with the HiSeq rapid sequenc-ing by synthesis (SBS) kit. The resulting readswere aligned, and gene-level unique molecularidentifier (UMI) counts were obtained by usingCell Ranger (Pipeline).The Cell Ranger single-cell software suite

v.2.0.1 (mouse studies) or v.3.0.1 (human studies)was used to perform sample demultiplexing, align-ment, filtering, and UMI counting (30). Clusteringand gene expression were visualized with theSeurat package (version 3.0) (31) on RStudio (32).Cells were first filtered to have >500 detectedgenes and less than 5% of total UMIs mappingto the mitochondrial genome. Clusters with veryfew cells were filtered before downstream anal-ysis. Data were scaled to mitigate the effects ofthe following variables: the number of genes de-tected per cell, the percentage of mitochondrialreads, and the cell cycle phase. Dimensionalityreduction was performed with the t-stochasticneighboring embeddingmethod (tSNE) by usingSeurat. Analysis of differential gene expressionamong clusters was performed by using theSeurat function FindMarkers with the Wilcoxtest. Violin plots, heatmaps, and individual tSNEplots for the given genes were generated by usingthe Seurat toolkit VlnPlot, DoHeatmap, andFeaturePlot functions, respectively.

Pseudotime analysis

Pseudotemporal analysis was performed on afiltered subset of clusters (groups 1 to 4 andadipocytes) from the p12 pups’ subcutaneousSVCs by using the Monocle R package (33). Order-ing genes were selected by using a cutoff of ex-pression in at least 10 cells and a combination ofintercluster differential expression and disper-sion with a q value cutoff of <1 × 10−10, whichproduced a list of 1027 genes. A split heatmapwas generated from selected genes showing sig-nificant change through pseudotime, high differ-ential expression, or known biologic identityby using the Monocle function plot_genes_branched_heatmap at branch_point 1.

RNA-seq library preparation and analysis

Subcutaneous SVCs from pooled C57BL/6 maleand female p10 to p16 pups were sorted as de-scribed above into DPP4+ and ICAM1+ popula-tions (Fig. 2) (GO analysis) or DPP4+, ICAM1+,and CD142+ populations (fig. S10) and collectedin TRIzol (Invitrogen). Total RNA was isolatedby using the RNeasy microkit (Qiagen). Three

independent biological replicates (BRs) were col-lected for each population. RNA concentrationandqualitywere assessed by usingNanodrop2000,Qbit, and Bioanalyzer RNA 2100 (Agilent; SantaClara, CA). All samples had an RNA integritynumber (RIN) greater than 7. Library prepara-tion and cDNA sequencing were performed byNovogene using paired-end 150–base pair reads(20 million reads per sample). FASTQ files werealigned by using STAR, and differential geneexpression was analyzed by using the R packageDESeq2.

Cell culture and differentiationMouse studies

DPP4+, ICAM1+, and CD142+ populations wereFACS purified; plated on Corning CellBind 384-,96-, 48-, or 24-well plates (Sigma-Aldrich); andcultured in DMEM/F12 containing 10% FBS andPrimocin (50 ng/ml) (InvivoGen catalog no.ant-pm-1). The cells were incubated for 24 to48 hours to facilitate attachment before the in-duction of either adipogenic or osteogenic differ-entiation. For adipogenic differentiation, cellswere plated at near confluence after sorting toensure the same number of cells per well foreach cell type at the start of adipogenic induc-tion. Adipogenic differentiation was carried outinDMEM/F12 containing 10%FBS and Primocin(50 ng/ml) with the addition of either a full adi-pogenic cocktail [20 nM insulin, 1 nM T3, 1 mMdexamethasone (Sigma catalog no.D4902), 0.5 mMisobutylmethylxanthine (Sigma catalog no. I7018),and 125 nM indomethacin] (complete) or aminimal adipogenic cocktail (20 nM insulin)(minimal). For the full adipogenic cocktail in-duction, cells were incubated for 2 days andthen transferred to adipogenic maintenance me-dium (20 nM insulin, 1 nMT3).Medium changeswere performed every 2 days, and cells wereanalyzed for all experiments within 4 to 6 days ofthe induction of differentiation. Osteogenic dif-ferentiationwas performedbyusing theMesenCultosteogenic stimulatory kit (Stem Cell Technologiescatalog no. 05504). Mediumwas changed every2 days, and the cells were analyzed after 8 daysof differentiation.

Human studies

DPP4+, ICAM1+, and CD142+ populations wereFACS purified; plated on Corning CellBind 384-,96-, or 48-well plates (Sigma-Aldrich); and firstcultured in PM-1 medium (Zenbio catalog no.PM-1) supplemented with 1 nM recombinantfibroblast growth factor 2 (FGF2) (Invitrogen,PHG0261) and Primocin (50 ng/ml). Cells wereplated at near confluence and incubated for 48to 72 hours to facilitate attachment before theinduction of adipogenic differentiation. Adipo-genic differentiation was carried out in DM-1medium (Zenbio catalog no. DM-1) containing3% FBS and Primocin (50 ng/ml) with the addi-tion of either a full adipogenic cocktail [20 nMinsulin, 1 nM T3, 1 mM dexamethasone (Sigmacatalog no. D4902), 0.5 mM isobutylmethylxan-thine (Sigma, I7018), and 1 nM rosiglitazonemaleate (Alexis Biochemicals, 350-103-G001)]

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(complete) or a minimal adipogenic cocktail(20 nM insulin, 1 nM rosiglitazone) (minimal).For the full adipogenic cocktail induction, cellswere incubated for 7 days and then transferredto adipogenic maintenance medium (20 nMinsulin, 1 nM T3). Medium was changed every7 days, and the cells were analyzed after 14 to18 days of differentiation.

Mouse and human studies

In some experiments, cultures were treated withrecombinant TGFb1 (10 ng/ml) (R&D Systemscatalog no. 240-B-002) or the TGFb inhibitor10 mM SB431552 (R&D Systems catalog no. 1614)throughout differentiation. TGFb1 was addedat the same time that adipogenic induction oc-curred, whereas SB431552 was added to cellswithin 24 hours of plating. The cells were in-cubated in a humidified incubator at 37°Cand 5% CO2.

Histology

Tissues were fixed with 2 to 4% paraform-aldehyde via transcardiac perfusion, after whichdissected adipose pads were fixed overnight. Thetissues were subsequently dehydrated through aseries of ethanol washes and then embedded inparaffin for thin sectioning. For cross-sectionalimaging of the inguinal adipose depots, a full-thickness section of the mouse flank (includingthe skin and adipose pad) was fixed and em-bedded in paraffin and then rotated and sectionedin the cross-sectional orientation. Immunohisto-chemistry analysis was performed by followingheat antigen retrieval methods, and samples werestained with the following antibodies: anti–redfluorescent protein (RFP) (rabbit; 1:500; Rock-land), anti-DPP4 (goat; 1:250; R&D Systems),anti-Pref1 (rabbit; R&D Systems), anti-Anxa3(rabbit; Biorbyt), andanti-Pi16 (rabbit;mybiosource).

Transplantation

DPP4+, ICAM1+, and CD142+ populations werepurified by FACS from the subcutaneous adiposeof pooledmale and female p10 to p16 ROSAmT/mG

pups (donor cells). mTomato+ donor cells werewashed with FACS buffer twice and then con-centrated by centrifugation to ~50,000 cells/mland mixed 1:1 with Matrigel (phenol free andgrowth factor reduced; Corning) on ice. WTC57BL/6 mouse pups aged 8 to 12 days wereanesthetized using an isoflurane nose cone,and abdominal hair was chemically removed be-fore the creation of an ~5-mm midline Y-shapedcutaneous incision to expose the bilateral in-guinal adipose pads. Tenmicroliters of the donorcell–Matrigel slurry was injected along the edgeof the inguinal fat pad in several 2-ml depots. Therecipient animals were closed with a 6-0 poly-propylene suture and placed back with theirlitter. After the indicated time, donor cells wereharvested from the recipient animals by dis-section of the entire inguinal adipose pad, fol-lowed by the FACS procedures as describedabove. BRs here mean separate pools of donorcells transplanted into separate individual re-cipient mice.

Quantification of adipocytedifferentiation and cell proliferationAdipogenesis was assessed by staining withBiodipy 493/503 (Invitrogen catalog no. D3922)for lipid droplet accumulation and by stainingwith Hoechst 33342 (Thermo Fisher catalog no.62249) for nucleus number at 4 to 6 days post-induction (mouse cells) and 14 to 18 days post-induction (human cells) in individual wells of a384-well plate (Sigma-Aldrich catalog no. CLS3770).The cells were imaged on a Keyence invertedfluorescencemicroscope (BZX-710) by using DAPI(excitation, 360/40 nm; emission, 460/50 nm;Keyence, OP-87762) and green fluorescent protein(excitation, 470/40 nm; emission, 525/50 nm;Keyence, OP-87763) filters. Individual wells wereimaged in their entirety at 20× magnification tocapture a 7-by-7 tiled/stitched grid (mouse studies)or at 10× magnification to capture a 5-by-5 tiled/stitched grid (human studies).

Image calculations

Tiling and stitching were performed with Key-ence BZ-XViewer software. Image quantificationwas performed in ImageJ as shown in fig. S23.Images were split into component channels.Nuclei (blue channel) were quantified by applyingGaussian Blur (3 Sigma), thresholding, perform-ing watershed calculation for segmentation, andcounting. Lipid accumulation was quantified byapplying Gaussian Blur (2 Sigma), thresholding,and quantifying the total area above the threshold.The level of adipogenesis, expressed as the adi-pogenic index, was assessed by dividing the totallipid area by the total number of nuclei to obtaina number with arbitrary units for comparingabsolute levels of adipogenesis. Relative adipo-genesis was calculated as the ratio of the adi-pogenic indices in treatment and control samples;here, the control is the untreated sample from thesame cell type and BR (for example, ICAM1–TGFbBR A/ICAM1 control BR A). This ratio repre-sents induction or suppression compared withthe baseline.

Statistics

All image calculations were first performed on aper-well basis with the same thresholds appliedto all wells within a single experiment. Oncethresholds were determined, calculations wereperformed on all images in an automatedmanner.The adipogenic index for BRs was calculated byaveraging the results from multiple technicalreplicates (2 to 8 wells per cell type per treat-ment per BR). Here, independent wells (i.e.,those with cells derived from the same biologicaland FACS pool but plated into separate wells ofthe same plate) are considered technical repli-cates. Statistical testing was performed on BRs.Cellular proliferation was assessed by plating

FACS-isolated DPP4+, ICAM1+, or CD142+ cellsinto a 96-well plate. Formouse studies, cells wereplated at 3600 cells per well (Fig. 2D) or 2000cells per well (Fig. 3C) in DMEM/F12 containing10% FBS and Primocin. For human studies, cellswere plated at 5000 cells per well in PM-1medium(Zenbio catalog no. PM-1) supplemented with

1 nM recombinant FGF2 (Invitrogen, PHG0261)and Primocin (50 ng/ml). For both human andmouse studies, the total nuclear content wasmeasured for each well by using the CyQuantdirect cell proliferation assay (Thermo Fishercatalog no. C35011). Where indicated, TGFb1(10 ng/ml) and SB431542 (10 mM) treatmentswere initiated after measuring the first timepoint. Proliferation data are displayed either asa curve showing data from independent wellsfrom a single representative BR (Fig. 2D and 3Cand fig. S12C) or as a bar graph from multipleBRs showing relative proliferation rates. Relativeproliferation rates were calculated for the humandata in order to provide a comparable baseline asfollows: The mean slope for DPP4, ICAM1, andCD142 cells was determined and called the pa-tient baseline. Data are displayed as the cell typeslope/patient baseline [for example, (DPP4 cellslope for patient 1)/(average slope for DPP4,ICAM1, and CD142 cells from patient 1)].

Quantitative reverse transcription PCR

Total RNA was isolated by using the RNeasymicrokit (Qiagen) and quantified by using aNanoDrop spectrophotometer. Five hundrednano-grams to 1 mg of RNAwas reverse-transcribed byusing the high-capacity cDNA synthesis kit (Ap-plied Biosystems) and then subjected to real-timepolymerase chain reaction (PCR) with SYBRGreen master mix (Applied Biosystems) on a7900 HT machine (Applied Biosystems). Tata-binding protein (encoded by Tbp) was used as aninternal normalization control. Data are presentedas the fold change relative to the control. ForFig. 3, E and G, data are calculated as the treatment/control ratio where the control is the untreatedsample from the same cell type and BR (for ex-ample, treatment sample ICAM1–TGFb BR A/ICAM1 control BR A). Data were analyzed usinganalysis of variance (ANOVA) followed by mul-tiple comparisons unless otherwise indicated.

Statistical methods

Statistical methods were not used to predeter-mine sample size. The experiments were notrandomized, and investigators were not blindedin experiments. All P values are reported withadjustment for multiple comparisons. Most stat-istical tests were performed by comparingDPP4+,ICAM1+, and CD142+ cells and therefore usedan ANOVA followed by multiple comparisons.Pairwise comparisons were determined a priori,were two sided, and were performed only if theANOVAwas significant, by using either theHolm-Sidak or Tukey post hoc test. Samples (from eachhuman donor or pool of mice) generated cellsfrom all three populations (DPP4+, ICAM1+, andCD142+). Therefore, two-way ANOVA or repeated-measures ANOVA was used to account for thematched nature of these data. For all parametrictests, quantile-quantile and residual plots weregenerated to test for deviation from the ANOVAassumptions of normally distributed residualswith equal variance. In cases with unequal var-iance between samples, the Brown-Forsythe cor-rection (one-way ANOVA) or Geisser-Greenhouse

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correction (repeated-measures ANOVA)was used.Where ANOVA would be the appropriate stat-istical test but the data were unbalanced, themixed effects model for statistical testing wasused instead. For fig. S14, multiple t tests fol-lowed by Holm correction were performed in-stead of ANOVA.Where indicated, BRs represent independent

samples (i.e., cells derived from different humandonors or from separate pools of mice). Forproliferation assays, cells from one human donoror pool of mice were plated into multiple wells,and this was repeated across multiple individualsor pools of mice.

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ACKNOWLEDGMENTS

We thank M. Lazar, E. Morrisey, S. Shapira, K. Ou, A. Wang,and members of the Seale lab for helpful advice and discussions.We thank L. Cheng and M. Lu for expertise in processinghistological samples. We thank the University of PennsylvaniaDiabetes Research Center (DRC) for use of the FunctionalGenomics Core (P30-DK19525). We thank R. Pellegrino da Silva,F. Mafra, and M. Gonzales for assistance with single-cell RNA-seqlibrary preparation and sequencing. Funding: This work wassupported by NIH grants RO1DK103008 and RO1DK107589 andADA grant 1-16-IBS-269 to P.S., NIH grant 5T32DK007314-37(T32) and the Measey Physician Scientist Fellowship Award toD.M., and NIH grant 5T32GM008216-29 to A.S. Authorcontributions: P.S., D.M., and A.S. were responsible forconceptualization, investigation, data curation and analysis,methodology, validation, visualization, and writing and review.Z.I. and C.O. performed experiments and assisted with dataanalysis. M.P.M. performed biostatistical analysis of the single-cellRNA-seq datasets. C.C. and I.P. provided fresh human adiposesamples. Competing interests: I.P. is a paid consultant forGalderma. She has no financial interest to declare in relation to thecontent of this article. Data and materials availability: Single-celland bulk RNA-seq matrix files are deposited in the GeneExpression Omnibus (GEO) under superseries accessionnumber GSE128889.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/364/6438/eaav2501/suppl/DC1Figs. S1 to S23

29 August 2018; accepted 27 March 201910.1126/science.aav2501

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Identification of a mesenchymal progenitor cell hierarchy in adipose tissue

Patrick SealeDavid Merrick, Alexander Sakers, Zhazira Irgebay, Chihiro Okada, Catherine Calvert, Michael P. Morley, Ivona Percec and

DOI: 10.1126/science.aav2501 (6438), eaav2501.364Science 

, this issue p. eaav2501; see also p. 328Sciencetherapeutic interventions that increase progenitor cell differentiation into adipocytes could ameliorate metabolic disease.progenitors reside in a fluid-filled network of collagen and elastin fibers surrounding adipose tissue. In principle, protein called DPP4 give rise to two distinct types of preadipocytes in response to different signals. The DPP4tissue in mice and humans (see the Perspective by Chau and Cawthorn). They found that progenitor cells expressing a

used single-cell RNA sequencing to define the hierarchy of mesenchymal progenitor cells that give rise to adiposeal.etthe number of adipocytes. The former process promotes metabolic disease, and the latter protects against it. Merrick

Fatty tissue can expand in two ways: through increases in the size of individual adipocytes or through increases inA singular focus on fat

ARTICLE TOOLS http://science.sciencemag.org/content/364/6438/eaav2501

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/04/24/364.6438.eaav2501.DC1

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