Toward a Consensus View of Mammalian Adipocyte Stem and Progenitor Cell Heterogeneity

The adipose stem and progenitor cell (ASPC) fraction is heterogeneous and comprises at least three subpopulations: adipose stem cells (ASCs), preadipocytes (PreAs), and adipogenesis regulators (Aregs).ASCs (DPP4+, CD55+) give rise to both PreAs (ICAM1+, VAP-1+) and Aregs (CD142+).In adult mice, Aregs (CD142+) inhibit adipogenesis through paracrine mechanisms, which are not yet fully understood.In visceral fat, a subpopulation of ASCs, known as fibro inflammatory progenitors (FIPs) (PDGFRβ+/LY6C+), exerts an antiadipogenic effect.Mesothelial cells populate the stromal vascular fraction of visceral but not subcutaneous adipose tissue and modulate the immune environment.Human and mouse ASCs exhibit a common gene expression signature, but human PreAs and Aregs are molecularly less distinct than in mouse.ASPC heterogeneity differences between human and mouse question the full relevance of mouse ASPCs as an appropriate human adipose biology model system. White adipose tissue (WAT) is a cellularly heterogeneous endocrine organ that not only serves as an energy reservoir, but also actively participates in metabolic homeostasis. Among the main constituents of adipose tissue are adipocytes, which arise from adipose stem and progenitor cells (ASPCs). While it is well known that these ASPCs reside in the stromal vascular fraction (SVF) of adipose tissue, their molecular heterogeneity and functional diversity is still poorly understood. Driven by the resolving power of single-cell transcriptomics, several recent studies provided new insights into the cellular complexity of ASPCs among different mammalian fat depots. In this review, we present current knowledge on ASPCs, their population structure, hierarchy, fat depot-specific nature, function, and regulatory mechanisms, and discuss not only the similarities, but also the differences between mouse and human ASPC biology. White adipose tissue (WAT) is a cellularly heterogeneous endocrine organ that not only serves as an energy reservoir, but also actively participates in metabolic homeostasis. Among the main constituents of adipose tissue are adipocytes, which arise from adipose stem and progenitor cells (ASPCs). While it is well known that these ASPCs reside in the stromal vascular fraction (SVF) of adipose tissue, their molecular heterogeneity and functional diversity is still poorly understood. Driven by the resolving power of single-cell transcriptomics, several recent studies provided new insights into the cellular complexity of ASPCs among different mammalian fat depots. In this review, we present current knowledge on ASPCs, their population structure, hierarchy, fat depot-specific nature, function, and regulatory mechanisms, and discuss not only the similarities, but also the differences between mouse and human ASPC biology. WAT (see Glossary) comprises multiple anatomic entities across the body (e.g., subcutaneous versus visceral) that are composed of loose connective tissue and that feature adipocytes as their fundamental unit. The connective tissue surrounding adipocytes contains the stromal vascular fraction (SVF) and harbors adipose stem and progenitor cells (ASPCs), which are the cells that give birth to mature adipocytes, together with immune cells and vasculature, which assure overall tissue homeostasis. ASPCs, defined as the SVF depleted of immune (CD45+), endothelial (CD31+), and red blood (TER119+, CD235a+) cells [or the lineage negative fraction (Lin–) of the SVF] are a heterogeneous cell population themselves [1.Rodeheffer M.S. et al.Identification of white adipocyte progenitor cells in vivo.Cell. 2008; 135: 240-249Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar,2.Sanchez-Gurmaches J. Guertin D.A. Adipocyte lineages: tracing back the origins of fat.Biochim. Biophys. 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This is why genome-wide assays, such as scRNA-seq, enable a fresh perspective on the heterogeneous nature of specific biological systems, such as ASPCs [18.Tang F. et al.mRNA-Seq whole-transcriptome analysis of a single cell.Nat. Methods. 2009; 6: 377-382Crossref PubMed Scopus (1592) Google Scholar]. However, scRNA-seq is not a trivial laboratory technique, especially when it comes to analyzing the resulting large data sets [19.Luecken M.D. Theis F.J. Current best practices in single-cell RNA-seq analysis: a tutorial.Mol. Syst. Biol. 2019; 15e8746Crossref PubMed Scopus (424) Google Scholar]. 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In 2018, scRNA-seq was used for the first time to resolve mouse adipose SVF heterogeneity [22.Schwalie P.C. et al.A stromal cell population that inhibits adipogenesis in mammalian fat depots.Nature. 2018; 559: 103Crossref PubMed Scopus (166) Google Scholar], promptly followed by several other adipose scRNA-seq analyses [23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24.Merrick D. et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364eaav2501Crossref PubMed Scopus (170) Google Scholar, 25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar, 26.Cho D.S. et al.Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.Life Sci. Alliance. 2019; 2e201900561Crossref PubMed Scopus (21) Google Scholar, 27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar, 28.Jackson-Jones L.H. et al.Stromal cells covering omental fat-associated lymphoid clusters trigger formation of neutrophil aggregates to capture peritoneal contaminants.Immunity. 2020; 52: 700-715Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. In this review, we discuss how these collective studies have significantly advanced our understanding of mammalian ASPC composition and function in WAT. Specifically, scRNA-seq studies of ASPCs identified three to four cell subpopulations and support a hierarchical relationship that orders these populations along an adipogenic commitment trajectory [22.Schwalie P.C. et al.A stromal cell population that inhibits adipogenesis in mammalian fat depots.Nature. 2018; 559: 103Crossref PubMed Scopus (166) Google Scholar, 23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24.Merrick D. et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364eaav2501Crossref PubMed Scopus (170) Google Scholar, 25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar, 26.Cho D.S. et al.Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.Life Sci. Alliance. 2019; 2e201900561Crossref PubMed Scopus (21) Google Scholar, 27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar, 28.Jackson-Jones L.H. et al.Stromal cells covering omental fat-associated lymphoid clusters trigger formation of neutrophil aggregates to capture peritoneal contaminants.Immunity. 2020; 52: 700-715Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. To compare and contrast the results of these different studies, we performed a novel integrative analysis of the respective scRNA-seq data sets (Figure 1, and Figure S1 in the supplemental information online). Based on the results of this analysis and for the sake of clarity and comparison, we propose a coarse-grain consensus nomenclature that considers the reported molecular and functional characteristics of each ASPC subpopulation: adipose stem cells (ASCs), preadipocytes (PreAs), and adipogenesis regulators (Aregs). These subpopulations are introduced and discussed later, following the proposed hierarchical model. In mouse, SCA1 surface protein, encoded by the Ly6a gene, is well established as the main marker to enrich ASPCs for a precursor cell population that has enhanced in vitro adipogenic capacity [29.Steenhuis P. et al.Cell surface expression of stem cell antigen-1 (Sca-1) distinguishes osteo-, chondro-, and adipoprogenitors in fetal mouse calvaria.Calcif. Tissue Int. 2008; 82: 44-56Crossref PubMed Scopus (16) Google Scholar]. However, not all SCA1+ cells give rise to adipocytes in vitro, implying that an even finer granularity of cell states or even cell types exists. Therefore, much effort has been devoted to uncover additional surface markers. For example, mesenchymal markers, such as CD34, CD29, CD24, and platelet-derived growth factor receptor (PDGFR)α/β, were also shown to enrich for adipogenic precursors, or a fraction of them [1.Rodeheffer M.S. et al.Identification of white adipocyte progenitor cells in vivo.Cell. 2008; 135: 240-249Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar,30.Cawthorn W.P. et al.Adipose tissue stem cells meet preadipocyte commitment: going back to the future.J. Lipid Res. 2012; 53: 227-246Crossref PubMed Scopus (261) Google Scholar, 31.Berry R. Rodeheffer M.S. Characterization of the adipocyte cellular lineage in vivo.Nat. 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Markers, such as CD29, CD34, CD13, CD44, CD73, CD90, CD142, CD9, CD10, and CD200 [33.Ong W.K. et al.Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots.Stem Cell Rep. 2014; 2: 171-179Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,34.García-Rubio J. et al.Cytometric analysis of adipose tissue reveals increments of adipocyte progenitor cells after weight loss induced by bariatric surgery.Sci. Rep. 2018; 815203Crossref PubMed Scopus (16) Google Scholar], have been used to further enrich for adipogenic cells in human, but there is no consensus yet on the exact molecular signature of hASPCs. Until recently, the identification of these markers was mostly based on flow cytometry and immunohistochemistry using hematopoietic, endothelial, and neural tissues as reference, which tends to introduce marker selection bias. However, over the past few years, the single-cell technology revolution has allowed researchers to delineate ASPCs at an unprecedented resolution (Figure 1, Figure 2). Numerous studies [22.Schwalie P.C. et al.A stromal cell population that inhibits adipogenesis in mammalian fat depots.Nature. 2018; 559: 103Crossref PubMed Scopus (166) Google Scholar, 23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24.Merrick D. et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364eaav2501Crossref PubMed Scopus (170) Google Scholar, 25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar, 26.Cho D.S. et al.Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.Life Sci. Alliance. 2019; 2e201900561Crossref PubMed Scopus (21) Google Scholar] recently resolved ASPC heterogeneity in mouse subcutaneous (inguinal WAT) and visceral (specifically epigonadal WAT) WAT (iWAT and eWAT, respectively). Despite differences in the utilized methodologies and/or mouse models (Table 1), all studies in mouse stratified ASPCs into two to three main subpopulations. Qualitative cross-comparison of the population-specific markers supports the notion that these populations are shared between subcutaneous and visceral depots, although intrinsic differences remain that are far from being fully understood. To empirically validate this ASPC classification, we integrated public data sets (highlighted in gray in Table 1) using the standard integration workflow of the Seurat program [35.Stuart T. et al.Comprehensive integration of single-cell data.Cell. 2019; 177: 1888-1902Abstract Full Text Full Text PDF PubMed Scopus (2791) Google Scholar,36.Butler A. et al.Integrating single-cell transcriptomic data across different conditions, technologies, and species.Nat. Biotechnol. 2018; 36: 411-420Crossref PubMed Scopus (3110) Google Scholar]. This large, integrated meta-data set provides a powerful means to explore the observed ASPC landscape across studies in an unbiased fashion. This is because it enables verification of the expression distribution and, thus, the specificity of previously established ASPC markers. In addition, it allows us to computationally explore parallels between the recently published scRNA-seq studies and to support the proposed population nomenclature. Specifically, our analyses revealed that the three populations (ASCs, PreAs, and Aregs) are robust since they: (i) are characterized by specific gene expression signatures; (ii) were first independently detected in each of the utilized data sets; (iii) can be projected onto one another upon data integration (Figure 1A, and Figure S1A,G in the supplemental information online); and (iv) are consistently clustered in various subclusters upon analysis of the integrated data set (Figure 1B, and Figure S1B,H, and the supplementary methods in the supplemental information online). Finally, we were able to confirm that the three populations presented in this review align with populations published across different studies (Figure 1C, and Figure S1C,D,I,J in the supplemental information online). Altogether, the integration shows that ASCs are a distinct population compared with PreAs and Aregs, which are closer in terms of gene expression but still feature distinct transcriptomic profiles. As a side note, mesothelial cells also populate the Lin– fraction of visceral SVF together with ASPCs [23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar,25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar,27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar,28.Jackson-Jones L.H. et al.Stromal cells covering omental fat-associated lymphoid clusters trigger formation of neutrophil aggregates to capture peritoneal contaminants.Immunity. 2020; 52: 700-715Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar], but are discussed separately in Box 1 since they are not considered to be part of the ASPC hierarchy. For human, only a couple of scRNA-seq studies have been published [24.Merrick D. et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364eaav2501Crossref PubMed Scopus (170) Google Scholar,27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar,37.Acosta J.R. et al.Single cell transcriptomics suggest that human adipocyte progenitor cells constitute a homogeneous cell population.Stem Cell Res Ther. 2017; 8: 250Crossref PubMed Scopus (31) Google Scholar], and these do not necessarily reach the same conclusions. This renders it difficult to establish a clear picture of hASPC heterogeneity. However, a closer look at the reported findings suggests that, based on shared gene signatures, at least two ASPC subpopulations are conserved between mouse and human, namely the ASCs and PreAs.Table 1Methodological Differences across scRNA-Seq Studies [22.Schwalie P.C. et al.A stromal cell population that inhibits adipogenesis in mammalian fat depots.Nature. 2018; 559: 103Crossref PubMed Scopus (166) Google Scholar, 23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24.Merrick D. et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364eaav2501Crossref PubMed Scopus (170) Google Scholar, 25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar, 26.Cho D.S. et al.Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.Life Sci. Alliance. 2019; 2e201900561Crossref PubMed Scopus (21) Google Scholar, 27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar,52.Spallanzani R.G. et al.Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors.Sci. Immunol. 2019; 4eaaw3658Crossref PubMed Scopus (89) Google Scholar]a,b.aAbbreviations: APC, adipose progenitor cells; Aregs, adipogenesis regulators; ASC, adipose stem cells; CP, committed preadipocytes; Fibro, fibroblasts; FIP, fibro inflammatory progenitors; HSC, hematopoietic stem cells; IP, interstitial progenitors; Meso, mesothelial cells; PreA, preadipocytes; T2DM PA, type 2 diabetes-dependent preadipocytes.bGray boxes indicate data sets used in the integration analysis (see Figure 1 in the main text and Figure S1 in the supplemental information online).cSubcutaneous.dVisceral.Box 1Mesothelial CellsMesothelial cells constitute the peritoneum both in mouse and human and belong exclusively to the visceral SVF. Not only do they line the walls of the abdominal cavity, but they are also found in small clusters within the visWAT itself, in the form of so-called 'milky spots' or 'fat-associated lymphoid clusters' [67.Michailova K.N. Usunoff K.G. 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Not surprisingly, mesothelial cells were found among visceral ASPCs using scRNA-seq ([23.Burl R.B. et al.Deconstructing adipogenesis induced by β3-adrenergic receptor activation with single-cell expression profiling.Cell Metab. 2018; 28: 300-309Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar,25.Hepler C. et al.Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.eLife. 2018; 7e39636Crossref PubMed Scopus (119) Google Scholar,28.Jackson-Jones L.H. et al.Stromal cells covering omental fat-associated lymphoid clusters trigger formation of neutrophil aggregates to capture peritoneal contaminants.Immunity. 2020; 52: 700-715Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar] in mouse, [27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. Metab. 2020; 2: 97-109Crossref PubMed Scopus (80) Google Scholar] in human) (Figure 1A,B,E, and Figure S1G,H,I and the supplemental methods in the supplemental information online). Thus, one should be aware that, for both human and mouse, cells of the mesothelial lineage exist within the visceral SVF and may cloud functional read-outs. Therefore, we argue that it may be best to sort these cells out before any downstream in vitro characterization of ASPCS.However, what is interesting about mesothelial cells in the context of adipose biology, besides being a heterogeneous population themselves [27.Vijay J. et al.Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.Nat. 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