Identification of a mesenchymal progenitor cell hierarchy in adipose tissue

Abstract

Metabolic health depends on the capacity of adipose tissue progenitor cells to undergo de novo adipogenesis. The cellular hierarchy and mechanisms governing adipocyte progenitor differentiation are incompletely understood. Through single-cell RNA sequence analyses, we show that the lineage hierarchy of adipocyte progenitors consists of distinct mesenchymal cell types that are present in both mouse and human adipose tissues. Cells marked by dipeptidyl peptidase-4 (DPP4)/CD26 expression are highly proliferative, multipotent progenitors. During the development of subcutaneous adipose tissue in mice, these progenitor cells give rise to intercellular adhesion molecule-1 (ICAM1)/CD54-expressing (CD54+) committed preadipocytes and a related adipogenic cell population marked by Clec11a and F3/CD142 expression. Transforming growth factor-β maintains DPP4+ cell identity and inhibits adipogenic commitment of DPP4+ and CD142+ cells. Notably, DPP4+ progenitors reside in the reticular interstitium, a recently appreciated fluid-filled space within and between tissues, including adipose depots.

Fig. 1.. Single-cell RNA-seq and cell trajectory analysis delineatethe lineage hierarchy of adipocyte progenitors.

(A) Unsupervised clustering of 11,423 cells (mean number of genes per cell = 1977)from the subcutaneous WAT of p12 pooled male and female C57BL/6J mouse 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 of representative marker genes. The y axis is the log-scale normalized read count. (C) Pseudotemporal cell ordering of groups 1 to 4 and adipocytes 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).

Fig. 2.. DPP4+ progenitors display enhanced proliferative and multilineage differentiation capacity.

Cells were isolated by using the following FACS strategy: Lin− (CD45−, CD31−) cells were stained with anti-DPP4, anti-ICAM1, and anti-CD142. Groups were gated as follows: CD142+ cells were selected first, followed by DPP4+ (CD142−, DPP4+) or ICAM1+ (CD142−, ICAM1+) cells. (A and B) Staining of adipocytes (with Bodipy lipid stain) (green) and quantification of adipogenesis in cell cultures from adult CD1 mice after exposure to the complete adipogenic differentiation cocktail (A) or insulin only (min) (B) [n = 3 biological replicates (BRs) per condition]. (C) mRNA levels of 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 levels of osteocyte-specific genes in cultures exposed to osteogenic differentiation inducers (n = 5 BRs). Statistical testing: not significant, P > 0.05;**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Dots represent BRs, and error bars indicate SEM. Scale bars, 50 μM.

Fig. 3.. TGFβ regulates the proliferation and adipogenic differentiation competency of multipotent DPP4+ progenitors.

(A) RNA-seq andGO analysis of signaling pathways enriched in DPP4+ versus ICAM1+ cells from pooled p12 pups (n = 3 BRs). Combined score = log P value multiplied by the z-score of deviation from the expected ranking.(B) mRNA levels of group 1, group 2, and adipocyte (adipo) marker genes in DPP4+ cells treated with vehicle control, TGFβ, or the TGFβ receptor inhibitor SB431542 (n = 4 BRs). (C) Quantification of cell growth in cultures treated with TGFβ or SB431542 (representative of 3 BRs).(D) Bodipy staining of adipocytes (green) differentiated with the complete induction cocktail with or without TGFβ treatment. Relative adipogenesis is the adipogenic index of TGFβ-treated cells relative to that of control cells (right) (n = 3 BRs). (E) Fold changes in mRNA levels of adipocyte-specific genes 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. Scale bars, 50 μM. Statistical testing: not significant (ns), P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Dots represent BRs, and error bars indicate SEM.

Fig. 4.. Human adipose contains analogous populations of DPP4+ progenitors and ICAM1+ preadipocytes.

(A) FACS isolation of cell populations from human adipose tissue (representative of eight individual donors). Populations were purified as follows: Lin− (CD45−, CD31−) cells were stained with anti-DPP4, anti-ICAM1, and anti-CD142. CD142+ cells were selected 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 of relative progenitor abundance from n = 8 human tissue donors. (C) Relative proliferation rates of human cells (n = 5 donors). (D) Bodipy staining of adipocytes (green) in cultures treated with complete (top) or minimal (insulin plus rosiglitazone) (bottom) induction cocktail. (E) Quantification of adipogenic differentiation from cells shown in (D) (n = 6 donors). (F) Unsupervised clustering of 11,338 cells (mean number of genes per cell = 1253) from the abdominal subcutaneous adipose tissue of a 31-year-old female donor (body mass index, 31.6) reveals five distinct cell groups represented on a tSNE map (relevant marker genes are in parentheses). (G) Individual gene tSNE and violin plots showing the expression levels and distribution of representative marker genes. The y axis is the log-scale normalized read 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 indicate SEM. Scale bars, 50 μM.

Fig. 5.. DPP4+ progenitors give rise to ICAM1+, CD142+ cells and mature adipocytes in vivo.

(A) Sort-purified CD142(−), DPP4(+) and (B) CD142(−), ICAM1(+) cells from mTomato(+) donor mice were analyzed before transplantation (day 0) (left) into the subcutaneous adipose of 10-day-old mTomato(−) recipient mice. Seven and 14 days after transplantation, mTomato(+) cells were recovered and analyzed for the expression of DPP4, ICAM1, and CD142 (shown is one representative transplant from n = 4 BRs). Fifty days after transplantation, mTomato+ cells were visualized by immunofluorescence in recipient iWAT (right) (representative image; n = 3 to 4 BRs). AF, autofluorescence (525/50 nM) showing host adipocytes. (C) Sort-purified CD142(−) cells were analyzed 14 days posttransplant for the expression of DPP4, ICAM1, and CD142 (shown is one representative transplant from n = 4 BRs). The left FACS plot depicts the CD142-positive gate, and the right FACS plot shows analysis of cells from the CD142-negative gate.

Fig. 6.. DPP4+ progenitor cells reside in the RI.

(A) Full-thickness flank tissue from a 2-day-old mouse pup was cross-sectioned. Murine skin and adipose were stained with hematoxylin and eosin (H&E) with labels localizing the dermis (D), dermal fibroblasts (DF), the panniculus carnosus (PC), the RI, and adipocytes (Ad). Anti-DPP4 (red), anti-PREF1 (green), and DAPI (blue) were used for immunofluorescence. (Inset 1) Magnification of the leading edge of the developing iWAT. (Inset 2) Cross-sectional magnification of the body of the iWAT. Arrows show examples of DP (DPP4+, PREF1+) cells. The arrowheads point to DPP4+, PREF1(−) cells. (B) Model depicting the lineage hierarchy relationships of the indicated cell types. (C) Model depicting the anatomical relationships of the indicated cell types at the leading edge of developing adipose tissue.