Site-Dependent Lineage Preference of Adipose Stem Cells

Abstract

Adult stem cells have unique properties in both proliferation and differentiation preference. In this study, we hypothesized that adipose stem cells have a depot-dependent lineage preference. Four rabbits were used to provide donor-matched adipose stem cells from either subcutaneous adipose tissue (ScAT) or infrapatellar fat pad (IPFP). Proliferation and multi-lineage differentiation were evaluated in adipose stem cells from donor-matched ScAT and IPFP. RNA sequencing (RNA-seq) and proteomics were conducted to uncover potential molecular discrepancy in adipose stem cells and their corresponding matrix microenvironments. We found that stem cells from ScAT exhibited significantly higher proliferation and adipogenic capacity compared to those from donor-matched IPFP while stem cells from IPFP displayed significantly higher chondrogenic potential compared to those from donor-matched ScAT. Our findings are strongly endorsed by supportive data from transcriptome and proteomics analyses, indicating a site-dependent lineage preference of adipose stem cells.

Keywords: adipogenesis; adipose stem cell; chondrogenesis; infrapatellar fat pad; osteogenesis; subcutaneous adipose tissue.

FIGURE 1

Comparison of proliferation capacity between ScASCs (n = 4) and donor-matched IPFSCs (n = 4) in (A) cell morphology; (B) cell population doubling (PD) time calculated by cell counting; (C) relative EdU incorporation; (D) CD146 surface marker expression level evaluated by flow cytometry analysis; (E) other surface marker gene expression levels evaluated by RNA-seq, in which a positive value in log2FC means higher expression in IPFSCs (in red) and a negative value means higher expression in ScASCs (in blue); and (F) stemness-related genes NANOG, REX1, NESTIN, and SOX2 and senescence-related genes CDKN1A and TP53 measured by qPCR. Data are shown as bar charts. * indicates a significant difference compared to the corresponding control group (p < 0.05).

FIGURE 2

Expression of transcription factors in ScASCs (n = 4) and donor-matched IPFSCs (n = 4) measured by qPCR. SOX9, PPARG, and RUNX2 were used to represent chondrogenesis, adipogenesis, and osteogenesis, respectively. Expanded cells were evaluated before induction (A) and after induction (B–E): (B) 24 h after centrifugation to form a pellet but before chondrogenic induction; (C) 18 days after chondrogenic induction; (D) 21 days after adipogenic induction; and (E) 21 days after osteogenic induction. Data are shown as bar charts. Expression of each target gene in undifferentiated IPFSCs (A) and differentiated ScASCs/IPFSCs (B–E) is plotted against undifferentiated ScASCs (A), which is set as “1”. * indicates a significant difference compared to the corresponding control group (p < 0.05).

FIGURE 3

Expression of differentiation genes in ScASCs (n = 4) and donor-matched IPFSCs (n = 4) before and after induction. For chondrogenic differentiation, typical markers, COL2A1 and ACAN, were evaluated using qPCR before (A) and after chondrogenic induction (B). Hypertrophic markers COL10A1 and MMP13 were also measured after chondrogenic induction (C). For adipogenic differentiation, typical markers, ADIPOQ and LPL, were evaluated using qPCR before (D) and after adipogenic induction (E). LEP expression was also measured after induction (E). Adiponectin (ADIPOQ) expression was confirmed using Western blot analysis with β-actin as an internal control (F). For osteogenic differentiation, typical markers, BGLAP and SPP1, were evaluated using qPCR before (G) and after osteogenic induction (H). Expression of DCN and SPARC were also measured after induction (H). Osteocalcin (OCN) expression was confirmed using ELISA analysis (I). Expression of each target gene in IPFSCs is plotted against the corresponding ScASCs, which is set as “1”, but for those in chondrogenic induction (B/C), expression in day 0 is plotted as “1”. Data are shown as bar charts. * indicates a significant difference compared to the corresponding control group (p < 0.05).

FIGURE 4

Differentially expressed gene analysis of ScASCs and donor-matched IPFSCs from four rabbits (n = 4). (A) Principal coordinates analysis on gene expression for RNA-Seq samples from ScASCs and IPFSCs. (B) MA plot showing the average and fold change (FC) of gene expression for differentially expressed genes between ScASCs and IPFSCs. Red: genes upregulated from ScASCs to IPFSCs; Black: genes downregulated from ScASC to IPFSCs; Blue background: all expressed genes. (C) Bar plot showing the ratio of observed number to the expected number of genes for transcription factor genes and for other genes grouped based on their expression changes between ScASCs and IPFSCs. (D) Heat map showing the expression changes between ScASCs and IPFSCs for differentially expressed transcription factors. Genes sorted first by their expression changes and then by names. Red rectangles: Homeobox (HOX) genes.

FIGURE 5

Expression changes between IPFSCs and ScASCs for selected groups of genes with function in chondrogenesis and/or adipogenesis: (A) Homeobox (HOX) gene family; (B) T (the brachyury gene)-box (TBX) gene family; (C) TGFβ (TGFB) gene family (D) SOX gene family; (E) Bone morphogenetic protein (BMP) gene family; (F) Wingless/integrase-1 (WNT) gene signals; (G) Collagen (COL) gene family; (H) Lysyl oxidase (LOX) gene family; (I) Genes coding for Basement membrane proteins; (J) Genes coding Matrix turnover enzymes; (K) Integrin (ITG) gene family; (L) Aquaporin (AQP) gene family; (M) Chondrogenic-related genes; and (N) Adipogenic-related genes. Green shaded FDR indicates favorable effect of the gene on chondrogenesis (M) or adipogenesis (N). A positive value in log2FC means higher expression in IPFSCs (in red), and a negative value means higher expression in ScASCs (in blue). Orange shaded FDR: > 0.05.

FIGURE 6

Proteomic results of cells and ECMs from ScASCs versus IPFSCs of four donor-matched rabbits. (A) Heatmap for global representation of protein differences (Z score used). The blue bar on the left indicates the region highly enriched in ECM components, the red bar indicates the region that contains primarily intracellular proteins. (B) Volcano plot with the Y-axis indicating -log10 p-value and the X-axis of the fold change (FC) in log2. Core ECM proteins are indicated in blue and Matrisome associated proteins are in green. (C) Gene enrichment analysis (top pathways, Reactome) is shown with the X-axis indicating the statistical significance based on false discovery rates using the Benjamani_hochberg method.