Characterization of human fibrocytes as circulating adipocyte progenitors and the formation of human adipose tissue in SCID mice

Abstract

SPECIFIC AIM An increase in fat mass associated with obesity results from recruitment and differentiation of adipocyte progenitor cells. Accumulating evidence suggests that circulating stem cells can differentiate into cells of mesenchymal lineage. However, it is unclear whether a progenitor adipocyte population exists in circulation that can become a tissue adipocyte. The aim of this study was to characterize a fibrocyte as a novel circulating progenitor that can differentiate into an adipocyte, with accumulation of intracellular lipids and expression of adipocyte-specific genes. PRINCIPAL FINDINGS 1. Circulating human fibrocytes differentiate into adipocytes and express adipocyte-specific markers. Adipogenic commitment is influenced by the cellular microenvironment and presence of TGF-β Fibrocytes comprise a small fraction of cells in the total PBMC pool (∼0.5–1%). To address the potential role of fibrocytes in adipogenesis, we cultured PBMCs and subsequently enriched for the CAPs by immuno-depletion of contaminating cells to achieve a population with distinct combination of markers, including CXCR4, collagen I, and CD45RO triple stained by FACS analysis. These cells showed homogeneous spindle-shaped morphology and are different from T and B cells. Monocyte and macrophage markers CD14 and CD68 also were not expressed. We next cultured enriched fibrocytes in the presence of media supplemented with growth factors known to be important for adipogenesis. After adipogenic induction cycles, fibrocytes transformed into cells of rounder shape, with associated intracellular lipid accumulation and positive staining for Oil Red O. This was confirmed by expression of specific genes and proteins known to be important during adipocyte differentiation. Real-time quantitative RT-PCR, immunostaining, and immunoblot analysis revealed up-regulation of mature adipocyte markers after fibrocyte adipogenesis, with a similar expression profile seen for adipocytes derived from human subcutaneous (s.c.) preadipocytes after identical treatment. We next examined whether lineage commitment and differentiation of fibrocytes to adipocytes is contingent upon specific signals influenced by the microenvironment. We found that extracellular matrix (ECM) and cell density were important determinants. We also assessed whether TGF-β1 influenced adipogenic potential of fibrocytes. At concentrations of 1 ng/mL and 10 ng/mL, TGF-β1 markedly reduced differentiation of fibrocytes to adipocytes associated with inhibition of PPARγ expression. 2. Gene microarray analysis confirms fibrocyte-to-adipocyte differentiation To further understand the epigenetic reprogramming related to fibrocyte-to-adipocyte differentiation, we performed cDNA microrray to assess changes in differential gene profiles of fibrocytes, visceral preadipocytes, and s.c. preadipocytes before and after differentiation. We found that many well-studied adipocyte markers were enriched in the differentiation of fibrocytes to adipocytes, including C/EBPα, resistin, lipoprotein lipase, and chemokines, such as CCL2 and CXCL8 (Fig. 1 ⤻ A). Significant overlap was seen between gene ontology clusters enriched during fibrocyte adipogenesis or during visceral and s.c. preadipocyte-to-adipocyte differentiation, such as those involved in lipoprotein metabolism and fatty acid biosynthesis (Fig. 1B⤻ ). This suggests a potential link in physiologic roles shared by the three cell types. Conversely, certain gene clusters were differentially regulated only during fibrocyte adipogenesis, such as those involved in cell motility, chemotaxis, or metalloproteinase activity. One conclusion emerging from these data is that although fibrocyte-derived adipocytes display metabolic characteristics of an adipocyte, they may possess unique functions for motility and chemoattractive activity that might allow the cell to participate in migration and trafficking relevant to a cell in circulation and homing to specific tissue site. Figure 1. Genome-wide expression profiling. A) Up-regulation of selected genes after fibrocyte adipogenesis. B) Gene clusters based on functional ontology or pathways were noted to be differentially regulated after adipogenesis for fibrocytes as well as s.c. and visceral preadipocytes (D-Chip Analyzer software) C) PATHWAYASSIST software demonstrating the interrelationships of primary proteins involved in adipocyte metabolism. Associations are drawn from search using established ResNet™ database. Color intensity seen for each gene is based on magnitude of expression change after differentiation. Download figure Download PowerPoint 3. Adipocytes differentiated from fibrocytes form human adipose tissue in SCID mice. Furthermore, CCR2 is expressed after fibrocyte-to-adipocyte differentiation, associated with increased chemotaxis in response to CCL2 We next wanted to determine whether fibrocyte-derived adipocytes could form adipose tissue in vivo. We used a SCID mouse chimeric model and embedded human fibrocyte-derived adipocytes in Matrigel and injected the mixture s.c. into SCID mice. After 4 wk, we noted formation of human adipose tissue at the site of fibrocyte-derived adipocytes implantation with associated neovascularization (Fig. 2 ⤻ ). Through staining using human-specific leptin antibody, we determined that the new adipose tissue was indeed derived from human adipocytes and not from maturation of surrounding endogenous preadipocytes. These results indicate that under a favorable microenvironment, fibrocyte-derived adipocytes can integrate themselves into tissue to form adipose tissue in vivo. Figure 2. Human fibrocyte-derived adipocytes form human adipose tissue in SCID mice. A, B) Mouse adipose tissue obtained from s.c. fat at a site not injected with fibrocyte-derived adipocytes, here...