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

Kurt M. Hong^*,, Marie D. Burdick, Roderick J. Phillips, David Heber^* and Robert M. Strieter^,,1

^* Center for Human Nutrition, David Geffen School of Medicine at UCLA, Los Angeles, California, USA;
Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, California, USA; and
Department of Medicine, Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA

¹Correspondence: Division of Pulmonary and Critical Care Medicine, Departments of Medicine, Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 900 Veteran Ave., 14-154 Warren Hall, Los Angeles, CA 90095-1786, USA. E-mail: rstrieter{at}mednet.ucla.edu

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-ß
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-ß1 influenced adipogenic potential of fibrocytes. At concentrations of 1 ng/mL and 10 ng/mL, TGF-ß1 markedly reduced differentiation of fibrocytes to adipocytes associated with inhibition of PPAR 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/EBP, 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.

View larger version (41K): [in this window] [in a new window]   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 ResNetTM database. Color intensity seen for each gene is based on magnitude of expression change after differentiation.

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.

View larger version (97K): [in this window] [in a new window]   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 stained with isotype control antibody and the same section stained with human-specific leptin antibody. C, D) Fibrocyte-derived adipocytes form human adipose tissue in SCID mice. Human adipose tissue in SCID mice stained with isotype control antibody and the same section stained with human-specific leptin antibody. Prominent cytoplasmic staining for leptin in human adipocytes is shown by arrows.

Finally, studies have shown that these progenitor cells express an array of chemokine receptors, including CCR7 and CXCR4. Surface expression of CCR2, the receptor for CCL2, was found to be markedly up-regulated in association with fibrocyte-to-adipocyte differentiation based on FACS analysis. We also examined the effect of adipogenic treatment on CCR2 transcript level using quantitative RT-PCR. Significant up-regulation of CCR2 expression was detected within 8 h of initiation of adipogenic induction; at 24 h maximal CCR2 transcript level was achieved, which was 7.3-fold higher than in untreated cells. Last, chemotaxis was performed to assess for specific migration in response to CCL2. When fibrocytes were treated with adipogenic media, migratory response CCL2 was seen at 1 and 3 wk.

CONCLUSIONS AND SIGNIFICANCE

The current study demonstrated that fibrocytes are a circulating population of progenitor cells that can give rise to adipocytes in the presence of specific environmental cues. Most work on adult stem cells has focused on populations of cells from bone marrow, such as mesenchymal stem cells (MSC) or multipotent adult progenitor cell (MAPC), a rare population that copurifies with MSC. Others have reported stem cells within the adipose tissue with the ability to differentiate into cells of mesodermal lineage. These resident cells express cell surface markers characteristic of MSC, but their precise origin is unknown. We provide for the first time several lines of evidence that the fibrocyte is a unique adult progenitor cell in circulation that can differentiate into an adipocyte and integrate themselves to form adipose tissue in vivo.

In our experiments, we saw significant up-regulation of specific mature adipocyte genes and proteins after fibrocyte differentiation to adipocyte, including FABP4, leptin, and PPAR. Fibrocyte lineage commitment and differentiation to an adipocyte was influenced by specific growth microenvironments. Permissive culture conditions have been described for adipogenesis of 3T3-L1 cells and human bone marrow-derived MSC. We demonstrated that low culture density fibronectin ECM, as well as the presence of fibrogenic cytokine TGFß, significantly inhibited fibrocyte adipogenesis. Although the precise mechanism is unknown, we speculate that these factors can differentially regulate fibrocyte cell morphology and attachment, cell cycle progression, and availability of local reservoir for growth factors.

The most compelling finding of the present study was the ability of human fibrocyte-derived adipocytes to form human adipose tissue in vivo after implantation into SCID mice. By immunostaining using human specific leptin antibody, we determined that the new adipose tissue was derived from human adipocytes and not from maturation of surrounding endogenous preadipocytes.

Finally, studies have shown that these progenitor cells express an array of chemokine receptors, including CCR7 and CXCR4. We now demonstrate that after fibrocyte differentiation to adipocyte, there is an increase in expression of CCR2 associated with increased chemotactic response to MCP-1(CCL2). It has been established that levels of some chemokines (e.g., CCL2) are up-regulated during obesity. CCL2 release is higher in obese subjects, associated with increased expression from visceral adipose tissue. We hypothesize that under homeostatic conditions, fibrocytes remain at low levels in the peripheral circulation. However, under a different microenvironmental niche related to positive energy balance, fibrocytes may increase in number and traffic to adipose tissue where levels of CCL2 are elevated.

In summary, our findings demonstrate for the first time that adult fibrocytes can differentiate into adipocytes with expression of key adipogenic markers consistent with other models of adipogenesis. Although this contrasts with the long-held view that adipocytes are derived only from resident tissue preadipocytes, it is possible that circulating fibrocytes could contribute to the growth and maintenance of adipose tissue. It is tempting to speculate that fibrocytes may prove to be an attractive therapeutic target in preventing the development of obesity related to metabolic syndrome (Fig. 3 ).

View larger version (46K): [in this window] [in a new window]   Figure 3. Summary schematic demonstrating differentiation of circulating adipogenic progenitors to adipocytes with the ability to form adipose tissue in vivo. Various microenvironmental stimuli can influence lineage specification and adipogenic commitment of fibrocytes. Expression of mature adipocyte genes/proteins are seen after fibrocyte adipogenesis, including expression of chemokine receptor CCR2. The associated increased chemotactic response to CCL2 may represent a mechanism whereby fibrocytes are recruited to expanding adipose tissue and contribute to pathogenesis of obesity-related metabolic syndrome.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4295fje;

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