Novel Pathway of Adipogenesis through Cross-Talk between Adipose Tissue Macrophages, Adipose Stem Cells and Adipocytes: Evidence of Cell Plasticity

INTRODUCTION: Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes. RESEARCH DESIGN AND METHODS: Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs. RESULTS: Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes. CONCLUSIONS: Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes

Novel pathway of adipogenesis through cross-talk between adipose tissue macrophages, adipose stem cells and adipocytes: evidence of cell plasticity.

IntroductionPrevious studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes.Research design and methodsHuman adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs.ResultsCo-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes.ConclusionsCross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes

Morphological changes exhibited during CD14 (+) cell differentiation to preadipocytes.

<p>Expression of markers CD14 (monocytes/macrophages) (light green), S-100 (preadipocytes/adipocytes) (red), and CD34 (ASCs) (dark green), Nile Red (preadipocytes/adipocytes) (orange). Before co-culture: (A) most of the ATMs are CD14 (+); (B–D) very few ATMs are CD14 (+)/S-100 (+); After co-culture: (E) ATMs become enlarged in size as they transform to preadipocytes (begin to express DLK/S-100 while maintaining CD14 expression); (F) Preadipocytes are S-100 (+) and DLK (+) and start loosing CD14 and CD34 expression; (G) As preadipocytes start differentiating to adipocytes, there is an increase cell size, lipid accumulation (Nile Red (+) cells) and C/EBPα and PPARγ gene expression. The brightness of the color inside the lowers bars indicates the changes in cell expression markers in correlation with the morphological changes exhibited during ATMs differentiation to preadipocytes.</p

Differentiation of adipose stem cells to preadipocytes/adipocytes.

<p>Isolated adipocytes and ATMs/ASCs were cultured separately for 24 hours to allow the cells to reach equilibrium and then were co-cultured for an additional 24 hours. The cells were then separated and ATMs/ASCs were cultured for another 2 days in regular medium followed by an additional 7 days in either the same medium or adipogenic medium (A) ATMs/ASCs without co-culture were cultured in regular medium for 2 days followed by an additional culture of 7 days in the same medium; (B) ASCs/ATMs with co-culture were cultured in regular medium for 2 days followed by an additional culture for 7 days in regular medium. (C) Fold change in gene expression of C/EBPα and PPARγ of ASCs/ATMs with and without co-culture, cell culture conditions are the same as described above (see 3A–B); (D) ASCs/ATMs without co-culture were cultured in regular medium for 2 days followed by an additional culture for 7 days in adipogenic medium; (E) ASCs/ATMs with co-culture were cultured for 2 days in regular medium followed by additional culture for 7 days in adipogenic medium; (F) Fold change in gene expression of C/EBPα and PPARγ of ASCs/ATMs with and without co-culture, cell culture conditions are the same as described above (see 3D–E).</p

FACS analysis of the ATM/ASC fraction alone and after co-culture with adipocytes.

<p>A monoclonal antibody to DLK was used to determine the presence of specific subsets of preadipocytes. (A) Before co-culture (total 24 h), (B) without co-culture (total 72 h), (C) with 24 h co-culture (total 72 h). IgG2a was used as a negative control.</p

Scheme of co-culture between adipocytes, and ATMs/ASCs isolated from adipose tissue.

<p>Characterization of the isolated adipocytes and ATMs present in the ATM/ASC fraction. Isolated adipocytes and ATMs/ASCs were cultured separately for 24 hours to allow the cells to reach equilibrium and then were co-cultured for an additional 24 hours. Finally, the cells were separated and cultured for another 24 hours or 48 hours. (<b>A</b>) At the end of these periods, the culture media and the cells were subjected to different studies. (<b>B</b>) Nile Red co-labeled with DAPI (light blue) indicates the presence of mature adipocytes (x100). (<b>C</b>) Immunofluorescence (40X) of adipocytes cultured for 24 hours, cells are CD14 (−). (<b>D</b>) ATMs/ASCs after 3 days in culture, labeled for CD14 (200X), (<b>E</b>) these cells are Nile Red (-).</p

Regulation of Adiponectin Secretion by Adipocytes in the Polycystic Ovary Syndrome: Role of Tumor Necrosis Factor-α

Context: Adipose tissue dysfunction associated with low-grade chronic inflammation and dysregulation of adipokine secretion might significantly contribute to the pathogenesis of polycystic ovary syndrome (PCOS)