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

Awakened by cellular stress: isolation and characterization of a novel population of pluripotent stem cells derived from human adipose tissue.

Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse) Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal). When compared to adipose stem cells (ASCs), microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell homing. Being highly resistant to severe cellular stress, Muse-AT cells have the potential to make a critical impact on the field of regenerative medicine and cell-based therapy

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

Isolation and morphologic characterization of Muse-ATs.

<p>(<b>A</b>) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O₂ (<b>See Methods</b>). (<b>B</b>) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (<b>C</b>) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (<b>D)</b> Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.</p

Muse-ATs express pluripotent stem cell markers.

<p>Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064752#pone.0064752-Zuk1" target="_blank">[16]</a> were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.</p