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

From the lost radium files: misadventures in the absence of training, regulation, and accountability

PURPOSE: Radium was the foundation of brachytherapy in the early decades of the 20th century. Despite being a most precious and perilous substance, it was mislaid with surprising frequency. This essay explores how it was lost, the efforts taken to recover it, and measures instituted to prevent mishandling. METHODS AND MATERIALS: Review of contemporary literature, government publications, archives, and lay press. RESULTS: Radium is a particularly dangerous substance because of its long half-life, its gaseous daughter (radon), and the high-energy emissions of its decay products. Despite the hazard, it was unregulated for most of the century. Any physician could obtain and administer it, and protocols for safe handling were generally lacking. Change came with appreciation of the danger, regulation, mandated training, and the institution of a culture of accountability. Unfortunately, careless management of medical radionuclides remains a global hazard. CONCLUSION: Responsible stewardship of radioactive material was not a high priority, for practitioners or the federal government, for much of the 20th century. As a result, large quantities of radium had gone astray, possibly subjecting the general public to continued radiation exposure. Lessons from the radium era remain relevant, as medical radionuclides are still mishandled

Goldilocks Mastectomy with Bilateral In Situ Nipple Preservation Via Dermal Pedicle

Summary:. Patients who don’t want or can’t have formal breast reconstruction after mastectomy surgery can be considered for a Goldilocks mastectomy, where the breast fullness is recreated from what is left behind after the gland tissue is removed from underneath the skin in a breast reduction pattern. A Goldilocks mastectomy does not require the use of implants or tissue transfer from other parts of the body and may be completed in a single surgery. This is best suited for larger breasted women who are willing to have much smaller breasts as a result. Previously, it was a challenge to be able to preserve the nipples when this operation was performed; however, this article describes a patient who had a bilateral Goldilocks mastectomy for right breast cancer who was able to save her nipples by keeping the blood flow in place from the surrounding skin. Conventional breast reconstruction after mastectomy is a challenge for larger breasted women. The Goldilocks mastectomy technique was designed to make best use of the redundant lower pole skin and subcutaneous fat to recreate a breast mound without a prosthetic implant or autologous tissue transfer. In its original description, the Goldilocks mastectomy did not include a means for nipple preservation. In this report, we describe the further refinement of the Goldilocks procedure that preserves the nipple areolar complex using a dermal pedicle. A patient with large pendulous breasts and right breast carcinoma underwent a bilateral Goldilocks nipple-sparing mastectomy and immediate reconstruction without an implant or flap

Review: Proposed Methods to Improve the Survival of Adipose Tissue in Autologous Fat Grafting

Summary:. In 2009, the American Society of Plastic Surgeons Task Force on Autologous Fat Grafting (AFG) determined that autologous fat grafting was a safe procedure with a relatively low rate of complications. This consensus opinion unleashed a wave of popularity as plastic surgeons discovered the procedures' efficacy in a wide variety of cosmetic and reconstructive indications. Frequently reported cosmetic applications include soft-tissue augmentation of breast, buttocks, hips, face, and hands, whereas reconstructive applications include adjunct for breast reconstruction contour problems, plantar fat pad improvement, and correction of various posttraumatic and surgical contour deformities. Recognition of other regenerative effects of fat grafting expanded the use AFG for improvement of hypertrophic scar tissue, postradiation sequelae, lipodystrophy, hyperpigmentation, senile skin changes, and actinic damage. The popularity of AFG is supported by a remarkably low risk of complications, minimal scars, and readily available donor sites. Despite recognition of the advantages of AFG, there still is no consensus regarding optimal techniques of harvest, graft preparation, and injection. Further, the yield of permanent volume falls within a very wide range. In this article, we review the basic science of fat grafting, proposed methods offered to improve engraftment, and reported outcomes of AFG procedures

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

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

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

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

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