Structural Analysis of Murine Versus Human Adipose Tissue Via Three-Dimensional Confocal Microscopy
Charles Blackshear, M.D., Ethan Shen, B.S., Ryan Chase Ransom, B.S., Natalie Chung, B.A., John Flacco, B.S., Dre Irizarry, M.D., Cristhian Montenegro, M.S., Stephanie Vistnes, B.A., Elizabeth Brett, M.S., Michael T. Longaker, M.D., MBA, Derrick C. Wan, M.D..
Stanford University, Stanford, CA, USA.
PURPOSE: Murine models are commonly employed to simulate aspects of human adipose physiology. Tremendous advances in our understanding of human obesity and endocrinology have been made possible by comparative studies in mice. However, fat depots — both within and between species — differ dramatically in their transcriptional properties. Understanding the fundamental differences between human and mouse adipose tissue, and thus the inherent limitations of murine models, begins with comparing their basic architecture. This study employs whole tissue mounting and confocal microscopy to characterize the three-dimensional architecture of the mouse inguinal fat pad, and two significant human fat depots, the lower abdomen and gluteofemoral regions.
METHODS: Abdominal and gluteofemoral adipose tissue specimens were obtained from three operative patients each, in accordance with Stanford Institutional Review Board policy. The patients ranged in age from 35 to 60 years and had no significant prior medical history. Bilateral inguinal fat pads were harvested from three Crl:CD1-Foxn1nu CD-1 Nude mice. The human samples were incubated in Human CD34 PE-Cy 5.5 Conjugate and Phosphate Buffered Saline (PBS) for 24 hours. Similarly, the mouse inguinal fat pad samples were incubated in purified anti-mouse CD34 antibody and PBS for 24 hours. The mouse and human specimens were separately incubated in a staining master mix containing Isolectin endothelial cell stain, LipidTox adipocyte stain (Thermo Fisher Scientific; Waltham, MA), and Hoechst nuclear stain for two hours and then whole-mounted. Laser scanning confocal microscopy was performed using the Leica TCS SP8 X. Three-dimensional volume rendering and analysis was performed on Imaris software (Bitplane AG; Zurich CH).
RESULTS: In general, there are significantly more vessels per adipocyte in mouse compared to human abdominal or gluteofemoral fat (p < 0.05, Fig 1). Quantification of ASC as normalized to adipocyte number shows similar ASC density between species (p = 0.31, Fig 2). Moreover, the ratio of ASC to blood vessels is significantly reduced in mouse compared to human fat (p < 0.05, Fig 2); however, the mean distance between ASC and blood vessels in human fat is significantly greater compared to mouse (p < 0.05, Fig 3). Expectedly, human adipocytes were generally larger and more heterogeneous in size. Frequency distribution of adipocyte volume in mouse and human samples demonstrates significantly greater diversity in human tissue (p < 0.05).
CONCLUSION: The basic architecture of human adipose tissue differs significantly from that of mice. These differences likely confer variance in functional properties between the two sources. Thus, caution should be exercised when drawing parallels between the two species, particularly when designing murine models of human disease.
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