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Research Papers

Cholesterol-Dependent Modulation of Stem Cell Biomechanics: Application to Adipogenesis

[+] Author and Article Information
Shan Sun

Department of Bioengineering,
University of Illinois at Chicago,
Chicago, IL 60607
e-mail: shansun88@gmail.com

Djanybek Adyshev

Department of Medicine,
University of Illinois at Chicago,
Chicago, IL 60607
e-mail: dadyshev@gmail.com

Steven Dudek

Department of Medicine,
University of Illinois at Chicago,
Chicago, IL 60607
e-mail: sdudek@uic.edu

Amit Paul

Department of Bioengineering,
University of Illinois at Chicago,
Chicago, IL 60607
e-mail: amitpaul88@gmail.com

Andrew McColloch

Department of Bioengineering,
University of Texas at Arlington,
Arlington, TX 76019
e-mail: andrew.mccolloch@mavs.uta.edu

Michael Cho

Department of Bioengineering,
University of Texas at Arlington,
Arlington, TX 76019
e-mail: michael.cho@uta.edu

1Corresponding author.

Manuscript received August 17, 2018; final manuscript received March 13, 2019; published online May 6, 2019. Assoc. Editor: Nathan Sniadecki.

J Biomech Eng 141(8), 081005 (May 06, 2019) (10 pages) Paper No: BIO-18-1375; doi: 10.1115/1.4043253 History: Received August 17, 2018; Revised March 13, 2019

Cell mechanics has been shown to regulate stem cell differentiation. We have previously reported that altered cell stiffness of mesenchymal stem cells can delay or facilitate biochemically directed differentiation. One of the factors that can affect the cell stiffness is cholesterol. However, the effect of cholesterol on differentiation of human mesenchymal stem cells remains elusive. In this paper, we demonstrate that cholesterol is involved in the modulation of the cell stiffness and subsequent adipogenic differentiation. Rapid cytoskeletal actin reorganization was evident and correlated with the cell's Young's modulus measured using atomic force microscopy. In addition, the level of membrane-bound cholesterol was found to increase during adipogenic differentiation and inversely varied with the cell stiffness. Furthermore, cholesterol played a key role in the regulation of the cell morphology and biomechanics, suggesting its crucial involvement in mechanotransduction. To better understand the underlying mechanisms, we investigated the effect of cholesterol on the membrane–cytoskeleton linker proteins (ezrin and moesin). Cholesterol depletion was found to upregulate the ezrin expression which promoted cell spreading, increased Young's modulus, and hindered adipogenesis. In contrast, cholesterol enrichment increased the moesin expression, decreased Young's modulus, and induced cell rounding and facilitated adipogenesis. Taken together, cholesterol appears to regulate the stem cell mechanics and adipogenesis through the membrane-associated linker proteins.

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Figures

Grahic Jump Location
Fig. 1

Composite images of hMSCs undergoing adipogenesis at day 7. hMSCs were captured and fluorescently visualized. Actins (red), lipid droplets (greed), and nuclei (blue). These images were recorded using a 60×/1.4 NA microscope objective. Bar = 20 μm.

Grahic Jump Location
Fig. 2

Membrane cholesterol accumulation during adipogenesis. The membrane cholesterol was stained with fresh filipin III (green) and imaged at day 0 (A) and day 7 (B), and overlaid with brightfield images. These images were recorded using a 60×/1.4 NA microscope objective. Bar = 20 μm.

Grahic Jump Location
Fig. 3

Flow cytometry analysis of membrane cholesterol expression during adipogenesis. The membrane cholesterol was stained with fresh filipin and analyzed by a flow cytometer with 360-nm argon laser excitation. Emission was collected using a 424/44 nm bandpass filter. Panel (a), undifferentiated control hMSCs; panels (b)–(d), at days 3, 7, and 10 of postdifferentiation. Panel (e) shows comparison of histograms of filipin at different time points and demonstrates a progressive shift to the right.

Grahic Jump Location
Fig. 4

Change of Young's moduli during adipogenesis: (a) Actual image of an AFM cantilever used to determine the cellular mechanical property and (b) AFM results indicate continuous decrease in the cell stiffness during the first 12 days of adipogenic differentiation. When cytochalasin D was used at day 7 or day 12, a significant decrease in the cell stiffness was observed. The dotted line represents data fitting to an exponential function. (c) Depletion of the membrane cholesterol by MβCD caused the elastic modulus to significantly increase (e.g., stiffer cells), while enrichment of the membrane cholesterol reduced it significantly (e.g., softer cells).

Grahic Jump Location
Fig. 5

Effect of cholesterol depletion on adipogenic differentiation. hMSCs were induced to undergo adipogenesis using the soluble factors and without (control; panels (a), (c), (e)) and with (panels (b), (d), (f)) depleting membrane cholesterol. At day 10, brightfield images of adipocyte-like cells are shown in response to the soluble factors only (a) and with MβCD treatment (b). Corresponding fluorescence images of LipidTox labeling ((c) and (d)) and oil red O staining ((e) and (f)) are consistent with the observation that membrane cholesterol depletion interferes with the intended adipogenesis. The oil red O staining was adopted from a published protocol [25]. Images were taken using a 10× microscope objective. Bar = 100 μm.

Grahic Jump Location
Fig. 6

Modulation of p-ezrin and p-moesin linker proteins by cholesterol. (a) The p-ezrin and p-moesin expressions were analyzed by Western blot. A cholesterol depletion is observed to upregulate p-ezrin but downregulate p-moesin. A cholesterol enrichment seems to induce opposite result. (b) AFM measurements showed the effect of ezrin and moesin on the cell stiffness when cells were treated with siRNA directed against either ezrin or moesin. Knockdown of the ezrin and moesin caused the elastic modulus to decrease or increase, respectively, indicating that ezrin is responsible for stiffening the cell and, in the opposite manner, moesin is required to soften the cells.

Grahic Jump Location
Fig. 7

Immunofluorescence images of ezrin and moesin in response to cholesterol treatment. Distribution of p-ezrin (green) in control hMSCs (a), in cells treated with MβCD (b) or cholesterol enriched (c). Distribution of p-moesin (green) in hMSCs under the same treatment as above ((d), control; (e), cholesterol depleted; and (f), cholesterol enriched). Actins and nuclei were costained with rhodamine-phalloidin (red) and DAPI (blue) in each image. Cholesterol appears to determine the cell morphology. Cholesterol-depleted hMSCs exhibited jagged-edge cell shape, while cholesterol enrichment induced rounded morphology and a reduction in the cell size. Bar = 50 μm.

Grahic Jump Location
Fig. 8

Proposed model for the role of cholesterol in adipogenesis. Based on our findings, we propose a working model in which cholesterol depletion or insertion can disrupt or reinforce the lipid rafts, respectively. Furthermore, cholesterol insertion leads to preferential upregulation of moesin, round cell morphology, a decrease in the cell stiffness and facilitated adipogenesis. In the opposite manner, cholesterol depletion leads to upregulation of ezrin, spread cell morphology, an increase in the cell stiffness and impeded adipogenesis.

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