Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8 (10)

Subcutaneous and Visceral Adipose-Derived Mesenchymal Stem Cells: Commonality and Diversity

Affiliations

Subcutaneous and Visceral Adipose-Derived Mesenchymal Stem Cells: Commonality and Diversity

Andreas Ritter et al. Cells.

Abstract

Adipose-derived mesenchymal stem cells (ASCs) are considered to be a useful tool for regenerative medicine, owing to their capabilities in differentiation, self-renewal, and immunomodulation. These cells have become a focus in the clinical setting due to their abundance and easy isolation. However, ASCs from different depots are not well characterized. Here, we analyzed the functional similarities and differences of subcutaneous and visceral ASCs. Subcutaneous ASCs have an extraordinarily directed mode of motility and a highly dynamic focal adhesion turnover, even though they share similar surface markers, whereas visceral ASCs move in an undirected random pattern with more stable focal adhesions. Visceral ASCs have a higher potential to differentiate into adipogenic and osteogenic cells when compared to subcutaneous ASCs. In line with these observations, visceral ASCs demonstrate a more active sonic hedgehog pathway that is linked to a high expression of cilia/differentiation related genes. Moreover, visceral ASCs secrete higher levels of inflammatory cytokines interleukin-6, interleukin-8, and tumor necrosis factor α relative to subcutaneous ASCs. These findings highlight, that both ASC subpopulations share multiple cellular features, but significantly differ in their functions. The functional diversity of ASCs depends on their origin, cellular context and surrounding microenvironment within adipose tissues. The data provide important insight into the biology of ASCs, which might be useful in choosing the adequate ASC subpopulation for regenerative therapies.

Keywords: adipose-derived mesenchymal stem cells; differentiation; migration; primary cilium; secretion; sonic hedgehog signaling.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Subcutaneous and visceral adipose-derived mesenchymal stem cells (ASCs) display comparable cell surface marker profiles, cell cycle distribution and cell proliferation. (A) Immunofluorescence staining of mesenchymal stem cell surface markers CD90 (green) and CD73 (red), and DNA (DAPI, blue) in subcutaneous ASCs (ASCsub) and visceral ASCs (ASCvis). Scale: 20 μm. (B) Flow cytometric analyses of positive cell surface markers CD90, CD73, CD146, and CD105, and negative markers CD14, CD31, CD106, and CD34 for mesenchymal stem cells (MSCs). Values represent the percentages of ASCs expressing the indicated protein. The results from eight independent experiments (donors) are presented as mean ± standard error of the mean (SEM). (C,D) Cell cycle distribution was analyzed using a FACSCaliburTM. Profile examples were shown (C). Cell cycle phases of ASCs were presented in percentage and the results were derived from four independent experiments (D). (E,F) ASCs were stained for pHH3 (S10) (green), α-tubulin (yellow), pericentrin (red) and DNA (blue), and representatives are shown (E). Scale: 10 μm. pHH3 positive cells were quantified in ASCsub and ASCvis (F). The results are from three independent experiments with ASCs from three different donors and presented as median ± min/max whiskers in box plots. n.s. > 0.05. (G) Cellular extracts from ASCs were prepared for Western blot analyses with indicated antibodies. β-actin served as loading control. (H) ASCs were seeded in 96-well plates for 0, 24, 48, 72, and 96 h. Cell viability was measured via CellTiter-Blue® assay. The results are presented as mean ± SEM and statistically analyzed, showing no significant difference (n.s.).
Figure 2
Figure 2
Both ASC subtypes display a comparable motility rate, but subcutaneous ASCs have a significantly higher directed migration capacity. (A,B) Wound healing/migration assays were performed with subcutaneous and visceral ASCs, and images were taken at indicated time points (0, 8, 16, 24 h) to document the migration front. (A) Representatives are shown. White dashed line depicts the migration front. Scale: 300 μm. (B) Quantification of the open area between both migration fronts at various time points. The cell-free area at 0 h was assigned as 100%. The results from three independent experiments are presented as mean ± SEM. *** p < 0.001. (C,D) Time-lapse microscopy was performed with subcutaneous or visceral ASCs for up to 12 h. Random motility of these cells was analyzed. (D) Representative trajectories of individual cells (n = 30) are shown. (C) Evaluated accumulated distance (left), velocity (middle), and directionality (right) from three independent experiments are shown as box plots with variations. Unpaired Mann–Whitney U-test, * p < 0.05, *** p< 0.001. (E,F) Invasion assay. ASCs were seeded into transwells and starved for 12 h. The cells were released into fresh medium for 24 h and fixed for quantification. (E) Quantification of invaded cells per field in percent. The results from three independent experiments are presented as mean ± SEM. Student’s t test was performed showing no significant difference (n.s. > 0.05). (F) Representatives of invaded ASCs are shown. Scale: 25 μm. (G,H) Homing assays. ASCs and breast cancer cells were seeded in separated chambers of a culture insert and cultured for 0, 8 and 15 h. (G) Evaluation of cell homing distance, the length between the nucleus and the outermost cell protrusion, in subcutaneous and visceral ASCs toward MCF-7 cells (left), MDA-MB-231 cells (middle), and ASC themselves (right). Each experiment was performed in triplicate, and the results are based on three independent experiments and presented as scatter plot showing mean ± SEM. (red dashed line indicates median value of ASCsub). ** p < 0.01, *** p < 0.001. (H) Representatives of ASCs on both migration fronts stained against phalloidin (red) and DAPI (blue) are depicted. White bars indicate cellular protrusion length. Scale: 50 μm.
Figure 3
Figure 3
Subcutaneous ASCs show a typical mesenchymal-like phenotype compared to their visceral counterparts. (A) Immunofluorescence staining of ASCsub and ASCvis. ASCs were stained for phalloidin (green), paxillin (red) and DNA (blue) to underline their cell morphology. Examples are shown. Scale: 25 μm. (B) Cellular extracts from ASCs were prepared for Western blot analyses with antibodies against β-actin, E-cadherin, fibronectin and vimentin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (C,D) Gene levels of mesenchymal associated transcription factors and cytoskeleton proteins ZEB1, SNAIL, TWIST, VIM, and EpCAM are shown for subcutaneous and visceral ASCs. The results are from three experiments, presented as RQ with minimum and maximum range. RQ, relative quantification of gene expression. Student’s t test, ∗ p < 0.05. (EK) The focal adhesion composition was analyzed by staining ASCs for focal adhesion kinase (FAK) (green), p-FAK (red), and DNA (DAPI, blue), or for p-paxillin (red), paxillin (green) and DNA (DAPI, blue) for fluorescence microscopy. Quantification of the mean fluorescence intensity of FAK (E), p-FAK (F), paxillin (G) p-paxillin (H), and focal adhesion area (I) in ASCsub versus ASCvis (at least 200 FAs per staining). The results are based on three independent experiments and presented as scatter plot showing mean ± SEM. Unpaired Mann–Whitney U-test, ** p < 0.01, *** p < 0.001. a.u., arbitrary units. Representatives are depicted (J,K). Scale: 25 μm.
Figure 4
Figure 4
Subcutaneous ASCs dynamically disassemble and reassemble their FAs. (A) Schedule of the nocodazole washout assay. (B) ASCs were incubated for 5 h with 10 µM nocodazole followed by washout, where the microtubules (MTs) were allowed to regrow for 0, 30 and 75 min. Cells were stained for paxillin (green), p-FAK (red), and DNA (DAPI, blue). Representatives of FA disassembly/reassembly are shown. Scale: 15 µm. (CE) Kinetics of FA disassembly during MT regrowth and FA reassembly after MT regrowth. Quantification of the mean fluorescence intensity of paxillin (C), p-FAK (D), and FA size (E) (270 FA per condition) is depicted. The results are based on three independent experiments and presented as scatter plots showing mean ± SEM. Unpaired Mann–Whitney U-test, ** p < 0.01, *** p < 0.001. a.u., arbitrary units.
Figure 5
Figure 5
Visceral ASCs are superior in osteogenic and adipogenic differentiation compared to subcutaneous ASCs. (A) Gene levels of stemness/self-renewal associated genes c-MYC, SOX2, KLF4, and NANOG are shown for subcutaneous and visceral ASCs. The results are from three experiments, presented as RQ with minimum and maximum range. Student’s t test, ∗ p < 0.05, ** p < 0.01. (BD) ASCsub and ASCvis cells were induced into osteogenic differentiation for up to 14 days. The percentage of differentiated ASCs was evaluated by Alizarin Red S staining. (B) The quantification data are presented as median ± min/max whiskers (red dashed line indicates median value of ASCsub, n = 300 cells for each condition, pooled from three experiments). Student’s t test, ∗p < 0.05. (C) Example images for Alizarin Red S staining are shown. Scale: 20 μm. (D) Expression levels of differentiation related genes PTCH1 (1st graph), RUNX2 (2nd graph), KLF4 (3rd graph), and c-MYC (4th graph) in differentiated subcutaneous and visceral ASCs. The results are from three experiments and presented as RQ with minimum and maximum range. Student’s t test, ∗ p < 0.05, ** p < 0.01. (EG) Analyses of cells with lipid vacuoles after 14 days of adipogenic differentiation. (E) The quantification shows the percentage of cells differentiated into adipogenic-like cells. Results are presented as median ± min/max whiskers in visceral ASCs (n = 200 cells for each condition, pooled from three experiments) and the red dashed line illustrates the median value of ASCsub. Student’s t test, ∗ p < 0.05. (F) Representative images of cells displaying lipid vacuoles stained for adiponectin (red) and DNA (DAPI, blue). (G) Gene levels of ADIPOQ, LEPTIN, and PPARγ after adipogenic differentiation are shown for subcutaneous and visceral ASCs. The results are from three experiments, presented as RQ with minimum and maximum range. Student’s t test, ∗ p < 0.05.
Figure 6
Figure 6
Visceral ASCs display high deciliation gene levels and enhanced activation of the sonic hedgehog (Hh) signaling pathway. (A) Primary cilia of ASCsub and ASCvis were stained for acetylated α-tubulin and Arl13b. Representatives are shown. Scale: 10 μm. Regions outlined in boxes are shown in a higher magnification. Inset scale: 10 µm. (B) The cilium length was evaluated. The results are based on six experiments using ASCs from six donors (n = 180 cilia for each group). (C) Ciliated ASCs were evaluated and the results are presented as mean ± SEM (n = 600 cells, pooled from six experiments). Unpaired Mann–Whitney U-test, * p < 0.05. (D) The gene levels of deciliation regulators PLK1, PLK4, and KIF2A. The data are based on three experiments and presented as RQ with minimum and maximum range. Student’s t test, ∗ p < 0.05, ** p < 0.01. (EG) Fluorescence intensities and expression levels of important genes related to the Hh pathway are shown for ASCs treated with SAG for 24 h. (E) Each point of the curve represents the mean fluorescence intensity (mean ± SEM) based on three experiments (n = 30 cilia). Unpaired Mann–Whitney U-test, * p < 0.05. (F) Representatives are shown for measurements of primary cilium staining of acetylated α-tubulin, Arl13b and Smoothened (Smo). Scale: 3 μm. (G) The gene levels of GLI1, PTCH1, NANOG, SMO, and TP53 are shown for ASCs treated or non-treated with 200 nM SAG for 24 h. The results are from three experiments, merged as biological group, and presented as mean ± SEM. Student’s t test, ∗ p < 0.05, ** p < 0.01.
Figure 7
Figure 7
Visceral ASCs secrete more pro-inflammatory cytokines. (A) The supernatants of subcutaneous and visceral ASCs in the third passage were collected after 72 h culture and used for evaluation of IL-6 (left), IL-8 (middle) and TNF-α (right) by enzyme-linked immunosorbent assay (ELISA). The results are from four experiments and presented as median ± min/max whiskers in box plots. Student’s t test, p < 0.05, ** p < 0.01. (B) The gene levels of IL-6, IL-8, IL-10, and TNFα. The data are based on three experiments and presented as RQ with minimum and maximum range. Student’s t test, p < 0.05. (C) Schematic illustration of the proposed similarities and dissimilarities between both ASC subtypes. The key dissimilarities between subcutaneous and visceral ASCs are their migration mode, differentiation capacity, and cytokine secretion, which affect a variety of different pathways, like the Hh signaling on the primary cilium.

Similar articles

See all similar articles

References

    1. Luong Q., Huang J., Lee K.Y. Deciphering White Adipose Tissue Heterogeneity. Biology. 2019;8:23 doi: 10.3390/biology8020023. - DOI - PMC - PubMed
    1. Natarajan S.K., Rasineni K., Ganesan M., Feng D., McVicker B.L., McNiven M.A., Osna N.A., Mott J.L., Casey C.A., Kharbanda K.K. Structure, Function And Metabolism Of Hepatic And Adipose Tissue Lipid Droplets: Implications In Alcoholic Liver Disease. Curr. Mol. Pharmacol. 2017;10:237–248. doi: 10.2174/1874467208666150817111727. - DOI - PMC - PubMed
    1. McNamara J., Huber K. Metabolic and Endocrine Role of Adipose Tissue During Lactation. Annu. Rev. Anim. Biosci. 2018;6:177–195. doi: 10.1146/annurev-animal-030117-014720. - DOI - PubMed
    1. Tchkonia T., Thomou T., Zhu Y., Karagiannides I., Pothoulakis C., Jensen M.D., Kirkland J.L. Mechanisms and metabolic implications of regional differences among fat depots. Cell Metab. 2013;17:644–656. doi: 10.1016/j.cmet.2013.03.008. - DOI - PMC - PubMed
    1. Ibrahim M.M. Subcutaneous and visceral adipose tissue: Structural and functional differences. Obes. Rev. 2010;11:11–18. doi: 10.1111/j.1467-789X.2009.00623.x. - DOI - PubMed

Publication types

Feedback