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, 9 (1), 35-50

Optimizing Adipogenic Transdifferentiation of Bovine Mesenchymal Stem Cells: A Prominent Role of Ascorbic Acid in FABP4 Induction

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Optimizing Adipogenic Transdifferentiation of Bovine Mesenchymal Stem Cells: A Prominent Role of Ascorbic Acid in FABP4 Induction

Sandra Jurek et al. Adipocyte.

Abstract

Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation.Abbreviations: AcA: acetic acid; AsA: ascorbic acid; ASC: adipose-derived stem cells; BSL: bovine serum lipids; DAPI: 4´,6-diamidino-2-phenylindole; DLK: delta like non-canonical notch ligand; DMEM: Dulbecco's modified Eagle's medium; DPBS: Dulbecco's phosphate-buffered saline; ENG: endoglin; FABP: fatty acid binding protein; FAS: fatty acid synthase; GLUT4: glucose transporter type 4; IBMX: 3-isobutyl-1-methylxanthine; LPL: lipoprotein lipase; MSC: mesenchymal stem cells; α-MEM: α minimum essential medium; NT5E: ecto-5'-nucleotidase; PDGFRα: platelet derived growth factor receptor α; PPARγ: peroxisome proliferator activated receptor γ; RPS19: ribosomal protein S19; SEM: standard error of the mean; THY1: Thy-1 cell surface antigen; TRT: treatment; TRT-Con: treatment negative control; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta.

Keywords: Adipocytes; adipose tissue; animal models; fatty acid binding protein; lipid droplets.

Figures

Figure 1.
Figure 1.
Verification of ASC identity. Representative phase contrast microscopic images of (a) pre-adipocytes before induction and (b) developed adipocytes after induction and 14 days in differentiation medium. Lipid droplets are amply present in differentiated adipocytes of graph b. The immunocytochemistry of undifferentiated pre-adipocytes identifies (c) the presence of NT5E (green) and ENG (red), as well as (d) recognition of THY1 (green) and ENG (red). For comparison, inverted light microscopic images using alizarin red staining are shown after 21 days (c) in control medium or (d) in osteogenic differentiation medium. The scale bar is representative of 100 µm in panels a and b (using a 20× objective), 25 µm in panels c and d (using a 63× objective), and 100 µm in panels e and f (using a 10× objective).
Figure 2.
Figure 2.
Influence of a gradual decrease in the concentrations of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine (IBMX) and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [22]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P < 0.05 to day 5.
Figure 3.
Figure 3.
Nile red staining of lipids in adipocytes after 2 d in induction medium followed by 7 d or 14 d of incubation in various adipocyte differentiation media. Nile red was imaged at (excitation/emission) 515 nm/590 nm for total lipids (coded to red) and 475 nm/570 nm (emission) for non-polar lipids (coded to green). The green-red overlay results in bright yellow colour for lipid droplets. Nuclei were stained with DAPI and imaged at 358 nm/461 nm (emission). Scale bar = 100 µm (20× objective). The graph A shows quantification of non-polar lipids (475 nm/570 nm) relative to DAPI fluorescence (358 nm/461 nm) for three independent experiments with two replicates while graph B shows corresponding DAPI fluorescence. The data symbolize means ± SEM. a,bMean values with different superscripts are significantly different (P < 0.05). TRT, treatment; Con, negative control; AsA, ascorbic acid; BSL, bovine serum lipids; FBS, foetal bovine serum.
Figure 4.
Figure 4.
Immunocytochemical classification of bovine subcutaneous adipose-derived stem cells at different stages of development analysed for the presence of different ASC and adipocyte markers. All images were made after 14 d in different types of differentiation media (TRT-1 through TRT-6; for code see Table 3). The colour code of the antigen labels represents the fluorophore colour of the imaged antigen (red or green) and nuclear stain (DAPI, blue), respectively. Scale bar = 25 µm (63 × objective).
Figure 5.
Figure 5.
Relative mRNA expression for NT5E, THY1, ENG, PDGFRα, FABP4, LPL, GLUT4 and PPARγ in bovine subcutaneous ASC after in vitro transdifferentiation. Quantitative reverse transcription PCR analysis of NT5E (A), THY1 (B), ENG (C), PDGFRα (D), FABP4 (E), LPL (F), GLUT4 (G) and PPARγ (H) are given as the mean ± SEM of four animals with three replicates. Pre, pre-adipocyte (RNA extracted before induction); Con, negative control; TRT = treatment; FBS, foetal bovine serum; AsA, ascorbic acid; BSL, bovine serum lipids. Letters (a-e) are used to denote statistical difference; columns within one graph are different if they do not share a common letter (P < 0.05).

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Grant support

This work was supported by the Alexander von Humboldt Foundation [Georg Forster Research Fellowship];City of Berlin [Elsa-Neumann Grant].
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