Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

Figures & data

Table 1. Characteristics and details of antibodies used, together with their antigens

Antigens	Primary antibodies	Incubation protocol (primary antibody)	Secondary antibodies^a	Visualization (excitation/emission)
NT5E	Rabbit polyclonal, (ab137595), 1:100	At room temperature for 1 h and then left overnight at 4°C in a humidified chamber	Goat anti rabbit IgG, labeled with Alexa Fluor 488	489 nm/508 nm
THY1	Rabbit monoclonal, (ab92574), 1:50
DLK1	Rabbit polyclonal, (ab21682), 1:200
FABP4	Rabbit polyclonal, (ab85875), 1:100
ENG	Mouse monoclonal, (MA5-11854), 1:50	At room temperature for 1 h and then left overnight at 4°C in a humidified chamber	Goat anti mouse IgG, labeled with Alexa Fluor 594	596 nm/620 nm
PDGFR-α	Rabbit polyclonal (orb6660), 1:50	At 37°C temperature for 1 h and then left overnight at 4°C in a humidified chamber	Goat anti rabbit IgG, labeled with Alexa Fluor 488	489 nm/508 nm
GLUT4	Rabbit polyclonal (ab33780), 1:30
FAS	Rabbit polyclonal (ab22759), 1:50

^aIncubation with secondary antibodies was at room temperature for 45 min.

Table 2. Primers used to amplify specific genes of the bovine subcutaneous ASC and differentiated adipocytes

Gene	Sense (5ʹ-3ʹ)	Anti-sense (3ʹ-5ʹ)	Amplicon size (bp)	RefSeq identifier
NT5E	TTTGGAGGCACCTTTGACC	AGAGGCTCATAACTGGGCAC	212	NM_174129.3
THY1	CAACTTCACCACCAAGGATG	TCTGGATCAGCAGGCTTATG	140	NM_001034765.1
ENG	CCTCAGCGTGAACAAATCC	CGTGAAAGACCAGTTTGGAG	89	NM_001076397.1
FABP4	TGGGATGGAAAATCAACCAC	TGGCTTATGCTCTCTCATAAAC	112	NM_174314.2
RPS19	GGAAAAGGACCAAGATGGGG	CGAACGAGGCAATTTATTAACC	136	NM_001037467.2
YWHAZ	AGAGAGAAAATAGAGACCGAGC	AGCCAAGTAGCGGTAGTAG	144	NM_174814.2
GLUT4	AGCCATGAGCTATGTCTCC	AAGATGAAGAAGCCAAGCAG	255	NM_174604.1
PPARγ	TAAAGAGCCTGCGAAAGCC	GCTTCACGTTCAGCAAACC	156	NM_181024.2
LPL	AGAGTAAAGGCAGGAGAGAC	CAGCCAGACTTTCTATTCAGG	134	NM_001075120.1
PDGFRα	CATCTTTGACAACCTGTACACC	TAGAGTCCACCATCATGCC	113	NM_001192345.3
GAPDH	AAGAAGGTGGTGAAGCAGG	GCATCGAAGGTAGAAGAGTGAG	116	NM_001034034.2

Table 3. Experimental concentrations of ascorbic acid (AsA), foetal bovine serum (FBS) and bovine serum lipids (BSL) in the differentiation media used in Experiment 2

Treatments	AsA (µg/mL)	FBS (%)	BSL (µL/mL)
TRT-Con	0	0	0
TRT-1	0	10	0
TRT-2	0	10	10
TRT-3	0	0	10
TRT-4	40	10	0
TRT-5	40	10	10
TRT-6	40	0	10

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. 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. 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. 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. 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)

Table 4. Cell nuclei index and lipid/nuclei ratio of bovine ASC kept for 7 or 14 d intervals in plastic ware with various coatings and differentiation media containing various concentrations of acetic acid (AcA)

Nuclei index
Factor^a					SEM	P-value
Coating^b	None	poly-L-lysine	Collagen-A	Gelatin-A
	11724^B	12846^A	11767^B	12612^A	242	< 0.001
AcA	0 mM	10 mM	20 mM
	12736^A	12411^A	11565^B		192	< 0.001
Day	7 d	14 d
	12892^A	11583^B			157	< 0.001
Lipid/Nuclei ratio
Factor					SEM	P-value
Coating	None	poly-L-lysine	Collagen-A	Gelatin-A
	0.035	0.029	0.035	0.033	0.004	0.741
AcA	0 mM	10 mM	20 mM
	0.029	0.034	0.036		0.004	0.438
Day	7 d	14 d
	0.026^B	0.041^A			0.003	< 0.001

Non-polar lipids were measured after Nile red staining (excitation/emission, 475 nm/570 nm), whereas nuclei were assessed by DAPI fluorescence (excitation/emission, 358 nm/461 nm).

^aFactor glucose was removed from the analyses after verification that the different glucose concentrations had no significant effect.

^A,BValues with similar capital letters in a single row do not differ statistically at P < 0.05.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–408.

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