Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor on the Proliferation and Osteogenic and Neural Differentiation of Adipose-Derived Stem Cells

Isolation and culture of ASCs

ASCs were obtained from 4-week-old female Sprague-Dawley rats (weight, 100–130 grams, n=9), as previously described (Zuk et al., 2001). Briefly, inguinal fat tissue was washed with sterile phosphate-buffered saline (PBS) and digested with 0.1% collagenase I in a water bath at 37°C for 1 h, and the cells were centrifuged at 800 rpm for 5 min. The cells were resuspended in either (1) unsupplemented basal medium (UN medium) only composed of Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, West Sussex, UK), 5% fetal bovine serum (FBS; Thermo Fisher Scientific, West Sussex, UK), and 1% penicillin/streptomycin (Invitrogen Corp., Carlsbad, CA, USA); or (2) EF medium, which consisted of basal medium containing 5 ng/mL EGF (Gibco Lab., NY, USA) and 10 ng/mL bFGF (Peprotech, NJ, USA). The cells were seeded into T25 flasks and cultured at 37°C in 5% CO₂. Approximately 1 day after initial plating, the media were removed and replaced with fresh media to remove nonadherent cells. On day 7 (D7), the cells were washed with PBS, incubated in 0.25% trypsin (Gibco Lab., NY, USA) at 37°C for 2 min, and subcultured at 1:3. The cells were routinely subcultured every 3 days thereafter. All experimental procedures were conducted according to Southeast University, Medical Faculty ethic committee approval.

Analysis of ASC morphology, proliferation, and cell cycle distribution

On D20, the morphology of ASCs cultured in UN or EF medium at 90% confluence was assessed by bright-field light microscopy (Zeiss Ti-S Germany, Oberkochen, Germany). Then, the same cells were incubated in 0.25% trypsin and plated into 12-well plates at 1×10³ cells/mL; the cell numbers were counted daily for 8 continuous days. The cell numbers were calculated in the log phase of the resulting growth curves. Cell cycle analysis was performed using propidium iodide (PI; Sunshine, Nanjing, China) staining and flow cytometry (Accuri C6; BD, Michigan, USA). The cells were detached, centrifuged, fixed with 70% cold (4°C) ethanol for 0.5 h, and 1 mL of 50 μg/mL PI and 20 μg/mlL RNase A (Sunshine, Nanjing, China) were added. The cells were incubated for 0.5 h in the dark and filtered through a 100-μm nylon mesh to remove clumps. Data analysis was performed using C-Flow software (BD, Michigan, USA) and FlowJo analysis software (FlowJo; Ashland, OR, USA). All experiments were performed in triplicate.

Osteogenic and neural differentiation

On D20, ASCs cultured in UN or EF medium were seeded into 12-wells plate at a density of 1×10³ cells/ mL. After 1–2 days, the media were replaced with osteogenic differentiation medium, which was replaced every 3 days for a period of 15 days. The osteogenic differentiation medium was UN or EF medium supplemented with 10 mmol/L glycerol phosphate disodium salt hydrate, 10 nmol/L dexamethasone, 50 μmol/L L-ascorbic acid sodium salt, 300 mg/L L-glutamine, and 10 nmol/L 1α,25-dihydroxyvitamin D3 (Sigma-Aldrich, St. Louis, MO, USA).

After 7 days induction, an alkaline phosphatase (AP) detection kit (Amresco, Solon, OH, USA) with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) as a substrate was used to assess osteogenic differentiation. The cells were fixed with 4% paraformaldehyde for 30 min, rinsed three times with PBS, and BCIP/NBT was added. The cells were incubated for 30 min, and the samples were rinsed once with water and observed using bright-field light microscopy. Neural differentiation was also assessed after 15 days induction using immunofluorescent staining and PCR, as described below.

Neural differentiation was performed in a similar manner over a period of 15 days using UN or EF medium supplemented with 100 ng/mL retinoic acid (RA; Sigma-Aldrich, St. Louis, MO, USA). Neural differentiation was assessed after 15 days induction using immunofluorescent staining and PCR, as described below.

Immunofluorescent analysis

Immunofluorescent analysis was employed to detect the expression of stemness markers in cells cultured in UN or EF medium. After 20 days of culture, the cells were fixed with 0.5% formaldehyde containing 0.2% Triton X-100 in PBS buffer at pH 7.4 for 5 min at room temperature, rinsed once with PBS, fixed again in 4% formaldehyde in PBS for 20 min, and rinsed three times with PBS. The samples were incubated with rabbit anti-rat primary antibodies against the stemness markers Oct4 and Sox2 (1:100, Santa Cruz Biotechnology, CA, USA) in 1% bovine serum albumin (BSA; Sunshine, Nanjing, China) at 4°C for at least 12 h.

After 15 days of induction in UN and EF osteogenic differentiation medium, ASCs were subjected to immunofluorescent staining using rabbit anti rat osteocalcin (Ocn) primary antibody (1:100; in 1% BSA; Santa Cruz Biotechnology, CA, USA) to detect differentiated osteogenic cells. After 15 days induction in UN and EF neural differentiation media, ASCs were subjected to immunofluorescent staining using rabbit anti-rat nestin and glial fibrillary acidic protein (Gfap) primary antibodies (1:100 in 1% BSA, Santa Cruz Biotechnology, CA, USA) to detect differentiated neural cells.

After washing with PBS, the samples were incubated with the appropriate secondary antibody (1:200 in 1% BSA; goat anti-rabbit Alexa-Fluor 488 or donkey anti-rabbit Alexa-Fluor 647; Invitrogen Corp., Carlsbad, California, USA) for 1 h at 37°C while protected from light. Then, the samples were washed twice with PBS, the nuclei were detected by staining with 10 μg/mL Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min while protected from light, and images were obtained using a Revolution XD confocal laser scanning microscope (Andor, Belfast, Northern Ireland).

RNA isolation, reverse transcription polymerase chain reaction, and real-time quantitative fluorescence PCR

ASCs cultured in EF or UN medium were harvested on D10, D20, and D30, and the expression of Oct4, Sox2, Klf4, Nanog, and Lin28a was measured to assess the changes in ASC stemness markers. After induction with osteogenic medium for 15 days, the expression of Alp, Ocn, core binding factor alpha (Cbfa), and collagen type I (Col) was quantified to assess osteogenic differentiation. After induction with neural medium for 15 days, the expression of Nestin, Gfap, and microtubule-associated protein-2 (Map2) was quantified to assess neural differentiation. Glycerol-3-phosphate dehydrogenase (Gapdh) was used as a control.

Total cellular RNA was isolated using the TRIzol method (Invitrogen Corp., Carlsbad, California, USA). DNase-treated total RNA (20 μL total volume) was incubated with 1 μL of 50 mM oligo(dT18) (TaKaRa, Dalian, China). After denaturation, 5× buffer, dNTPs, RNase inhibitor, and PrimeScript Reverse Transcriptase (TaKaRa, Dalian, China) were added, as specified by the manufacturer's protocol. Reverse transcription was performed for 60 min at 42°C, followed by 5 min at 85°C to inactivate the reverse transcriptase.

Reverse transcription polymerase chain reaction (RT-PCR), and real-time quantitative fluorescence PCR (qRT-PCR) were performed using standard protocols. Specific primers were designed for each gene based on the GenBank sequences; the primer sequences are listed in Table 1. RT-PCR was carried out at 95°C for 1 min, 95°C for 15 sec, 57°C/60°C for 40 sec, and 72°C for 40 sec (28–37 cycles). qRT-PCR was performed on the Applied Biosystems 7500 Sequence Detection System (BD, Michigan, USA). Briefly, 1 μL cDNA was added to 10 μL of 2× SYBR Green PCR master mix (TaKaRa, Dalian, China) and 200 nM of each primer in a total volume of 20 μL. The reactions were amplified over 40 cycles of 95°C for 15 sec and 60°C for 1 min. Afterward, a thermal denaturation protocol was performed to determine the number of products present in each reaction. The reactions were typically run in triplicate. The cycle number at which the reaction crossed an arbitrarily placed threshold (Ct) was determined for each gene. Target gene expression was normalized to the expression of Gapdh in each sample. Data were analyzed using the 2^−ΔΔCt method(Livak et al., 2001).

Statistical analysis

All data were expressed as the mean &plumn; standard deviation (SD). Differences were compared using the Student t-test; p values <0.05 were considered statistically significant (*p<0.05, **p<0.01).

EGF and bFGF enhance the proliferation of ASCs

ASCs were isolated from adipose tissue, according to a previously published protocol, and the influence of EFG and bFGF on cell morphology, proliferation, and cell cycle distribution was analyzed. ASCs grew in colonies and continuously expanded until they approached confluency; the cells displayed stable and rapid growth in vitro. The expression of ASC surface markers was confirmed by flow cytometry (data not shown). ASCs cultured in UN medium displayed a heterogeneous morphology; most of the cells had a broad, flattened morphology and extended pseudopods (Fig. 1a). In contrast, ASCs cultured in EF medium displayed evident central nucleoli, their cytoplasm contained elongated and spindle-shaped processes, and short protrusions appeared on the surface of the cell bodies (Fig. 1b).

To investigate the biological effects of EGF and bFGF on ASCs, the number of cells was recorded every day for 8 days and cell cycle distribution was analyzed by flow cytometry. ASCs displayed logarithmic growth curves in both UN and EF media. After an initial lag or stationary period, the cells expanded rapidly in a logarithmic manner, until they reached a plateau. However, ASCs grew significantly faster in the presence of EGF and bFGF (Fig. 1c). On day 8, the number of cells in the EF group was three-fold higher than the UN group.

The cell cycle analysis (Fig. 1d, e) indicated that, compared to UN medium, the ratio of G1-phase ASCs in the EF medium decreased from 79.49% to 71.88% (p<0.05), and the ratio of G2/M phase increased from 8.21% to 12.30% (p<0.05). These data demonstrate that EGF and bFGF stimulate the growth of ASCs and enhance their proliferative capacity.

EGF and bFGF alter the pluripotency of ASCs

Alhough EGF and bFGF can stimulate ASC proliferation, the effect of these growth factors on the maintenance of stemness should be carefully investigated. Oct4 and Sox2, markers of undifferentiated stem cells, are critically involved in self-renewal (Driessens et al., 2011; Wang et al., 2012b). Immunofluorescent staining was used to detect the expression of both Oct4 and Sox2 in ASCs cultured in UN and EF media for 20 days (Fig. 2a, b, c, d). The level of Oct4 expression in ASCs cultured in EF medium was lower than in UN medium (Fig. 2 a, b). However, the expression of Sox2 was higher in ASCs cultured in EF medium (Fig. c, d).

RT-PCR and qRT-PCR were used to assess the pluripotency of ASCs cultured in EF medium on D10, D20, and D30. As shown in Figure 2e, the levels of Sox2, Klf4, and Nanog initially increased by D20 and were further upregulated by D30. The expression of Sox2, Klf4, and Nanog increased 3.45-fold, 9.47-fold, and 3.89-fold by D30 compared to D10 (Fig. 2f). In contrast, the expression of Oct4 and Lin28a was downregulated 2.38-fold and 1.76-fold, respectively (Fig. 2f). These results indicate that culture in medium supplemented with 5 ng/mL EGF and 10 ng/mL bFGF may affect the pluripotent state of ASCs.

EGF and bFGF impair the osteogenic differentiation potential of ASCs

The results in this study demonstrated that EGF and bFGF increased the expression of Sox2 and decreased the expression of Oct4 in ASCs, which indicates that EGF and bFGF may induce ASCs to undergo neural lineage differentiation and impair their ability to undergo osteogenic differentiation. To investigate this, osteogenic differentiation was induced by culturing ASCs in UN or EF medium containing glycerol phosphate disodium salt hydrate, dexamethasone, L-ascorbic acid sodium salt, L-glutamine, and 1α, 25-dihydroxyvitamin D3. As shown in Figure 3, a and b, ALP activity significantly reduced after 7 days in the cells cultured in osteogenic EF medium, compared to ASCs cultured in osteogenic UN medium. By D15, the level of Ocn, a late osteogenic marker, was markedly attenuated in ASCs cultured in osteogenic EF medium (Fig. 3c, d). RT-PCR confirmed that ASCs cultured in osteogenic EF medium expressed significantly lower levels of the osteogenic markers Alp, Ocn, Col, and Cbfa (Fig. 3e). qRT-PCR revealed that expression of Alp, Ocn, Col, and Cbfa decreased 1.4-fold, 3.8-fold, 4.1-fold, and 6.2-fold in ASCs cultured in osteogenic EF medium, compared to ASCs cultured in osteogenic UN medium (Fig. 3f). The results suggested that the osteogenic potential of ASCs was impaired by culture in EGF and bFGF medium.

EGF and bFGF enhance the neural differentiation potential of ASCs

Next, the ability of ASCs cultured in UN or EF medium to differentiate into the neural lineage after the addition of RA was investigated. Morphologically, ASCs cultured in medium containing RA retracted continuously to form a sphere-shaped group of cells (Fig. 4a). Subsequently, the cells migrated from the sphere and formed simple bipolar-shaped cells. ASCs cultured in UN medium containing RA for 15 days expressed Nestin, an early neuronal marker, but did not express detectable levels of Gfap (Fig. 4b, d, f). Conversely, ASCs cultured in EF medium containing RA displayed retractile cell bodies with highly branched, complex multipolar structures and expressed Gfap and low levels of Nestin (Fig. 4c, e, g). Immunofluorescent staining confirmed that these markers were continuously expressed for at least 2 weeks, which indicated that ASCs cultured in EF neural differentiation medium terminally differentiated into neural lineage cells. In contrast, the mature neural proteins Gfap and Map2 were barely detectable in the cells cultured in UN neural differentiation medium. Nevertheless, the ASCs derived from EF neural differentiation medium expressed Gfap and Map2, but not Nestin. Quantification revealed that expression of Nestin decreased 9-fold, whereas Gfap and Map2 were upregulated 21.9-fold and 18.9-fold, respectively, in ASCs derived from EF neural differentiation medium, compared to UN neural differentiation medium (Fig. 4i).

These data suggested that ASCs cultured in neural differentiation medium containing EGF and bFGF successfully transdifferentiated into mature neural cells, whereas the cells induced in UN neural differentiation medium only generated neural progenitors. These results show that ASCs cultured in EGF and bFGF will differentiate into neural cells rather than osteocyte lineage cells when treated with RA.