Huade Technology丨Effects of EGF and FGF-2 on the Proliferation and Differentiation of Neural Crest Stem Cells in Porcine Subcutaneous Fat

Huade Technology丨Effects of EGF and FGF-2 on the Proliferation and Differentiation of Neural Crest Stem Cells in Porcine Subcutaneous Fat

Dong, B.; Cui, X.; Bai, R.; Zheng, M.; Zhang, L.; Lyu, X.; Ren, Y. Effects of EGF and FGF-2 on the proliferation and differentiation of neural crest stem cells derived from porcine subcutaneous fat [J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(12): 4878–4886.
Available from: https://kns.cnki.net/kcms2/article/abstract?v=ZIcs-hjNidHqDCHVR3xoWE_cbdabOsBfdGpgfAOsXhm5x76Zt7aqUc-EyXV0iRoRbi3DUamh1MVfUIeGwN_Iu6PCNlA45giz_kbcbk3itpsb88_8FaNCEhmsp9HK1taUOuk4venT292rf7B3-t5KFfSE4kVv7lAtuMTeTozAvPs=&uniplatform=NZKPT

Abstract

[Objective] This study aims to investigate the effects of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) on the proliferation and differentiation of neural crest stem cells (NCSCs) derived from porcine subcutaneous fat, with the goal of optimizing the culture conditions for these cells. 

[Methods] Primary NCSCs were isolated and cultured in vitro from porcine subcutaneous fat, and the expression of the NCSC marker p75 NTR was confirmed by immunofluorescence staining. Different concentrations of EGF and FGF-2 (0 and 0, 10 and 10, 10 and 20, 20 and 10, 20 and 20, 30 and 30 ng/mL) were applied to passaged porcine subcutaneous fat NCSCs. The cell proliferation rate was measured using a CCK-8 assay kit to determine the optimal concentrations of EGF and FGF-2 for cell growth. The experiment was divided into a blank control group and an optimal concentration group. The growth curves of both groups were plotted, and after adipogenic induction, Oil Red O staining was performed to compare the amount of lipid droplet formation in the two groups. 

[Results] Immunofluorescence staining results showed that primary porcine subcutaneous fat NCSCs tested positive for p75 NTR. The CCK-8 cell proliferation assay indicated that the combination of 20 ng/mL EGF and 20 ng/mL FGF-2 had the most significant promoting effect on the proliferation of passaged porcine subcutaneous fat NCSCs. The growth curve revealed that cells in both groups entered the logarithmic growth phase between days 5 and 9, while cell proliferation slowed from days 10 to 15, gradually reaching the plateau phase. Oil Red O staining results demonstrated that the optimal concentration group exhibited significantly more lipid droplet formation in the cytoplasm compared to the control group. 

[Conclusion] The addition of 20 ng/mL EGF and 20 ng/mL FGF-2 to the culture medium promotes the proliferation and differentiation of porcine subcutaneous fat NCSCs.

Introduction

This study aims to investigate the effects of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) on the proliferation and differentiation of neural crest stem cells (NCSCs) derived from porcine subcutaneous fat, with the goal of optimizing their in vitro culture conditions. NCSCs possess pluripotency and regenerative potential, making them widely applicable in tissue engineering and disease treatment. By elucidating the synergistic effects of EGF and FGF-2 and determining their optimal concentrations, this research provides a theoretical foundation for establishing an efficient in vitro culture system for NCSCs, thereby advancing their applications in regenerative medicine. 

Materials and Methods

Primary neural crest stem cells (NCSCs) were isolated and cultured from neck adipose tissue of healthy one-day-old Danish binary piglets. The cells were identified by immunofluorescence staining targeting the specific marker protein p75NTR. Primary cells were passaged and cultured in medium supplemented with varying concentrations of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). The cell proliferation rate was measured using a CCK-8 assay kit, which determined 20 ng/mL as the optimal concentration. Growth curves were plotted for both the EGF- and FGF-2-treated group and the blank control group. The amount of lipid droplet formation after adipogenic induction was compared between the two groups.

Results
Morphological observation of neural crest stem cells (NCSCs) derived from porcine subcutaneous adipose tissue

Porcine subcutaneous adipose-derived NCSCs were isolated using the tissue explant adherence method. Two days after the adipose tissue was cultured by adherence, a small number of elongated spindle-shaped cells were observed migrating out from the tissue (Fig. 1A). After 5 days of adherence culture, the number of cells increased and extended outward (Fig. 1B). By 9 days of adherence culture, the cell number surged, arranged densely in a swirling pattern (Fig. 1C), with the darker areas at the periphery representing the subcutaneous adipose tissue.

Identification of Porcine Subcutaneous Adipose-Derived Neural Crest Stem Cells (NCSCs)

Immunofluorescence staining for the NCSC-specific protein marker p75 NTR showed positive results (Fig. 2).

The Effects of EGF and FGF-2 on the Proliferation of Porcine Subcutaneous Adipose-Derived Neural Crest Stem Cells (NCSCs)

As shown in Table 1, after 24 hours of treatment with EGF and FGF-2, the D450 nm values of groups T1 to T5 were significantly higher than those of group C (P < 0.01), while no significant differences were observed among groups T1 to T5 (P > 0.05). After 48 hours of treatment, the D450 nm values of groups T1 to T5 remained significantly higher than those of group C (P < 0.01). Furthermore, the D450 nm value of group T5 was significantly higher than that of group T2 (P < 0.05) and extremely significantly higher than that of group T4 (P < 0.01). After 72 hours of treatment, the D450 nm values of groups T1, T3, and T5 were significantly higher than those of group C (P < 0.01).

Results of Growth Curve Determination for Porcine Subcutaneous Adipose-Derived Neural Crest Stem Cells (NCSCs)

After inoculating porcine subcutaneous adipose-derived NCSCs in vitro for 1–3 days, both the T and C groups showed rapid proliferation. Starting from day 5, the T group entered the logarithmic growth phase and reached its peak on day 9. In contrast, the proliferation rate of the C group was significantly lower than that of the T group, though it also peaked on day 9. Subsequently, the proliferation rates of both groups declined gradually, entering the plateau phase (Fig. 3).

Results of Adipogenic Induction in Porcine Subcutaneous Adipose-Derived Neural Crest Stem Cells (NCSCs)

One day before lipogenesis induction, the T and C groups of cells from pig subcutaneous fat were fusiform or polygonal, and the number of cells in the T group was greater than that in the C group under the same magnification (Figures 4A, 4B). Two days after lipogenesis induction, cells in the T group showed vacuolation, with many small dark droplets appearing around the vacuoles in the cytoplasm, while the C group showed more extensive vacuolation, with dark droplets scattered around the vacuoles (Figures 4C, 4D). Four days after lipogenesis induction, the morphology of the T group cells showed no significant difference from that at two days prior, but the vacuoles in the cytoplasm had almost disappeared, replaced by dark droplets of varying sizes, some even distributed in the interstitial space between cells. The morphology of the C group cells did not differ significantly from that at two days prior; dark droplets of varying sizes were scattered in the cytoplasm, but the number of small droplets was less than that in the T group (Figures 4E, 4F). At six days post-induction, there was little morphological difference between the T and C groups, but the number of dark droplets in the cytoplasm of both groups had significantly increased, appearing like clusters of grapes, although the number of small droplets in the T group was much greater than that in the C group (Figures 4G, 4H). Both the T and C groups of cells were stained with Oil Red O, and the dark droplets in the cytoplasm were stained red, indicating lipid droplets, with the T group showing a significantly higher number of red-stained lipid droplets compared to the C group (Figure 5).

Discussion and Conclusion
The addition of 20 ng/mL EGF and 20 ng/mL FGF-2 to the culture medium significantly promoted the proliferation and differentiation of porcine subcutaneous adipose-derived neural crest stem cells (NCSCs). These findings provide important insights for further optimizing the in vitro culture conditions of porcine subcutaneous adipose-derived NCSCs and support their potential application in regenerative medicine and therapeutic development.