Basic Study
Copyright ©The Author(s) 2021.
World J Stem Cells. Nov 26, 2021; 13(11): 1783-1796
Published online Nov 26, 2021. doi: 10.4252/wjsc.v13.i11.1783
Figure 1
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Figure 1 Crystal violet and MTT assays in different samples of adipose-derived stem cells cultures. A: Representative microphotographs of crystal violet staining of human adipose-derived stem cells (ASCs) at 1 d and 8 d of culture. ASCs: Control ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Scale bar: 100 μm; B and C: Proliferation rate (crystal violet assay, CV) (B) and cell viability (MTT assay) at 1, 4 and 8 d of culture (C). Absorbance values were determined at 570 nm. Values are expressed as mean ± SD of three independent experiments. Values are referred to the control ASC population, at each corresponding time point. aP < 0.05 vs ASCs at day 1; Two-way ANOVA, followed by Tukey’s multiple comparisons test. sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium.
Figure 2
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Figure 2 Nestin immunoreactivity in different samples of adipose-derived stem cells cultures. Nestin (red fluorescence) detected after 1 d and 8 d of culture of human adipose-derived stem cells (ASCs). ASCs: Control ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Blue fluorescence indicates DAPI staining of cell nuclei. Scale bar: 50 μm. sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium.
Figure 3
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Figure 3 Western blot analysis of neural marker expression in different samples of adipose-derived stem cells cultures. A: Immunoblot analysis of whole-cell lysates at day 1, 4 and 8 of culture for NSE, PGP9.5, nestin, GFAP and GAPDH as internal control; B-E: ASCs: Control adipose-derived stem cells (ASCs) cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Quantitative data are illustrated in histograms. Values are expressed as mean ± SD obtained from three independent experiments. aP < 0.05 vs ASCs of corresponding time point; Two-way ANOVA, followed by Tukey’s multiple comparisons test. NSE: Neuron specific enolase; PGP9.5: Protein gene product 9.5; GFAP: Glial fibrillary acidic protein; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium.
Figure 4
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Figure 4 Immunofluorescence quantification for neural markers in each adipose-derived stem cells group at day 1 (white columns) and day 8 (dark columns). A: Nestin; B: NSE; C: PGP9.5; D: GFAP. ASCs: Control adipose-derived stem cells (ASCs) cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Bars represent CTCF mean values ± SD, obtained from at least three independent experiments. aSignificant differences (P < 0.01) vs ASCs; bSignificant differences (P < 0.01) between CM/ASCs and DMEM/F12. CTCF: Corrected total cell fluorescence; NSE: Neuron specific enolase; PGP9.5: Protein gene product 9.5; GFAP: Glial fibrillary acidic protein; sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium.
Figure 5
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Figure 5 Neuronal specific enolase immunoreactivity in different samples of adipose-derived stem cells cultures. Neuronal specific enolase (red fluorescence) detected after 1 d and 8 d of culture of human adipose-derived stem cells (ASCs). ASCs: Control ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Blue fluorescence indicates DAPI staining of cell nuclei. Scale bar: 50 μm. sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium; NSE: Neuronal specific enolase.
Figure 6
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Figure 6 Protein gene product 9. 5 immunoreactivity in different samples of adipose-derived stem cells cultures. Protein gene product 9.5 (green fluorescence) detected after 1 d and 8 d of culture of human adipose-derived stem cells (ASCs). ASCs: Control ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Blue fluorescence indicates DAPI staining of cell nuclei. Scale bar: 50 μm. sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium; PGP: Protein gene product.
Figure 7
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Figure 7 Glial fibrillary acid protein immunoreactivity in different samples of adipose-derived stem cells cultures. Glial fibrillary acid protein (red fluorescence) detected after 1 d and 8 d of culture of human adipose-derived stem cells (ASCs). ASCs: Control ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); sfASCs: ASCs cultured in serum-free DMEM; F12/ASCs: ASCs cultured in serum-free DMEM/F12; CM/ASCs: ASCs cultured in serum-free DMEM/F12 conditioned from ARPE-19. Blue fluorescence indicates DAPI staining of cell nuclei. Scale bar: 50 μm. sfASCs: Serum-free adipose-derived stem cells; CM: Conditioned medium; GFAP: Glial fibrillary acid protein.