Table 1.
Conventional media used in this study.
Table 2.
Sequences of primers used in this study.
Fig 1.
Culture of 201B7 cells in conventional media.
201B7 cells were cultured in different conventional media for 7 days, and then subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP). The expression levels of AFP were significantly higher in the cells in WE, DF12 and fetal liver than those in ReproFF (P < 0.05, one-way analysis of variance, n = 3). FF, ReproFF; L15, Leibovitz's-15; DMEM, Dulbecco's Modified Eagle's Medium; RPMI, Roswell Park Memorial Institute 1640; WE, William’s E; DF12, Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; MEM, Minimum Essential Medium; GMEM, Glasgow’s Minimum Essential Medium; IMDM, Iscove’s Modified Dulbecco’s Medium; CMRL, Connaught Medical Research Laboratories 1066; BME, Basal Medium Eagle; McCoy, McCoy’s 5A; MCDB, MCDB 131; fetal, fetal liver. Data are presented as the mean ± standard deviation (error bars). *, The expression levels of AFP were higher in the cells in WE and DF12 and fetal liver than those in ReproFF with P <0.05 (one-way analysis of variance).
Fig 2.
Culture of 201B7 cells in conventional media following that in HDI.
(A) 201B7 cells were cultured in HDI for 2 days, followed by L15, WE, or DF12 for 5 days. (B) Cells were imaged on days 0, 2, and 7. (C) Numbers of the cells were counted on days 0, 2, and 7. Cells survived more in HDI-WE (thin-solid line) and HDI-DF12 (thick-solid line) than HDI-L15 (broken line). The numbers of the cells were significantly higher in the cells in HDI-WE and HDI-DF12 than those in HDI-L15 (*: P < 0.05, one-factor analysis of variance, n = 3). (D) RNA was isolated, and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP). The expression levels of AFP were significantly higher in the cells in HDI-WE and HDI-DF12 than those in ReproFF (**: P < 0.05, one-factor analysis of variance, n = 3). HDI: hepatocyte differentiation inducer, L15: Leibovitz’s-15, WE: William’s E, DF12: Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham, FF, ReproFF; HDI-L15, HDI followed by L15; HDI-WE, HDI followed by WE; HDI-DF12, HDI followed by DF12. Data are presented as the mean ± standard deviation (error bars). Original magnification, ×200; scale bar, 100 μm. *, the numbers of the cells were higher in HDI-WE and HDI-DF12 than HDI-L15 with P <0.05 (one-factor analysis of variance), n = 3; **, the expression levels of AFP was higher in the cells in HDI-WE and HDI-DF12 than those in ReproFF with P <0.05 (one-factor analysis of variance), n = 3.
Fig 3.
Culture of 201B7 cells in media without glucose and arginine, but supplemented with galactose and ornithine, followed by that in conventional media.
(A) 201B7 cells were cultured in media without glucose and arginine, but supplemented with galactose and ornithine HDI, mWE, or mDF12), for 2 days, followed by 7 days in L15, WE, or DF12. Cells cultured for 9 days in L15, WE, or DF12 only were used to compare with those in HDI, mWE, and mDF12. (B and C) RNA was isolated and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP) (B) and albumin (ALB) (C). HDI, hepatocyte differentiation inducer; WE, William’s E; DF12, Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; mWE, modified William’s E; mDF12, modified Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; FF, ReproFF; HDI-L15, HDI followed by L15; mWE-L15, mWE followed by L15; mDF12-L15, mDF12 followed by L15; HDI-WE, HDI followed by WE; mWE-WE, mWE followed by WE; mDF12-WE, mDF12 followed by WE; HDI-DF12, HDI followed by DF12; mWE-DF12, mWE followed by DF12; mDF12-DF12, mDF12 followed by DF12. Data are presented as the mean ± standard deviation (error bars). *, P < 0.05 compared with FF (one-way analysis of variance); n = 3.
Fig 4.
Culture of 201B7 cells in conventional media followed by that in media without glucose and arginine, but supplemented with galactose and ornithine.
(A) 201B7 cells were cultured in L15, WE, or DF12 for 7 days, followed by 2 days in media without glucose and arginine, but supplemented with galactose and ornithine (HDI, mWE, or mDF12). Cells cultured for 9 days in L15, WE, or DF12 only were used to compare with those in HDI, mWE, and mDF12. (B and C) RNA was isolated, and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP) (B) and albumin (ALB) (C). HDI, hepatocyte differentiation inducer; WE, William’s E; DF12, Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham; mWE, modified William’s E; mDF12, modified Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; FF, ReproFF; L15-HDI, L15 followed by L15; L15-mDF12, L15 followed by mDF12; WE-HDI, WE followed by HDI; WE-mWE, WE followed by mWE; WE-mDF12, WE followed by mDF12; DF12-HDI, DF12 followed by HDI; DF12-mWE, DF12 followed by mWE; DF12-mDF12, DF12 followed by mDF12. Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with FF; n = 3.
Fig 5.
Time-course culture of 201B7 cells in hepatocyte differentiation inducer (HDI).
(A) 201B7 cells were cultured in HDI (black bar) for 0, 3, 6, 12, 24, and 48 h, followed by another 7 days of culture in William’s E (WE) medium (gray bar). (B and C) RNA was isolated, and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP) (B) and albumin (ALB) (C). Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with ReproFF (FF); n = 3.
Fig 6.
Repeated cycles of 12-h culture of 201B7 cells in hepatocyte differentiation inducer (HDI) either before or after culture in William’s E (WE) medium.
(A) 201B7 cells were cultured in three conditions: HDI for 12 h followed by WE for 5 days; WE only for 12 h + 5 days; and WE for 5 days followed by HDI for 12 h. (B and C) After one, two, or three cycles of these culture conditions, RNA was isolated and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP) (B) and albumin (ALB) (C). The thick solid line indicates HDI followed by WE only, broken line indicates WE only, and thin solid line indicates WE followed by HDI. Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with 0 time of repeat; n = 3.
Fig 7.
Repeated cycles of 2d culture of 201B7 cells in hepatocyte differentiation inducer (HDI) either before or after culture in William’s E (WE) medium.
(A) 201B7 cells were cultured in three conditions; HDI for 2 days followed by WE for 5 days, WE only for 7 days, and WE for 5 days followed by HDI for 2 days. (B and C) After one, two, or three cycles of these culture conditions, RNA was isolated, and subjected to quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP) (B) and albumin (ALB) (C). The thick solid line indicates HDI followed by WE, thick broken line indicates WE only, and thin solid line indicates WE followed by HDI. Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with 0 time of repeat; n = 3.
Fig 8.
Morphology of 201B7 cells cultured in repeated combinations of hepatocyte differentiation inducer (HDI) and William’s E (WE) medium.
(A) 201B7 cells were cultured in ReproFF, HDI followed by WE, WE only, or WE followed by HDI. The cells were cultured in HDI for either 12 h or 48 h and then cultured for another 5 days in combination with HID. After three cycles of the culture combinations, the cells were imaged (B). Original magnification, ×400; scale bar, 100 μm.
Fig 9.
Expression of liver-specific genes in 201B7 cells in presence of HDI.
(A) 201B7 cells were cultured in three conditions: hepatocyte differentiation inducer (HDI) for 12 h followed by William’s E (WE) medium for 5 days; WE only for 12 h + 5 days; and WE for 5 days followed by HDI for 12 h. (B) 201B7 cells were cultured in three conditions: HDI for 2 days followed by WE for 5 days; WE only for 7 days; and WE for 5 days followed by HDI for 2 days. RNA was isolated and subjected to quantitative polymerase chain reaction to analyze the expression of glucose-6-phosphatase (G6P) (C), cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) (D), and aldehyde dehydrogenase 2 (ALDH2) (E). Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with ReproFF (FF); n = 3.
Fig 10.
Immunostaining and Indocyanine Green uptake by cells grown in HDI.
(A) 201B7 cells were cultured in three conditions: hepatocyte differentiation inducer (HDI) for 2 days followed by William’s E (WE) for 5 days (HDI-WE); WE only for 7 days (WE); and WE for 5 days followed by HDI for 2 days (WE-HDI). The cells cultured in ReproFF (FF) were considered undifferentiated controls. (B) The cells were immunostained with antibodies against α-feto protein (AFP) and albumin (ALB). The cells were subjected to Indocyanine Green uptake (ICG). Original magnification was ×400 and scale bars represent 50 μm for both immunostaining and ICG uptake.