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Rapid and high-efficiency generation of mature functional hepatocyte-like cells from adipose-derived stem cells by a three-step protocol.

Xu F, Liu J, Deng J, Chen X, Wang Y, Xu P, Cheng L, Fu Y, Cheng F, Yao Y, Zhang Y, Huang M, Yu D, Wei Y, Deng H - Stem Cell Res Ther (2015)

Bottom Line: The generation of functional hepatocytes is a major challenge for regenerative medicine and drug discovery.Upon transplantation into mice with carbon tetrachloride (CCl4)-induced acute fulminant liver failure, iHeps restore the liver function and prolong survival.The work could contribute to the development of alternative strategies to obtain nonhepatic cell-derived mature hepatocytes with potential for biomedical and pharmaceutical applications.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, section3, Renmin South Road, Chengdu, 610041, P.R. China. xufen1224@163.com.

ABSTRACT
The generation of functional hepatocytes is a major challenge for regenerative medicine and drug discovery. Here we show a method that facilitates generation of induced functional hepatocytes (iHeps) from adipose-derived stem cells (ADSCs) within 9 days. iHeps express hepatocytic gene programs and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity. Upon transplantation into mice with carbon tetrachloride (CCl4)-induced acute fulminant liver failure, iHeps restore the liver function and prolong survival. The work could contribute to the development of alternative strategies to obtain nonhepatic cell-derived mature hepatocytes with potential for biomedical and pharmaceutical applications.

No MeSH data available.


Related in: MedlinePlus

Characterization of iHeps in vitro. a Immunofluorescence analysis of ALB and AAT in iHeps. More than 90 % of iHeps efficiently expressed both ALB and AAT at day 9. b Secretion of ALB increased during the hepatogenic induction period as measured by ELISA. c The cumulative urea amounts gradually increased in the iHeps culture system during the hepatogenic induction period. d Gene expression profile analysis of ADSCs, iHeps, and rHeps cells by cDNA microarray. Hierarchical clustering showed that iHeps were grouped together with rHeps. e Analysis of basic hepatic function in iHeps, including PAS staining, ac-LDL, and ICG uptake. f mRNA levels of CYP genes were determined by quantitative PCR in iHeps and rHeps before inducer treatment. Data normalized to the level of GAPDH. g mRNA levels of the induced CYP enzymes were measured by quantitative PCR. CYP1A1and CYP1A2 was induced by 3-methylcholanthrene. CYP2A1 and CYP3A1 were induced by phenobarbital. CYP2E1 was induced by acetone. Fold induction in iHeps and rHeps was normalized to levels in cells without inducer treatment, respectively. h CYP metabolic activity in iHeps. CYP enzymes were induced in iHeps for 48 hours. Fresh rHeps were directly used as a positive control. The metabolic products of phenacetin (acetaminophen, assay for CYP1A2 activities), coumarin (7-hydroxycoumarin, assay for CYP2A1 activities), and chlorzoxazone (6-hydroxychlorzoxazone, assay for CYP2E1 activities) were determined by liquid chromatography–tandem mass spectrometry. Scale bar: 100 μm (a, e). AAT alpha-1-antitrypsin, ac-LDL acetylated low-density lipoprotein, ADSC adipose-derived stem cell, ALB albumin, CYP cytochrome P, ICG indocyanine green, iHep induced functional hepatocyte, PAS Periodic acid–Schiff, rHep primary rat hepatocyte, UD undetectable
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Fig3: Characterization of iHeps in vitro. a Immunofluorescence analysis of ALB and AAT in iHeps. More than 90 % of iHeps efficiently expressed both ALB and AAT at day 9. b Secretion of ALB increased during the hepatogenic induction period as measured by ELISA. c The cumulative urea amounts gradually increased in the iHeps culture system during the hepatogenic induction period. d Gene expression profile analysis of ADSCs, iHeps, and rHeps cells by cDNA microarray. Hierarchical clustering showed that iHeps were grouped together with rHeps. e Analysis of basic hepatic function in iHeps, including PAS staining, ac-LDL, and ICG uptake. f mRNA levels of CYP genes were determined by quantitative PCR in iHeps and rHeps before inducer treatment. Data normalized to the level of GAPDH. g mRNA levels of the induced CYP enzymes were measured by quantitative PCR. CYP1A1and CYP1A2 was induced by 3-methylcholanthrene. CYP2A1 and CYP3A1 were induced by phenobarbital. CYP2E1 was induced by acetone. Fold induction in iHeps and rHeps was normalized to levels in cells without inducer treatment, respectively. h CYP metabolic activity in iHeps. CYP enzymes were induced in iHeps for 48 hours. Fresh rHeps were directly used as a positive control. The metabolic products of phenacetin (acetaminophen, assay for CYP1A2 activities), coumarin (7-hydroxycoumarin, assay for CYP2A1 activities), and chlorzoxazone (6-hydroxychlorzoxazone, assay for CYP2E1 activities) were determined by liquid chromatography–tandem mass spectrometry. Scale bar: 100 μm (a, e). AAT alpha-1-antitrypsin, ac-LDL acetylated low-density lipoprotein, ADSC adipose-derived stem cell, ALB albumin, CYP cytochrome P, ICG indocyanine green, iHep induced functional hepatocyte, PAS Periodic acid–Schiff, rHep primary rat hepatocyte, UD undetectable

Mentions: Immunofluorescent staining showed that more than 90 % of iHeps expressed both ALB and AAT at day 9 (Fig. 3a), suggesting a highly-efficient hepatogenic differentiation. To assess the metabolic activity of iHeps, we quantified ALB production and urea synthesis. We found that iHeps had a remarkable capability for secreting the plasma protein ALB at a level close to rHeps at day 9 (Fig. 3b). The cumulative urea amounts also gradually increased in the iHep culture system during the induction period (Fig. 3c). Genome-wide expression profile analysis revealed that iHeps were clustered closely with cultured rHeps (Fig. 3d). Furthermore, gene set enrichment analysis showed that pathways enriched in rHeps were significantly enriched in iHeps, including those involved in glucose metabolism, lipid metabolism, amino acid metabolism, and phase I and phase II detoxification (see Additional file 2). These data indicate that ADSCs undergo hepatic differentiation by transcriptional alterations. In accordance with the expression of hepatic genes, iHeps displayed numerous hallmark functions of mature hepatocytes, such as glycogen storage, ac-LDL intake, and ICG uptake (Fig. 3e). The drug metabolic capacity is one of the most important functions that distinguish hepatocytes. CYP450 enzymes of hepatocytes are the main enzymes accounting for drug metabolism. Their activities and responses to specific inducers are used to evaluate drug metabolism of hepatocytes. We quantitatively confirmed the expression of CYP enzymes in iHeps, CYP1A1, CYP1A2, CYP2A1, CYP2C7, CYP2C12, CYP2E1, and CYP3A1. Results showed that iHeps already expressed these genes at remarkable levels without addition of chemical inducers (Fig. 3f). Furthermore, chemical inducers (3-methylcholanthrene, phenobarbital, and acetone) could markedly induce mRNA expression levels of CYP1A1, CYP1A2, CYP2A1, CYP2E1, and CYP3A1, except for CYP2C7 and CYP2C12 (Fig. 3g). Importantly, in another assay for CYP activities, iHeps displayed CYP enzyme-dependent metabolism of phenacetin, coumarin, and chlorzoxazone (Fig. 3h). These results offer the possibility of using iHeps for toxicity screening during drug discovery.Fig. 3


Rapid and high-efficiency generation of mature functional hepatocyte-like cells from adipose-derived stem cells by a three-step protocol.

Xu F, Liu J, Deng J, Chen X, Wang Y, Xu P, Cheng L, Fu Y, Cheng F, Yao Y, Zhang Y, Huang M, Yu D, Wei Y, Deng H - Stem Cell Res Ther (2015)

Characterization of iHeps in vitro. a Immunofluorescence analysis of ALB and AAT in iHeps. More than 90 % of iHeps efficiently expressed both ALB and AAT at day 9. b Secretion of ALB increased during the hepatogenic induction period as measured by ELISA. c The cumulative urea amounts gradually increased in the iHeps culture system during the hepatogenic induction period. d Gene expression profile analysis of ADSCs, iHeps, and rHeps cells by cDNA microarray. Hierarchical clustering showed that iHeps were grouped together with rHeps. e Analysis of basic hepatic function in iHeps, including PAS staining, ac-LDL, and ICG uptake. f mRNA levels of CYP genes were determined by quantitative PCR in iHeps and rHeps before inducer treatment. Data normalized to the level of GAPDH. g mRNA levels of the induced CYP enzymes were measured by quantitative PCR. CYP1A1and CYP1A2 was induced by 3-methylcholanthrene. CYP2A1 and CYP3A1 were induced by phenobarbital. CYP2E1 was induced by acetone. Fold induction in iHeps and rHeps was normalized to levels in cells without inducer treatment, respectively. h CYP metabolic activity in iHeps. CYP enzymes were induced in iHeps for 48 hours. Fresh rHeps were directly used as a positive control. The metabolic products of phenacetin (acetaminophen, assay for CYP1A2 activities), coumarin (7-hydroxycoumarin, assay for CYP2A1 activities), and chlorzoxazone (6-hydroxychlorzoxazone, assay for CYP2E1 activities) were determined by liquid chromatography–tandem mass spectrometry. Scale bar: 100 μm (a, e). AAT alpha-1-antitrypsin, ac-LDL acetylated low-density lipoprotein, ADSC adipose-derived stem cell, ALB albumin, CYP cytochrome P, ICG indocyanine green, iHep induced functional hepatocyte, PAS Periodic acid–Schiff, rHep primary rat hepatocyte, UD undetectable
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595267&req=5

Fig3: Characterization of iHeps in vitro. a Immunofluorescence analysis of ALB and AAT in iHeps. More than 90 % of iHeps efficiently expressed both ALB and AAT at day 9. b Secretion of ALB increased during the hepatogenic induction period as measured by ELISA. c The cumulative urea amounts gradually increased in the iHeps culture system during the hepatogenic induction period. d Gene expression profile analysis of ADSCs, iHeps, and rHeps cells by cDNA microarray. Hierarchical clustering showed that iHeps were grouped together with rHeps. e Analysis of basic hepatic function in iHeps, including PAS staining, ac-LDL, and ICG uptake. f mRNA levels of CYP genes were determined by quantitative PCR in iHeps and rHeps before inducer treatment. Data normalized to the level of GAPDH. g mRNA levels of the induced CYP enzymes were measured by quantitative PCR. CYP1A1and CYP1A2 was induced by 3-methylcholanthrene. CYP2A1 and CYP3A1 were induced by phenobarbital. CYP2E1 was induced by acetone. Fold induction in iHeps and rHeps was normalized to levels in cells without inducer treatment, respectively. h CYP metabolic activity in iHeps. CYP enzymes were induced in iHeps for 48 hours. Fresh rHeps were directly used as a positive control. The metabolic products of phenacetin (acetaminophen, assay for CYP1A2 activities), coumarin (7-hydroxycoumarin, assay for CYP2A1 activities), and chlorzoxazone (6-hydroxychlorzoxazone, assay for CYP2E1 activities) were determined by liquid chromatography–tandem mass spectrometry. Scale bar: 100 μm (a, e). AAT alpha-1-antitrypsin, ac-LDL acetylated low-density lipoprotein, ADSC adipose-derived stem cell, ALB albumin, CYP cytochrome P, ICG indocyanine green, iHep induced functional hepatocyte, PAS Periodic acid–Schiff, rHep primary rat hepatocyte, UD undetectable
Mentions: Immunofluorescent staining showed that more than 90 % of iHeps expressed both ALB and AAT at day 9 (Fig. 3a), suggesting a highly-efficient hepatogenic differentiation. To assess the metabolic activity of iHeps, we quantified ALB production and urea synthesis. We found that iHeps had a remarkable capability for secreting the plasma protein ALB at a level close to rHeps at day 9 (Fig. 3b). The cumulative urea amounts also gradually increased in the iHep culture system during the induction period (Fig. 3c). Genome-wide expression profile analysis revealed that iHeps were clustered closely with cultured rHeps (Fig. 3d). Furthermore, gene set enrichment analysis showed that pathways enriched in rHeps were significantly enriched in iHeps, including those involved in glucose metabolism, lipid metabolism, amino acid metabolism, and phase I and phase II detoxification (see Additional file 2). These data indicate that ADSCs undergo hepatic differentiation by transcriptional alterations. In accordance with the expression of hepatic genes, iHeps displayed numerous hallmark functions of mature hepatocytes, such as glycogen storage, ac-LDL intake, and ICG uptake (Fig. 3e). The drug metabolic capacity is one of the most important functions that distinguish hepatocytes. CYP450 enzymes of hepatocytes are the main enzymes accounting for drug metabolism. Their activities and responses to specific inducers are used to evaluate drug metabolism of hepatocytes. We quantitatively confirmed the expression of CYP enzymes in iHeps, CYP1A1, CYP1A2, CYP2A1, CYP2C7, CYP2C12, CYP2E1, and CYP3A1. Results showed that iHeps already expressed these genes at remarkable levels without addition of chemical inducers (Fig. 3f). Furthermore, chemical inducers (3-methylcholanthrene, phenobarbital, and acetone) could markedly induce mRNA expression levels of CYP1A1, CYP1A2, CYP2A1, CYP2E1, and CYP3A1, except for CYP2C7 and CYP2C12 (Fig. 3g). Importantly, in another assay for CYP activities, iHeps displayed CYP enzyme-dependent metabolism of phenacetin, coumarin, and chlorzoxazone (Fig. 3h). These results offer the possibility of using iHeps for toxicity screening during drug discovery.Fig. 3

Bottom Line: The generation of functional hepatocytes is a major challenge for regenerative medicine and drug discovery.Upon transplantation into mice with carbon tetrachloride (CCl4)-induced acute fulminant liver failure, iHeps restore the liver function and prolong survival.The work could contribute to the development of alternative strategies to obtain nonhepatic cell-derived mature hepatocytes with potential for biomedical and pharmaceutical applications.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, section3, Renmin South Road, Chengdu, 610041, P.R. China. xufen1224@163.com.

ABSTRACT
The generation of functional hepatocytes is a major challenge for regenerative medicine and drug discovery. Here we show a method that facilitates generation of induced functional hepatocytes (iHeps) from adipose-derived stem cells (ADSCs) within 9 days. iHeps express hepatocytic gene programs and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity. Upon transplantation into mice with carbon tetrachloride (CCl4)-induced acute fulminant liver failure, iHeps restore the liver function and prolong survival. The work could contribute to the development of alternative strategies to obtain nonhepatic cell-derived mature hepatocytes with potential for biomedical and pharmaceutical applications.

No MeSH data available.


Related in: MedlinePlus