RESUMEN
Human embryonic stem cells (hESCs) may provide a cell source for functional hepatocytes for clinical applications and drug development. Initially, the hESC population was enriched to be more than 85% definitive endoderm (DE) as assessed by the expression of CXCR4, SOX17, and FOXA2. We then successfully converted DE into hepatic progenitors with 93% of the cells being positive for alpha-feto protein within 9 days. The percentage of albumin positive cells gradually increased to 90% at days 20-22 after differentiation. Moreover, our hESC-derived hepatocytes (hEH) developed a complete biotransformation system including phase I and II metabolizing enyzmes and phase III transporters. Nuclear receptors, which are critical in regulating the expression of metabolizing enzymes, were also expressed by our hEH. Using ultraperformance liquid chromatography-tandem mass spectrometry technology, we identified seven metabolic pathways of the drug bufuralol including four newly-reported ones in our hEH, which are the same as those in freshly isolated human primary hepatocytes (hPH). In addition, the results of the metabolism of four drugs indicate that our hEH have the capacity to metabolize these drugs at levels that are comparable to hPH. In conclusion, we have generated a relatively homogenous population of hepatocytes from hESCs, which appear to have complete metabolic function that is comparable to primary liver cells. These results represent a significant step towards the efficient differentiation of mature hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , HumanosRESUMEN
Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultra performance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Hepatocitos/trasplante , Células Madre Pluripotentes Inducidas/citología , Fase II de la Desintoxicación Metabólica/fisiología , Fase I de la Desintoxicación Metabólica/fisiología , Antagonistas Adrenérgicos beta/metabolismo , Albúminas/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/genética , Etanolaminas/metabolismo , Expresión Génica , Hepatocitos/citología , Humanos , Huésped Inmunocomprometido , Células Madre Pluripotentes Inducidas/metabolismo , Hígado , Ratones , Ratones SCID , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Espectrometría de Masas en Tándem , Trasplante HeterólogoRESUMEN
Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field.