Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro

Abstract

Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro.

FIG. 1.

Characterization of differentiated human amniotic epithelial cells (hAEC). hAEC were differentiated over a 4-week period. Albumin and glycogen continued to be accumulated while the transcription factor hepatocyte nuclear factor-4 alpha (HNF-4α) was found only in the nuclei of stimulated cells; nuclei stained with DAPI is shown in the inset (A). Compared with hAEC, the differentiated cells showed increased expression of ABCA2, CYP7A1 involved in cholesterol metabolism, and ABCB11, encoding a bile acid export pump. Expression of genes involved in fat (SLC27A2) and xenobiotic metabolism (EPHX1) were reduced relative to hAEC. Data were normalized to 18S rRNA, expressed as 2^−ΔΔCt relative to naïve hAEC and analyzed by the unpaired t-test (B). Scale bar=100 μm. Color images available online at www.liebertpub.com/scd

FIG. 2.

Functional characteristics of the differentiated cells. Differentiated cells were positive for low-density lipoprotein (LDL) receptors and LDL was present in the cytoplasm in contrast to naïve hAEC. Only differentiated cells took up indocyanine green (ICG) and effluxed the dye (A). Total and oxidized glutathione levels remained similar (B). Differentiated cells showed increased mRNA expression of the drug metabolizing enzyme CYP3A4 and had elevated CYP3A4 enzymatic activity after rifampicin stimulation compared with hAEC (C). Upon stimulation with ammonium chloride, there was increased mRNA expression of the urea cycle gene ASS1 and increased urea synthesis in the differentiated cells relative to naïve hAEC. Data were analyzed by the unpaired t-test (D). Scale bar=100 μm. Color images available online at www.liebertpub.com/scd

FIG. 3.

Characterization of encapsulated hepatocyte-like cells (enc HLC). HLC were encapsulated and cultured for 7 days to determine effects on viability and function. Comparisons were made with monolayer cultures extended for 7 days (ex HLC). Enc HLC remained viable as shown by 6-carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) staining and quantitated by flow cytometry (A). HNF-4α localized to the nuclei of enc HLC; DAPI-stained nuclei shown in the inset. Enc HLC had stored albumin and glycogen after 7 days in culture (B). Scale bar=100 μm. Relative to ex HLC, mRNA expression of ABCA1 decreased while SLC27A2 increased in enc HLC; data were analyzed by ANOVA followed by Tukey's post hoc test (C). Color images available online at www.liebertpub.com/scd

FIG. 4.

Functional characteristics of enc HLC. Enc HLC cultured for 7 days took up LDL and ICG (A). Both total and oxidized glutathione levels in enc HLC grown for 7 days were lower than monolayer HLC cultures extended for 7 days (ex HLC) (B). Rifampicin stimulation (+) and encapsulation led to increased CYP3A4 mRNA expression. HNF-4α, which regulates CYP3A4 mRNA expression was highly elevated in the enc HLC. CYP3A4 enzymatic activity was highest in the enc HLC. *P<0.05 and ***P<0.001, respectively (C). Despite some changes in the mRNA expression of the urea cycle genes ARG1 and CPS1, urea output increased in enc HLC (D). mRNA expression data were normalized to 18S rRNA, expressed as 2^−ΔΔCt relative to HLC. Comparisons were made by ANOVA. Scale bar=100 μm. Color images available online at www.liebertpub.com/scd