The Use of Foxa2-Overexpressing Adipose Tissue-Derived Stem Cells in a Scaffold System Attenuates Acute Liver Injury

Abstract

Background/aims: For the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model.

Methods: A microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection).

Results: Foxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamide-induced acute liver injury model.

Conclusions: Foxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects.

Keywords: Foxa2; Liver failure; Mesenchymal stem cell; Scaffold; acute.

Fig. 1

Hepatic differentiation ability in two-dimensional (2D) culture. (A) Gene expression of hepatic differentiation markers (AFP, ALB, and CK18) in transfected ADSCs after 2 weeks measured by qRT-PCR. (B) Amount of cytoplasmic glycogen after the overexpression of various gene in 2D culture measured by periodic acid-Schiff (PAS) staining (PAS staining: blue, nuclei; purple, glycogen in cytoplasm; magnification: ×400). The arrows indicate cytosol. AFP, alpha fetoprotein, ALB, albumin; CK18, cytokeratin 18; ADSCs, adipose tissue-derived stem cells; qRT-PCR, real-time quantitative polymerase chain reaction; Foxa2, forkhead box protein. *p<0.05.

Fig. 2

Comparison of hepatic differentiation ability in three-dimensional (3D) culture using a scaffold. (A) qRT-PCR expression of hepatic differentiation markers (AFP, CK18, CD26, and CX32) in Foxa2-transfected ADSCs after 2 weeks in 3D culture. (B) H&E staining, periodic acid-Schiff (PAS) and cytochrome P450 immunofluorescence in Foxa2-overexpressing ADSCs in 3D culture (a and b: H&E staining, blue - nuclei, pink - cytosol, ×400; c and d: PAS staining, blue - nuclei, purple - glycogen in cytoplasm, ×1,000; e and f: blue - nuclei, green - cytochrome 450 in cytosol, ×1,000). (C) Quantification of cytochrome P450 intensity and glucose expression. qRT-PCR, real-time quantitative polymerase chain reaction; AFP, alpha fetoprotein; CK18, cytokeratin 18; CD26, dipeptidyl peptidase 4; CX32, connexin 32; ADSCs, adipose tissue-derived stem cells; Foxa2, forkhead box protein. *p<0.05.

Fig. 3

Comparison of hepatocyte differentiation in a scaffold during in vivo culture. (A) In vivo culture design of scaffold preparation and implantation into nude mice. (B) Scanning electron microscopy of ADSCs in the PLGA scaffold. (C) qRT-PCR expression of hepatic differentiation markers (AFP, CK18, CD26 and CX32) in Foxa2-transfected ADSCs after 2 weeks in nude mice. (D) H&E staining, periodic acid-Schiff (PAS) staining and immunofluorescent staining for cytochrome P450 activity after Foxa2 gene overexpression in in vivo culture (a and b: H&E staining, blue - nuclei, pink - cytosol, ×400; c and d: PAS staining, blue - nuclei, purple - glycogen in cytoplasm, ×1,000; e and f: blue - nuclei, green - cytochrome 450 in cytosol, ×1,000). (E) Quantification of cytochrome P450 intensity and glucose expression. ADSCs, adipose tissue-derived stem cells; PLGA, poly(lactic-co-glycolic acid); qRT-PCR, real-time quantitative polymerase chain reaction; AFP, alpha fetoprotein; CK18, cytokeratin 18; CD26, dipeptidyl peptidase 4; CX32, connexin 32; Foxa2, forkhead box protein. *p<0.05.

Fig. 4

The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury in a thioacetamide-induced acute liver failure model. Hematoxylin and eosin staining and necrotic area percentage. (A) The scheme of experimental design. (B) H&E staining and necrotic area percentage. (C) Expression of several hepatic enzymes (albumin, ALT, AST and total bilirubin) in serum. (D) The gene expression of inflammation markers (IL-1β and MCP1) was compared among the thioacetamide (TAA) injection group, ADSCs/scaffold implanted group and Foxa2-overex-pressing ADSCs/scaffold implanted group in mouse livers. Foxa2, forkhead box protein; ADSCs, adipose tissue-derived stem cells; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IL, inter-leukin; MCP1, monocyte chemoattractant protein-1. *p<0.05.

Fig. 5

Stem cell homing. (A) Mouse and human β2-microglobulin mRNA expression was compared between the TAA-only group and the ADSCs scaffold group. (B) Immunofluorescence stain using human anti-mitochondria antibody was performed in mouse liver (×200) and implanted scaffold (×630) (immunofluorescence: blue - nuclei, green - human mitochondria). H&E staining (blue - nuclei, pink - cytosol, ×400) and periodic acid-Schiff (PAS) staining (blue - nuclei, purple - glycogen in cytoplasm, ×630) of implanted scaffold. TAA, thioacetamide; ADSCs, adipose tissue-derived stem cells; Foxa2, forkhead box protein.

Fig. 6

Paracrine effects of cytokines. Growth factors and inflammatory cytokines were evaluated using a magnetic Luminex assay kit. On the 2nd day, differentiation medium #1 was used. On the 5th and 7th days, differentiation medium #2 was used as a control treatment. (A) Growth factors. (B) Anti-inflammatory cytokines. (C) Pro-inflammatory cytokines. Foxa2-overexpressing adipose tissue-derived stem cells (ADSCs) expressed various cytokines relative to ADSCs on day 5. HGF, hepatocyte growth factor; Foxa2, forkhead box protein; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; VEGF-A, vascular endothelial growth factor-A; IL-6, interleukin 6; TNF-α, tumor necrosis factor-α; MCP1, monocyte chemotactic protein-1.