Human adipose-derived mesenchymal stem cells promote recovery of injured HepG2 cell line and show sign of early hepatogenic differentiation

Abstract

Currently, orthotopic liver transplantation is the gold standard therapy for liver failure. However, it is limited by the insufficient organ donor and risk of immune rejection. Stem cell therapy is a promising alternative treatment for liver failure. One of the most ideal sources of stem cells for regenerative medicine is adipose-derived stem cells (ADSCs). In this study, primary ADSCs seeded on cell culture insert were indirectly co-cultured with injured HepG2 to elucidate the role of ADSCs in promoting the recovery of injured HepG2 in non-contact manner. HepG2 recovery was determined by the surface area covered by cells and growth factor concentration was measured to identify the factors involved in regeneration. Besides, HepG2 were collected for q-PCR analysis of injury, hepatocyte functional and regenerative markers expression. For the ADSCs, expression of hepatogenic differentiation genes was analyzed. Results showed that non-contact co-culture with ADSCs helped the recovery of injured HepG2. ELISA quantification revealed that ADSCs secreted higher amount of HGF and VEGF to help the recovery of injured HepG2. Furthermore, HepG2 co-cultured with ADSCs expressed significantly lower injury markers as well as significantly higher regenerative and functional markers compared to the control HepG2. ADSCs co-cultured with injured HepG2 expressed significantly higher hepatic related genes compared to the control ADSCs. In conclusion, ADSCs promote recovery of injured HepG2 via secretion of HGF and VEGF. In addition, co-cultured ADSCs showed early sign of hepatogenic differentiation in response to the factors released or secreted by the injured HepG2.

Keywords: Adipose-derived stem cells; Hepatocytes; Hepatogenic differentiation; Liver; Non-contact culture; Regeneration.

Fig. 1

Tri-lineage differentiation of ADSCS after induction for 21 days. a Adipogenic differentiation- positive Oil Red O staining (x200 magnification); b Osteogenic differentiation- positive Alizarin Red staining (x100 magnification); c Chondrogenic differentiation—positive Alcian blue staining (x100 magnification)

Fig. 2

a Morphological assessment of HepG2 cells in injured HepG2 and ADSC-injured HepG2 co-culture groups after 24, 48 and 72 h (100× magnification). b Recovery rate of hepatocytes. “#”: Significant difference (p < 0.05) compared to control HepG2 and injured HepG2 groups at that particular time point. “a”: Significant difference (p < 0.05) compared to time point 24 h. “b”: Significant difference (p < 0.05) compared to time point 48 h

Fig. 3

HGF (a), VEGF (b) and bFGF (c) secretion by injured HepG2, ADSC control and ADSC-injured HepG2 co-culture groups after 24, 48 and 72 h. Data are denoted as mean ± S.E.M of n = 6. “*”: Significant difference (p < 0.05) compared to time point 24 h. “#”: Significant difference (p < 0.05) compared to time point 48 h. “a”: Significant difference (p < 0.05) between two groups

Fig. 4

Injury markers: a IL-1β, b MMP-1, c CYP7A1, and d ALT; Functional markers: e FoxA3, and f ALB; Regeneration markers: g FoxM1, h IL-6 and i IL-8 mRNA relative gene expression by control HepG2, injured HepG2 and HepG2 in ADSC-injured HepG2 co-culture group. Data are denoted as mean ± S.E.M. of n = 6. “*”: Significant difference (p < 0.05) compared to control HepG2 group. “#”: Significant difference (p < 0.05) compared to injured HepG2 group

Fig. 5

Hepatogenic trans-differentiation markers expression in ADSCs from different experiment groups. a ALB, b AFP, c CK18, d CYP7A1, e TDO2 and f ALT mRNA relative gene expression of ADSC control and ADSC-Injured HepG2 co-culture group. Data are denoted as mean ± S.E.M. of n = 6. “*”: Significant difference (p < 0.05) compared to ADSC control group

Fig. 6

Periodic acid-Schiff staining of ADSCs from different experiment groups. a ADSC control group, b ADSC- Injured HepG2 co-culture group. (100× magnification)