Characteristics of SOX9-positive progenitor-like cells during cholestatic liver regeneration in biliary atresia

Abstract

Background: The progression of Biliary Atresia (BA) is associated with the number of reactive ductular cells (RDCs) whose heterogeneity in origin and evolution in humans remains unknown. SOX9-positive liver progenitor-like cells (LPLCs) have been shown to participate in RDCs and new hepatocyte formation during cholestatic liver regeneration in an animal model, which implies the possibility that hepatocyte-reprogrammed LPLCs could be a source of RDCs in BA. The present study aimed to elucidate the characteristics of SOX9-positive LPLCs in BA for exploring new possible therapeutic targets by manipulating the bi-differentiation process of LPLCs to prevent disease progression.

Methods: Twenty-eight patients, including 24 patients with BA and 4 patients with Congenital Choledochal Cyst as the control group, were retrospectively recruited. Liver biopsy samples were classified histologically using a 4-point scale based on fibrosis severity. LPLCs were detected by SOX9 and HNF4A double positive staining. Single immunohistochemistry, double immunohistochemistry, and multiple immunofluorescence staining were used to determine the different cell types and characteristics of LPLCs.

Results: The prognostic predictors of BA, namely total bile acid (TBA), RDCs, and fibrosis, were correlated to the emergence of LPLCs. SOX9 and HNF4A double-positive LPLCs co-stained rarely with relevant markers of portal hepatic progenitor cells (portal-HPCs), including CK19, CK7, EPCAM, PROM1 (CD133), TROP2, and AFP. Under cholestasis conditions, LPLCs acquired superior proliferation and anti-senescence ability among hepatocytes. Moreover, LPLCs arranged as a pseudo-rosette structure appeared from the periportal parenchyma to the portal region, which implied the differentiation from hepatocyte-reprogrammed LPLCs to RDCs with the progression of cholestasis.

Conclusions: LPLCs are associated with disease progression and prognostic factors of BA. The bipotent characteristics of LPLCs are different from those of portal-HPCs. As cholestasis progresses, LPLCs appear to gain superior proliferation and anti-senescence ability and continually differentiate to RDCs.

Keywords: Biliary atresia; Hepatic progenitor cells; Liver progenitor-like cells; Reactive ductular cells; SOX9.

Fig. 1

Correlation of LPLCs with cholestatic progression in BA. A Histological staining of LPLCs and RDCs for the different fibrosis progression groups. B–D Correlation analyses showing a positive relationship between LPLCs and cholestatic-related factors for BA: B, TBA; C, fibrosis; and D, RDCs. E Correlation analyses showing a correlation between the fibrosis stage and different types of RDCs. Atypical RDCs were inconsistently observed in the cirrhosis stage

Fig. 2

Bipotent characteristics of LPLCs differ from those of portal-HPCs. LPLCs co-stained with the relevant markers of portal-HPCs, including (A) epithelial cell marker (panCK), (B) hepatoblast marker (AFP), (C–E) hepatic progenitor markers (EPCAM, PROM1, and TROP2), and (F) RDCs markers (CK7 and CK19)

Fig. 3

LPLCs exhibit superior proliferation and anti-senescence abilities as compared to SOX9-negative hepatocytes with the progression of cholestasis. Multiple immunofluorescence staining of LPLCs with (A) proliferation marker (Ki67) and (B) anti-senescence marker (p21). Overall percentage (C) and cell cluster percentage (D) of Ki67+ LPLCs and Ki67+ SOX9-negative hepatocytes in different fibrosis stages. Overall percentage (E) and cell cluster percentage (F) of p21+ LPLCs and p21+ SOX9-negative hepatocytes in different fibrosis stages

Fig. 4

LPLCs differentiated into RDCs in the periportal region with the progression of cholestasis. A Pseudo-rosette formation in the periportal parenchyma appears to have bipotent characteristics during cholestatic liver damage, which co-stains with the LPLC markers SOX9 and HNF4A and with other hepatocyte and cholangiocyte markers, namely HepPar1, MRP2, and PKCζ. Pseudo-rosette hepatocytes (HepPar1-positive) appeared (B) in the periportal parenchyma with weak SOX9 staining and (C) in the adjacent portal region with increasing SOX9 staining and ductular-like structure in the fibrosis stage and (D) occurred inside the portal region with SOX9-positive ductular structure in the cirrhosis stage