Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride

Abstract

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl₄) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl₄ (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl₄ induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for α-smooth muscle actin (α-SMA) and collagen I were decreased. While α-SMA, collagen α1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-β were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of α-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl₄. hHSPCs mediate downregulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro.

Keywords: Adult human hepatic stem/progenitor cells; cell transplantation; hepatic fibrosis.

Figure 1

Phase contrast microscopy (A, B) and expression of hepatocyte and biliary epithelial cell markers (C, D) of hHSPCs. (A) During the first 10-20 days in culture, the cells that are attached on the dish occur in cluster and appear round. (B) In subculture, the cells are spreading and look flattened and larger. Original magnification 100 ×. (C) Western blots demonstrate expression of albumin and CK19 by hHSPCs. Upon exposure to DMSO and HGF, albumin expression is elevated while CK19 expression is lost. (D) RT-PCR shows that hHSPCs express the hepatocyte marker genes Albumin, CK8 and weakly AFP, as well as the cholangiocyte marker genes CK7 and CK19.

Figure 2

A: Liver fibrosis revealed by Sirius red staining for collagens. Top row: Representative images showing the development of advanced fibrosis induced by CCl₄, which includes formation of nodules (cirrhosis) and linking septa without apparent nodular formation (bridging fibrosis). Bottom row: Representative images showing fibrosis in mice treated with CCl₄ and were also given hHSPCs. Short septa and focal fibrotic tissue are visible. Original magnification 100 ×. B: Histomorphometry of Sirius red stained collagens. The amount of collagens in the liver parenchyma was halved in mice received the hHSPC transplantation. N = 10 mice/group.

Figure 3

Expression of α-SMA and collagen I proteins (A, B) and changes in mRNA levels (C) in the liver of CCl₄ mice received hHSPC transplantation. (A, B) Upper panel: Immunoblots of four mice; lower panel: corresponding histograms of data of the blots. Both α-SMA and collagen I levels were reduced by nearly halves in the mice with the transplanted hHSPCs. Values are expressed as fold change relative to CCl₄-treated mice; N = 4 mice/group. (C) While α-SMA, collagen α1(I) and TIMP-1 mRNA levels were downregulated, MMP-13 mRNA level was upregulated. The gene expression for TGF-β and MMP-2 was not altered. Values are expressed relative to CCl₄-treated mice; N = 10 mice/group.

Figure 4

Expression of α-SMA and collagen I proteins (A, B) and changes in mRNA levels (C) in HSCs co-cultured with hHSPCs. (A, B) Upper panel: Immunoblots of three separate cultures; lower panel: corresponding histograms of data of the blots. Both α-SMA and collagen I levels in HSCs were reduced by halves in the presence of hHSPCs. Values are expressed relative to the control HEK293/HSC culture. (C) Control: HSCs were cultured alone in the bottom compartment of the Transwell, while the upper compartment contained only the medium. Negative control: HSCs in the bottom compartment were co-cultured HEK293 cells in the upper compartment. hHSPC: HSCs were co-cultured with hHSPCs in the upper compartment. Gene expression for α-SMA, collagen α1(I) and TIMP-1 in HSCs were downregulated by the presence of hHSPCs. The mRNA level of MMP-1 was higher, while the levels of TGF-β and MMP-2 were not altered. Values are expressed relative to HSC control.

Figure 5

Effects of hHSPCs on proliferation of HSCs. Control: HSCs were cultured alone in the bottom compartment of the Transwell, while the upper compartment contained only the medium. Negative control: HSCs co-cultured with HEK293 cells. hHSPCs: hHSPCs were co-cultured with HSCs. hHSPCs when co-cultured with HSCs at the ratio of 3/1 elicited a 25% inhibition of HSC proliferation. Values are expressed relative to HSC control.