Therapeutic efficiency of human amniotic epithelial stem cell-derived functional hepatocyte-like cells in mice with acute hepatic failure

Abstract

Background: Hepatocyte transplantation has been proposed as an effective treatment for patients with acute liver failure (ALF), but its application is limited by a severe shortage of donor livers. Human pluripotent stem cells (hPSCs) have emerged as a potential cell source for regenerative medicine. Human amniotic epithelial stem cells (hAESCs) derived from amniotic membrane have multilineage differentiation potential which makes them suitable for possible application in hepatocyte regeneration and ALF treatment.

Methods: The pluripotent characteristics, immunogenicity, and tumorigenicity of hAESCs were studied by various methods. hAESCs were differentiated to hepatocyte-like cells (HLCs) using a non-transgenic and three-step induction protocol. ALB secretion, urea production, periodic acid-Schiff staining, and ICG uptake were performed to investigate the function of HLCs. The HLCs were transplanted into ALF NOD-SCID (nonobese diabetic severe combined immunodeficient) mouse, and the therapeutic effects were determined via liver function test, histopathology, and survival rate analysis. The ability of HLCs to engraft the damaged liver was evaluated by detecting the presence of GFP-positive cells.

Results: hAESCs expressed various markers of embryonic stem cells, epithelial stem cells, and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAESC-derived hepatocytes possess the similar functions of human primary hepatocytes (hPH) such as producing urea, secreting ALB, uptaking ICG, storing glycogen, and expressing CYP enzymes. HLC transplantation via the tail vein could engraft in live parenchymal, improve the liver function, and protect hepatic injury from CCl₄-induced ALF in mice. More importantly, HLC transplantation was able to significantly prolong the survival of ALF mouse.

Conclusion: We have established a rapid and efficient differentiation protocol that is able to successfully generate ample functional HLCs from hAESCs, in which the liver injuries and death rate of CCl₄-induced ALF mouse can be significantly rescued by HLC transplantation. Therefore, our results may offer a superior approach for treating ALF.

Keywords: Acute liver failure; Cell transplantation; Hepatocyte-like cells; Human amniotic epithelial stem cells; Human primary hepatocytes.

Fig. 1

Characteristics of cellular morphology and expressions of markers in hAESCs. a Phase-contrast microscopic images of cultured hAESCs without (i) or with EGF (10 ng/ml) (ii). b RT-PCR analysis for the expressions of markers in hAESCs. Human AESCs specifically expressed various markers of CD73, CD105, CK7, E-cadherin, Oct4, and Nanog, but not CD90. Water was used as negative control. c Detection of surface markers in hAESCs (blue) and in isotype controls (red) by flow cytometry. Human AESCs expressed CD29, CD73, CD105, and HLA-ABC, but not CD90, CD34, CD45, and HLA-DR. d RT-PCR analysis for expressions of the major histocompatibility proteins. There were no expression of HLA-DR or low expression of HLA-ABC in hAESCs during passages 0 to 15. Water was used as negative control and ovarian cancer cell SK-VO-3 were used as positive control. e Immunofluorescence staining for the markers. All of hAESCs expressed the antigens of Oct4, SSEA-4, Nanog, and E-cadherin

Fig. 2

Tumorogenesis of hAESCs in vivo and in vitro. a Clonogenicity in soft agar cultures. No colonies were observed within 30 days in the hAESC group. HepG2 cells were used as a positive control. b Tumorogenesis in NOD-SCID mice. There was no any tumor formation after 5 months of hAESC injections, in which the cells were injected into the right back and left thigh muscle of NOD-SCID mice. Embryonic stem cells were used as a positive control

Fig. 3

Differentiation of hAESCs into hepatocyte-like cells. a Schematic diagram of hAESCs differentiation into hepatocyte-like cells. b Images of the sequential morphological changes from hAESCs to hepatocyte-like cells. (I) Morphology at the preinduction stage of hAESCs. (II) The cell morphology has become polygonal in shape after 7-day hepatic lineage commitment medium induction. (III) At day 14, the cell morphology had become cuboidal in shape. (IV) The morphology of hPH. c Immunocytochemical examination of endoderm cells and hepatic precursor cells. The vast majority of induced cells expressed the definitive endoderm marker FOXA2 and SOX17 at day 2 and AFP at day 7 after induction of the differentiation procedure. d Expressions of the hepatocyte markers in the various stages of differentiation. After 0, 7, and 14 days of induction, the expressions of hepatocyte markers CK7, CK18, AFP, AAT, CYP3A4, ALB, and CK19 of hAESCs differentiated cells and hPH were analyzed by RT-PCR

Fig. 4

Functional analysis of HLCs derived from hAESCs. a Immunofluorescence analysis of AAT and ALB in hAESCs and HLCs. There were no expressions of AAT and ALB in hAESCs, and the HLCs specifically expressed both AAT and ALB after induction of 14 days. b Expressions of hepatocyte-specific markers. The mRNA expressions of CYP3A4, CYP1A2, CYP7A1, and CYP2B6 were detected by RT-qPCR in hAESCs, HLCs, and hPH, and the results showed that HLCs specifically expressed all of these markers. c Secretion of albumin. The secretion of albumin was determined by ELISA in the culture medium of hPH, hAESCs, and HLCs, and the results showed that the HLCs specifically secreted albumin after induction of 14 days. The hPH cells were used as the positive control. d Biochemical analysis of urea. The urea production was also secreted in culture medium of HLCs after induction of 7 and 14 days. The hPH cells were used as the positive control. e PAS staining and ICG uptake. The glycogen storage and ICG uptake function of hAESCs, HLCs, and hPH were analyzed by PAS staining (I, II, III) and ICG uptake (IV, V, VI), respectively, and the results showed that HLCs can be specifically stained by PAS and uptaken ICG. Significance was measured via a two-way ANOVA. *P < 0.05, *P < 0.01, ***P < 0.001

Fig. 5

hAESC-derived hepatocyte-like cells improve the liver functions and reduce hepatic damage in ALF mice. a Experimental schematic of CCl₄-induced acute liver failure in the NOD-SCID mouse model. The experiments were conducted in three groups including the olive oil group, CCl₄+PBS group, and CCl₄+HLCs group. b Examinations of liver function. Some liver functional parameters including TBIL, ALT, AST, ALB, and ALP were determined in olive oil, CCl₄+PBS, and CCl₄+HLC group mice after 3 days and 7 days of HLC transplantation. c H&E staining of liver tissue. HLC injection led to notable improvements in the liver tissue structure at the day 3. d Measurements of necrosis area. The necrosis area of five random, nonoverlapping fields was quantitatively measured in liver tissues of CCl₄-induced ALF mice from different experimental groups. Significance was measured via a two-way ANOVA. *P < 0.05, *P < 0.01, ***P < 0.001

Fig. 6

The distribution and therapeutic effects of HLCs in lethal acute liver failure mice. a Representative images of GFP-expressing HLCs. b The distribution of GFP marked HLCs in vivo of acute liver failure mice on day 3 after transplantation. HLCs mainly distributed in the lung and liver. c Immunohistochemical staining and immunostaining analysis of GFP-expressing HLCs in liver tissue. The results showed that GFP-positive cells were detected in injured liver after 7 days of infusion. d Human-specific Alu gene were analyzed by RT-PCR using genomic DNA extracted from HLCs, normal mice livers, and HLC-infused CCL₄-infused mice livers. e Survival curves of CCl₄-induced ALF in mice. The CCl₄-induced ALF NOD-SCID mice were administrated intravenously with 2 × 10⁶ HLCs (CCl₄+HLCs group) or PBS (CCl₄+PBS group), and the death rates were determined within 14 days. The olive oil-treated mice (olive oil group) were used as a normal control (n = 8 in each group)