Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury

Abstract

: Liver fibrosis represents the end stage of chronic liver inflammatory diseases and is defined by the abnormal accumulation of extracellular matrix in the liver. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Liver transplantation has been the most effective treatment for these diseases, but the procedure is limited by the shortage of suitable donors. Mesenchymal stromal cells (MSCs) have shown great potential in the treatment of chronic inflammatory diseases associated with fibrosis. This study aimed to evaluate the therapeutic effect of MSC-based cell transplantation as an alternative treatment for liver fibrosis. A CD34-positive subpopulation of human placental amnion membrane-derived stem/progenitor cells (CD34⁺ AMSPCs) was isolated through the depletion of CD34-negative stromal fibroblasts (CD34^- AMSFCs) facilitated by CD34 fluorescence-activated cell sorting, enriched and expanded ex vivo. These cells express pluripotency markers and demonstrate multidirectional differentiation potentials. Comparative analysis was made between CD34⁺ AMSPCs and CD34^- AMSFCs in terms of the expressions of stemness surface markers, embryonic surface antigens, and multilineage differentiation potentials. A mouse model of liver fibrosis was established by thioacetamide (TAA) administration. When injected into the spleen of TAA-injured mice, human placental amnion membrane-derived MSCs (hAM-MSCs) can engraft into the injury site, ameliorate liver fibrosis, and restore liver function, as shown by pathological and blood biochemical analysis and downregulated gene expressions associated with liver damage. CD34⁺ AMSPCs represent a more primitive subset of hAM-MSCs and could be a suitable candidate with a potentially better safety profile for cell-based therapy in treatment of liver diseases associated with fibrosis.

Significance: In this study, a CD34⁺ subpopulation of stem/progenitor cells derived from neonatal placental amnion membrane, denoted as CD34⁺ AMSPCs, were identified, enriched, and characterized. These cells are highly proliferative, express mesenchymal stromal cells and pluripotent stem cell markers, and demonstrate multidirectional differentiation potentials, indicating their promising application in clinical regenerative therapies. CD34⁺ AMSPC transplantation ameliorated liver fibrosis in mice with drug-induced liver injury. These cells represent a potential therapeutic agent for treating liver diseases associated with fibrosis.

Keywords: Antifibrotic; Human amnion membrane-derived CD34+ MSC; Thioacetamide-induced liver fibrosis; Transplantation.

Figure 1.

Comparison of cell phenotype between CD34⁺ amnion membrane-derived stem/progenitor cells and CD34⁻ amnion membrane-derived stromal fibroblast cells. Flow cytometry analysis of common mesenchymal stromal cell makers (CD29, CD44, CD73, CD90, and CD105), stem cell transcription factors (Nanog and Oct-3/4), embryonic antigens (SSEA-1, SSEA-3, SSEA-4, SSEA-5, and GloboH), stem cell biomarkers (CD34, CD133, CD117, CD146, and CD201), and other cell surface markers and receptors (CD31, CD56, CD271, EGFR, and PDGFR). n = 3; ∗, p < .05. Abbreviations: CD34⁺, CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, CD34⁻ amnion membrane-derived stromal fibroblast cells; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor.

Figure 2.

Mesenchymal differentiation capacities of CD34⁺ amnion membrane-derived stem/progenitor cells and CD34⁻ amnion membrane-derived stromal fibroblast cells. After 14 days’ induction in respective differentiation medium, histochemical staining and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were performed to confirm the differentiation and quantify the relative gene expressions representative to each lineage. (A): Adipogenic: intracelluar oil droplets were seen under Oil Red O staining; osteogenic: calcified extracellular matrix was observed by Von Kossa staining; chondrogenic: chondrosphere was formed and photographed. (B): Relative expression of adipogenic, osteogenic, and chondrogenic lineage-specific genes by RT-qPCR. n = 3; ∗, p < .05. Abbreviations: AP, alkaline phosphatase; BM-MSC, bone marrow mesenchymal stromal cells; cbfA, core binding factor-α; CD34⁺, CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, CD34⁻ amnion membrane-derived stromal fibroblast cells.

Figure 3.

Comparison of multidirectional differentiation capacities between CD34⁺ AMSPCs and CD34⁻ amnion membrane-derived stromal fibroblast cells. Immunohistostaining analysis was done to detect markers for cell types from three germ layers. TuJ1: marker for neuron differentiation (ectoderm); troponin T and major histocompatibility complex: markers for cardiomyogenic differentiation (mesoderm); secretory albumin and cytokeratin 18: markers for hepatic differentiation (endoderm). Integrated morphometry analysis was done to quantify the fluorescence intensity. Scale bar = 100 μm. ∗, p < .05. Abbreviations: AMSPCs, amnion membrane-derived stem/progenitor cells; CD34⁺, CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, CD34⁻ amnion membrane-derived stromal fibroblast cells; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MHC, major histocompatibility complex; TUJ1, neuron-specific class III β tubulin.

Figure 4.

Human mesenchymal stromal cell transplantation alleviated thioacetamide (TAA)-induced liver fibrosis. (A): The degree of liver injury from TAA-treated mice scored as 0–4 by histopathological criteria (Masson trichome staining), modified from Nanji et al. [43] as follows: 0 representing no fibrosis, 1 representing fibrosis confined to enlarged portal zones, 2 representing periportal or portal septa with intact architecture, 3 representing architectural distortion (septal fibrosis, bridging) without obvious cirrhosis, and 4 representing probable or definite cirrhosis. (B): The collagen accumulation in the mice quantified by hydroxyproline assay. (C): Histopathological analysis of the livers from CD34⁺, CD34⁻, and adipose tissue-derived mesenchymal stem cell-transplanted mice by Masson's trichrome staining. One-way analysis of variance, followed by Tukey’s multiple-comparisons test, was used to analyze the data; statistical significances were identified by adjusted p values; p < .05 was considered statistically different. Scale bar = 100 μm. ♦♦♦♦ and ####, p < .0001; ♦♦♦, ∗∗∗_, and △△△, p < .001. Abbreviations: adipose, adipose tissue-derived mesenchymal stem cells transplantation; CD34⁺, CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, CD34⁻ amnion membrane-derived stromal fibroblast cells; Control: nontransplantation.

Figure 5.

Human mesenchymal stromal cell transplantation improved the hepatic function of thioacetamide-injured mice. Liver function was evaluated by biochemical parameters. (A): Glutamate-oxaloacetate transaminase; (B): glutamate-pyruvate transaminase; (C): albumin; (D): total bilirubin. One-way analysis of variance, followed by Tukey’s multiple-comparisons test, was used to analyze the data; statistical significances were identified by adjusted p value; p < .05 was considered as statistically different. ♦♦♦♦ and ∗∗∗∗, p < .0001; ♦♦♦ and ∗∗∗, p < .001; ♦♦, ∗∗_, and ##, p < .01; ♦, ∗_, and #, p < .05. Abbreviations: Adipose, transplantation with adipose tissue-derived mesenchymal stem cells; CD34⁺, transplantation with CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, transplantation with CD34⁻ amnion membrane-derived stromal fibroblast cells; Control, nontransplantation; GOT, glutamate oxaloacetate transaminase; T-bil, total bilirubin.

Figure 6.

Engraftment of human mesenchymal stromal cells in the thioacetamide (TAA)-injured mice liver. The frozen sections of normal liver (A–C), CD34⁺ amnion membrane-derived stem/progenitor cells (AMSPC) liver (D–F), CD34⁻ amnion membrane-derived stromal fibroblast cell (AMSFC) liver (G–I), and adipose tissue-derived mesenchymal stem cells (ADMSC) liver (J–L) were shown under a bright field (A, D, G, J), a fluorescent field (B, E, H, K), and a merged field (C, F, I, L). PKH26-labeled CD34⁺ AMSPCs, CD34⁻ AMSFCs, and ADMSCs engrafted into TAA-injured mice tissues were shown in (E), (H), and (K). The dashed lines in (J) and (K) showed the exemplified bridging fibrosis area of the stem/progenitor cells homing. Scale bar = 100 μm. Abbreviation: TP, transplantation.

Figure 7.

The effects of human mesenchymal stromal cell transplantation on the mRNA expressions of Col I (A), α-smooth muscle actin (B), transforming growth factor-β (C), and hepatocyte growth factor (D) in the livers of thioacetamide-injured mice. The relative mRNA levels were measured by quantitative reverse-transcription polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as endogenous control. One-way analysis of variance, followed by Tukey’s multiple-comparisons test, was used to analyze the data; statistical significance was identified by adjusted p value; p < .05 was considered statistically different. ♦♦♦♦, ∗∗∗∗, ####, and △△△△, p < .0001; ♦♦♦ and ∗∗∗, p < .001; ♦♦, p < .01; ♦, ∗, #, and △, p < .05. Abbreviations: α-SMA, α-smooth muscle actin; Adipose, transplantation with adipose tissue-derived mesenchymal stem cells; CD34⁺, transplantation with CD34⁺ amnion membrane-derived stem/progenitor cells; CD34⁻, transplantation with CD34⁻ amnion membrane-derived stromal fibroblast cell; Control, nontransplantation; HGF, hepatocyte growth factor; TGF-β, transforming growth factor-β.