Korean J Transplant.  2020 Jun;34(2):100-108. 10.4285/kjt.2020.34.2.100.

Hepatocyte and mesenchymal stem cell co-transplantation in rats with acute liver failure

• Ho CM
• Chen YH
• Chien CS
• Ho SL
• Chen HL
• Hu RH
• Lee PH

• KMID: 2503886
• DOI: http://doi.org/10.4285/kjt.2020.34.2.100

Abstract

Background
Cell therapy is considered a potential alternative to liver transplantation in acute liver failure (ALF). We aimed to evaluate the add-on therapeutic benefit of hepatocyte and mesenchymal stem cell (MSC) cotransplantation over hepatocyte-only transplantations in a rat model of ALF.
Methods
ALF was induced by D-galactosamine in Sprague-Dawley rats. Freshly isolated donor hepatocytes were derived from Tg (UBC-emGFP) rats and MSCs were collected from the bone marrow cells of DsRed rats. Donor hepatocytes (1×107/mL) were intraportally transplanted 24 hours after treatment with D-galactosamine over a 70-second interval, and donor MSCs (0.5, 1, or 2×106/0.5 mL) were intraportally transplanted 1 hour after the hepatocyte transplantation was complete. Animals were sacrificed after 7 and 14 days and subjected to donor cell identification, liver histology, serologic testing, and immunohistopathological examination.
Results
MSCs were observed in the periportal area, 1 and 2 weeks after transplantation. Transplanted hepatocytes did not actively proliferate when compared to hepatocyte-only transplantation. Morphologically, transplanted MSCs did not appear to differentiate into hepatocytes even 2 weeks after transplantation. Cotransplantation of MSCs was associated with lower macrophage infiltration, and reduced type I collagen, hepatocyte growth factor, tumor necrosis factor-α, and interleukin 10 expression, with similar gene expression profiles for epidermal growth factor and interleukin 6, when compared to hepatocyte-only transplantation.
Conclusions
Hepatocyte and MSC cotransplantation is feasible and safe in rat models of ALF. MSCs were found to survive the process and could be located within the periportal niches 2 weeks after treatment, without enhancing transplanted hepatocyte proliferation or differentiating into hepatocytes, while ameliorating the inflammatory response.

Keyword

Acute liver failure; Cell transplantation; Mesenchymal stem cells

Figure

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  Fig. 1 Model of acute liver failure (ALF) used in the cotransplantation study. (A) Experimental design for the cotransplantation of rat hepatocytes and mesenchymal stem cells (MSCs) in a rat model of ALF. (B-E) Characterization of MSC. The MSCs were spindle shaped (B) and expressed CD44c (C) but not CD45 (D). They were fluoro-red (+) (E) since they were derived from bone marrow aspirates of DsRed transgenic Sprague-Dawley rats. IP, intraperitoneal injection; BF, blank field; DAPI, 4', 6-diamidino-2-phenylindole; RFP, red fluorescent protein.

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  Fig. 2 Histopathological changes after hepatocyte and mesenchymal stem cell (MSC) cotransplantation in acute liver injury. (A) Control liver. Acute liver injury developed, 1 day after D-galactosamine treatment. Ductular reactions were noted by oval cell marker (OV-6), expression near the portal vein. Prominent CD163+ macrophage and low G-6-P expression were also observed. Fibrosis was limited to the periportal region. (B) One, 2 and 4 weeks after cotransplantation, γ-glutamyltransferase (γGT), was expressed in the ductular cells along with obvious signs of liver recovery. (C) Tracing of transplanted donor cells after cotransplantation. Donor hepatocytes were labelled green and MSCs were labelled red. CD163 marker expression was used to determine macrophage infiltration after cotransplantation and is shown on the right. Arrows indicate the donor cells. CD31 marks the endothelial inner lining of the portal vein. GFP, green fluorescent protein; RFP, red fluorescent protein. Magnification, all ×200.

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  Fig. 3 Serological marker and gene expression changes. Serological changes after cotransplantation of hepatocyte and mesenchymal stem cells (MSCs) in rats with acute liver injury. Markers of injury index (A) and synthetic function index (B). Gene expression of growth factors (C), inflammation (D) and fibrosis (E) after cotransplantation. Control: acute liver injury 1 day after D-galactosamine treatment; Baseline expression: glyceraldehyde 3-phosphate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; H, hepatocyte; HGF, hepatocyte growth factor; EGF, epidermal growth factor; SCF, stem cell factor; IL, interleukin; TNF-α, tumor necrosis factor-α; TGF-β1, transforming growth factor-β1.

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