Human adipose–derived mesenchymal stem cells attenuate liver ischemia–reperfusion injury and promote liver regeneration Reza F. Saidi, MD,a Barur Rajeshkumar, PhD,b Ahmad Shariftabrizi, MD,c Alexei A. Bogdanov, PhD,d Shaokuan Zheng, PhD,d Karen Dresser, MS,c and Otto Walter, MD,c Providence, RI, and Worcester, MA

Background. Ischemia-reperfusion injury (IRI) of the liver is a well-known cause of morbidity and mortality after liver transplantation. Effective treatment strategies aimed at decreasing hepatic IRI injury and accelerating liver regeneration could offer major benefits in liver transplantation, especially in the case of partial allografts. Human adipose–derived mesenchymal stem cells (HADMSCs) are an attractive source for regenerative medicine because of their anti-inflammatory and regenerative properties. We hypothesized that HADMSCs attenuate IRI and promote liver regeneration. Methods. Mice were subjected to 60 minutes of partial IRI with or without 70% partial hepatectomy. Animals were treated with HADMSCs. Liver IRI was evaluated with serum levels of alanine aminotransferase, serum interleukin-6, and histopathology. Liver samples were stained for specific markers of liver regeneration. Results. Histology, serum interleukin-6, and alanine aminotransferase release revealed that treatment with HADMSCs attenuated liver injury compared with control patients. Improved animal survival and increased number of regenerating cells were observed in HADMSC-treated animals who underwent IRI and partial hepatectomy compared with the control group. Conclusion. HADMSC represents a potential therapeutic strategy to decrease IRI and promote regeneration in liver transplantation. (Surgery 2014;156:1225-31.) From the Division of Organ Transplantation, Department of Surgery,a Alpert Medical School of Brown University, Providence, RI; and Department of Medicine,b Department of Pathology,c and Department of Radiology,d University of Massachusetts Medical School, Worcester, MA

LIVER TRANSPLANTATION (LT) has evolved as the therapy of choice for patients with end-stage liver disease regardless of its etiology. Liver ischemiareperfusion injury (IRI) is the leading cause of hepatocellular injury, mortality, and morbidity after LT.1 IRI negatively affects liver regeneration after LT. Pathogenic mechanisms involved in IRI have been investigated extensively,2-9 but interaction among signaling pathways involved in IRI are highly complex and are not well described. IRI represents the inflammatory process that includes activation of innate immunity and cytokine release

Accepted for publication May 12, 2014. Reprint requests: Reza F. Saidi, MD, FICS, FACS, Division of Organ Transplantation, Department of Surgery, Alpert Medical School of Brown University, 593 Eddy Street, APC 921, Providence, RI 02903. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.05.008

followed by hepatocyte and sinusoidal endothelial damage. Strategies to attenuate IRI and enhance liver regeneration can improve outcomes of LT. Mesenchymal stem cells (MSCs) are known to have regenerative and anti-inflammatory properties.2-4 MSCs are found in multiple tissues, including bone marrow (BM), umbilical cord blood, the periodontal ligaments, and adipose tissue.2 Among these, human adipose tissue-derived MSCs (HADMSCs) are an attractive source for regenerative medicine because they are abundant and can be harvested with minimally invasive techniques.3,4 MSCs can differentiate along multiple cell lineage pathways in a regulated and reproducible manner and also can be transplanted safely and effectively to either an autologous or an allogeneic host because these cells have low immunogenicity. BMderived mesenchymal stem cells have been shown to attenuate live IRI by inhibiting hepatocellular apoptosis and stimulating regeneration.5 The hypothesis of the present study is that HADMSCs attenuate IRI and promote liver regeneration. SURGERY 1225

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MATERIALS AND METHODS Animals. This study was approved by the Institutional Animal Care and Use Committee at The University of Massachusetts Medical School. Wildtype (C57BL6) mice were used. All mice used were 8–10 weeks old, fed a pellet diet with water ad libitum, and kept on a 12-hour light/12-hour dark cycle. For all procedures, mice were anesthetized with an intraperitoneal injection of 0.05 mL/10 g body weight of a ketamine cocktail that consisted of ketamine (13 mg/mL), xylazine (2.6 mg/mL), and acepromazine (0.15 mg/mL) in 0.9% NaCl. All animals were allowed to adapt to the laboratory environment for 7 days with free access to water and standard laboratory chow. Animals were housed under standard environmental conditions with a 12-hour light/dark cycle. Isolation and culturing of HADMSCs. HADMSCs were isolated and cultured in our laboratory as described previously.6 Adipose tissue from the perinephric fat of kidneys used for transplantation was washed with Dulbecco’s modified Eagle medium/F12 medium supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin and cut into 1-mm3 pieces. Adipose tissue was digested in 2 mL of Hank’s balanced salt solution containing 1 mg/mL collagenase type 1 (Worthington Biochemical Corporation, Lakewood, NJ) with shaking for 1 hour at 378C. After we removed undigested tissues by passing the samples through a nylon mesh with a pore size of 100 mm, the stromal vascular fraction (SVF) was precipitated by centrifugation of the filtrate at 1,200 rpm for 5 min at room temperature. The SVF was washed three times by resuspension in DMEM/F12 medium and further centrifugation, and the number of nucleated SVF cells was counted by staining with Turk’s solution. SVF cells (1.0 3 105) were seeded in 20-cm2 T-flasks coated with human fibronectin (Sigma-Aldrich, St. Louis, MO) and cultured in 5 mL of media at 378C under a humidified atmosphere of 5% CO2/95% air. After 24 hours, nonattached cells and the medium were removed, and the culture was continued by replacement of fresh medium every other day. We used HADMSCs that were expanded for 5–10 passages. MSC were characterized by their adherence, fibroblast-like morphology, and capacity to differentiate into the mesenchymal cell lineages of adipocytes, chondrocytes, and osteoblasts. HADMSC were analyzed by immunohistochemistry via a panel contained in a group of antibodies for the positive (CD105, CD29, CD44, CD90) and negative (CD45) selection of this lineage.

Surgery November 2014

Livery injury techniques. To mimic the clinical scenarios relevant to LT dealing with IRI and liver regeneration, animals were subjected to one of the following procedures. For procedures involving hepatic IRI, mice (n = 7) were subjected to partial warm hepatic IRI. The portal triad was dissected and a microvascular clamp placed, conferring ischemia to the median and the left lobes for 60 minutes, followed by reperfusion. In all IRI studies, HADMSCs were administered intravenously (in the tail vein) 30 minutes before ischemia at a dose of 1–2 million. Sham animals just had a laparotomy. The control group received primary human fibroblasts (1 million). Our pilot study showed that preischemic (30 minutes) administration of HADMSCs was the most effective timing. Postischemic administration of cells was ineffective (data not shown). Mice were killed at predetermined time points after reperfusion for serum and liver sampling (6, 12, 18, and 24 hours). For procedures involving combined IRI and 70% partial hepatectomy (PH), we incorporated both of the aforementioned hepatic IR and PH procedures. The portal triad was dissected and a microvascular clamp placed, conferring ischemia to the median and the left lobes. PH (70%) was performed by resecting the right and caudate lobes and leaving only ischemic tissue in place. After these operations, mice were killed, and livers and blood samples were collected at different time points postoperatively (6, 12, 18, and 24 hours). A separate group (n = 7) was watched for survival analysis. Blood also was collected from the vena cava for serum preparation at the time of sacrifice. HADMSCs were administered intravenously (via the tail vein) 30 minutes before ischemia at a dose of 1–2 million. Control animals received primary human fibroblasts (1 million). Markers of hepatocellular injury and IRI. Serum levels of alanine aminotransferase (ALT) were determined (IDDEX Veterinary Services, Sacramento, CA). Serum interleukin-6 (IL-6) levels were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s instructions. Histologic analysis. After euthanasia, livers were fixed with 10% buffered formalin for paraffin embedding, or in OCT compound for frozen sections with no fixation. Five-micron, paraffinembedded sections were stained with hematoxylin and eosin for conventional morphologic evaluation. Suzuki classification7 consisted of three

Surgery Volume 156, Number 5

parameters of hepatic ischemia reperfusion injury: sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal necrosis. Each parameter was graded numerically as follows: congestion: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. The same criteria were used in the graduation of the vacuolization, and for necrosis, the numerical graduation was as follows: 0 = nonnecrotic cells, 1 = single-cell necrosis, 2 =

Human adipose-derived mesenchymal stem cells attenuate liver ischemia-reperfusion injury and promote liver regeneration.

Ischemia-reperfusion injury (IRI) of the liver is a well-known cause of morbidity and mortality after liver transplantation. Effective treatment strat...
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