ARTICLE IN PRESS
Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure Rajesh Ramanathan, MD,a Giuseppe Pettinato, PhD,b John T. Beeston, RN,a David D. Lee, MD,c Xuejun Wen, MD, PhD,b Martin J. Mangino, PhD,a and Robert A. Fisher, MD,d Richmond, VA, Jacksonville, FL, and Boston, MA
Introduction. Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Methods. Intraperitoneal D-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. Results. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Conclusion. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. (Surgery 2015;j:j-j.) From the Departments of Surgerya and Chemical and Life Science Engineering,b Virginia Commonwealth University, Richmond, VA; the Department of Surgery,c Mayo Clinic Jacksonville, Jacksonville, FL; and the Transplant Institute,d Beth Israel Deaconess Medical Center, Boston, MA
CELL TRANSPLANTATION holds great promise as an alternative to whole-organ liver transplantation for patients suffering from some forms of acute,
R.R. and G.P. contributed equally to this report. Supported by the Virginia Commonwealth University HumeLee Transplant Center’s Irvin Grant for supplies and resident salary support. There was no industry funding for this study. Conflicts of interest: None of the authors have any personal conflict of interests to report. Presented at the 10th Annual Academic Surgical Congress in Las Vegas, Nevada, February 3–5, 2015. Accepted for publication April 22, 2015. Reprint requests: Robert A. Fisher, MD, FACS, Transplant Institute, Beth Israel Deaconess Medical Center, 110 Francis Street, 7th Floor, Boston, MA 02215. E-mail: [email protected]. edu. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.04.014
life-threatening liver disease. Despite its promise and preliminary success in human trials, widespread application of cell transplantation for liver failure has been hindered by the scarcity of human donor livers for primary cell isolation.1,2 Stem cell-derived hepatocytes are an alternate source for cell transplantation. Induced pluripotent stem cells (iPSC) and mesenchymal stem cells (MSC) are adult-derived cell sources that can be expanded to generate large numbers of cells. These cells display the potential for multilineage differentiation, are devoid of ethical controversy, may be less immunogenic, and are a potential autologous cell source enabling immunosuppression minimization or cessation.3-6 The iPSC and MSC have been differentiated successfully into hepatocyte-like cells. We recently developed a novel, suspension-based, embryoid body approach for differentiating multipotent stem cells from different sources into hepatocyte-like cells.7 SURGERY 1
ARTICLE IN PRESS 2 Ramanathan et al
Because the transplantation of stem cell-derived hepatocytes results in transiency in function, we hypothesized that incorporating endothelial cells (ECs) into clusters of stem cell-derived hepatocytes may improve engraftment and prolong function.8 In this study, we utilized an animal xenotransplant model of lethal acute liver failure (ALF) to investigate the efficacy of human stem cell-derived hepatocyte-like cells in reversing ALF. Using an identical differentiation protocol, we evaluated the in vivo function of hepatocyte-like cells derived from human iPSC, human MSC, and human iPSC + EC. METHODS Rat model of ALF. The Institutional Animal Care and Use Committee approved the use of rats for experimentation in this study. The goal for the model of ALF was reproducible mortality within 3 days of onset of liver failure. Sterile D-galactosamine (Sigma-Aldrich, St. Louis, MO) dissolved in Hank’s balanced salt solution (HBSS) was used to induce ALF. Anesthesia was induced by placement of the rats in an isofluorane induction chamber. Intraperitoneal injection of 800–1,000 mg/kg of D-galactosamine was evaluated during the developmental stage of this model of ALF to determine the dose responsiveness. The model was initially developed in male Lewis rats (LEW/SsNHsd; Harlan Laboratories, Indianapolis, IN) and repeated and translated in male nude athymic rats (Crl:NIH-Foxn1rnu [Charles River Laboratories, Wilmington, MA] and Hsd:RH-Foxn1rnu [Harlan Laboratories]). The Lewis rats were housed in our institutional vivarium with 12-hour light–dark cycles and received standard chow and water ad libitum; the athymic nude rats were housed in a specialized barrier facility and received irradiated chow and water. All rats were weighed daily and signs of hepatic encephalopathy scored using published neurologic criteria.9 Liver injury was quantified by measuring rat serum alanine transferase (ALT) by tail venipuncture (VetScan 2.0, Abaxis, Union City, CA). Animals were monitored for 14 days after transplantation, and when humanely killed, liver and spleen samples were retrieved. Animals were killed at earlier time points if they met the following humane criteria: moribund state, weight loss of $30% from before induction of ALF, or increase in ALT to >15 times baseline. The data were analyzed to determine the optimal dosage required to achieve a reproducible increase in ALT and mortality within 72–96 hours after induction of ALF.
Surgery j 2015
Xenotransplant model. To develop the model, primary hepatocytes isolated from mouse livers were transplanted into the spleens of athymic nude rats 16–18 hours after induction of ALF. ALF was induced in 270–350 g male, athymic nude rats with 950–975 mg/kg of sterile D-galactosamine dissolved in HBSS at a concentration of 97.5 mg/ mL. Isolation of hepatocytes. Primary mouse hepatocytes were isolated from 6- to 8-week-old male mice (C57BL/6NHsd, Harlan Laboratories) weighing 25–30 g. We used 22 mice to establish and optimize the technique. An additional 16 mice were used to obtain donor hepatocytes for culture and transplantation. Under ketamine anesthesia, the inferior vena cava was cannulated with a 24-gauge angiocath (Becton-Dickson, Franklin Lakes, NJ) and the liver flushed and digested retrograde. The flush and digestion solutions were oxygenated for 10 minutes before perfusion and warmed to 378C. The flush solution consisted of 6.67 g/L NaCl, 0.16 g/L KCl, 2.1 g/L NaHCO3, 0.34 g/L KH2PO4, 0.11 g/L MgCl2, and 0.14 g/L MgSO4. The digestion solution consisted of the flush solution with 1.2 mmol/L calcium chloride and 1 mg/mL Type 2 Collagenase (Worthington Biochemical Corp, Lakewood, ND). Both solutions were administered for 7 minutes each at a flow rate of 6 mL/min. After digestion, the peritoneal and diaphragmatic attachments of the liver were transected and the liver was removed in a 50-mL, conical tube with sterile Krebs-Henseleit buffer (25 mmol/L NaHCO3, 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L NaH2PO4, 10 mmol/L glucose and 1.3 mmol/L CaCl2) at 48C. Using a sterile, cotton-tipped applicator, the liver capsule was ruptured and cells released. The solution was strained through a sterile nylon mesh, and the resultant solution was washed twice at 50g for 2 minutes with cold Krebs-Henseleit buffer. The final pellet was re-suspended in 5 mL of cold, hepatocyte maintenance medium (HMM) consisting of enriched Iscove’s Modified Dulbecco’s Medium (Invitrogen, Grand Island, NY) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1 3 penicillin/streptomycin (100 U/mL penicillin + 0.1 mg/mL streptomycin), 2 g/L glucose, 1 g/L galactose, 0.5 g/L nicotinamide, 0.110 g/L sodium pyruvate, 10 mg linoleic acid, 0.5 mL of insulin–transferrin–selenium, 50 mg/L dexamethasone, and 10 mg/L epidermal growth factor (all from Sigma-Aldrich). The resulting sample was analyzed for yield and viability using Trypan blue exclusion. Using our isolation protocol, the mean yields were 1–5 3 107 hepatocytes per
ARTICLE IN PRESS Ramanathan et al 3
Surgery Volume j, Number j
Fig 1. Isolated mouse hepatocytes displaying strong green autofluorescence, Magnification = 20x (A) and close to 90% viability by Trypan blue staining (B).
Table I. Posttransplant survival and human albumin detection in the rat serum at 3 and 14 days after cell transplant Variable
n
iPSC iPSC + EC MSC Mouse hepatocytes Control
14 8 11 9 6
Survivors to 14 d 10 6 8 4 1
(71%)* (75%)* (73%)* (44%) (14%)
Mean survival (d) 11.2 11.2 11.6 8.1 5.4
± ± ± ± ±
Human albumin at 3 d
Human albumin at 14 d
58% (7/12)* 63% (5/8)* 22% (2/9) — (0/2)
0% (0/10) 50% (3/6)* 0% (0/8) — (0/1)
4.7* 4.5* 4.2* 5.6 4.5
*P < .05 versus control. EC, Endothelial cells; iPSC, induced pluripotent stem cells; MSC, mesenchymal stem cell.
mouse. The isolated hepatocytes showed strong autofluorescence and viability between 75 and 90% (Fig 1). The sample was diluted to approximately 1.2 3 106 cells/mL using warm HMM. For immediate transplantation, 2 3 106 cells in 500 mL of HMM were used per rat. Nine athymic nude rats were transplanted with 2 3 106 mouse hepatocytes per rat. Rats that were transplanted with mouse hepatocytes demonstrated survival to 14 days in 4 of 9 rats compared with 1 of 6 rats among the controls (Table I). Cell transplantation. Under isofluorane anesthesia, the caudal pole of the spleen in the recipient rat was eviscerated through a left subcostal incision. After ligating the caudal spleen hilum, cells and media were injected via the caudal spleen into the body of the spleen, and the caudal spleen parenchyma was ligated with 6-0 silk ties. The spleen was placed back in the abdomen, and the muscle and skin were closed with 4-0 Vicryl suture. The animals were monitored for #14 days after transplantation. They were weighed and examined daily. Animals were killed in accordance with predefined humane care criteria. Experimental groups. Cell transplantation was performed using hepatocyte-like cells derived from
3 cell types: iPSC, MSC, and iPSC + EC. The iPSC (WiCell Research Institute, Madison, WI), the MSC (ScienceCell, Carlsbad, CA) and the iPSC combined with human adipose-derived EC (ScienceCell) were differentiated into hepatocyte-like cells using a 4-stage, embryoid body-based, differential protocol in suspension developed based on the in vivo differentiation process of human hepatocytes.7 Cells differentiated using this protocol demonstrated in vitro hepatocyte morphology, albumin synthesis, urea metabolism, and sequential mRNA expression and protein expression of the hepatocyte markers SOX-17, FOXA2, Hhex, GATA4, HNF-4a, AFP, albumin, and CK-18. For transplantation, 100 clusters (approximately 1.2 3 106 cells) of iPSC-derived, MSC-derived and iPSC + EC-derived hepatocyte-like cells were injected into the body of the spleen via the caudal spleen pole through an 18-gauge needle. The control group consisted of transplantation with 500 mL of HMM vehicle. Endpoints. The primary endpoint used was mortality with complete survival defined as survival to 14 days after cell transplantation. Secondary endpoints included serum ALT, presence of human albumin in the rat serum, and histologic
ARTICLE IN PRESS 4 Ramanathan et al
evidence of human cells in the rat liver and spleen. Concentration of serum ALT in whole blood was measured using VetScan 2.0 (Abaxis). The tail vein was used to obtain blood samples before cell transplantation, at 48–72 hours after transplantation, and at killing when possible. The presence of human albumin in the rat serum was evaluated using a Human Albumin ELISA Quantitation Set that was non-cross reactive with rat albumin (Bethyl Laboratories, Montgomery, TX). Liver and spleen samples were recovered at time of death and fixed with 10% neutral buffered formalin. The cells were embedded in paraffin and sectioned with hematoxylin and eosin staining for histologic assessment. The paraffin-embedded slides were deparaffinized using xylene substitute and ethanol. Immunohistochemistry and immunofluorescence were performed on liver and spleen sections to identify the presence of human hepatocyte markers and human ECs. Immunohistochemistry. For detection of human albumin, after nonspecific binding was blocked with 2% donkey serum (Sigma-Aldrich), the slides were incubated with a non–cross-reactive goat antibody to human albumin primary antibody (Bethyl Laboratories; 1:500) for 60 minutes. The secondary antibody used was HRP-conjugated donkey antibody to goat immunoglobulin (Ig)G (Santa Cruz Biotechnology, Dallas, TX; 1:200) for 60 minutes. For detection of ECs, a non–crossreactive mouse antibody to human platelet EC adhesion molecule (PECAM)-1 (Santa Cruz) was used as the primary antibody. PECAM-1, also known as CD31, is expressed in multiple hematopoietic cell types and has high expression on human ECs. A goat antibody to mouse IgG1-HRP (AbD Serotec, Raleigh, NC) was used as the secondary antibody for 60 minutes. Metal-enhanced diaminobenzidine substrate was used to activate the horseradish peroxidase in both cases (ThermoScientific, Waltham, MA). The slides were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Immunofluorescence. After deparaffinization, antigen retrieval was achieved through boiling the slides at 1008C for 10 minutes. Permeabilization was performed with 0.3% (v/v) Triton-X 100 for 1 hour and nonspecific blocking was performed with 0.5% (v/v) goat serum (Sigma-Aldrich) for 1 hour. The slides were incubated with the following primary antibodies overnight at 48C: mouse anti-human HNF-3b, goat anti-human albumin, rabbit anti-human C-MET, and mouse antihuman PECAM-1 (Santa Cruz). The following secondary antibodies were used: Cy2-AffiniPure goat anti-mouse IgG (Fc Subclass 1 Specific),
Surgery j 2015
Cy2-AffiniPure goat anti-mouse IgG (Fc Subclass 2 Specific), Cy3-AffiniPure donkey anti-goat IgG (H + L), and Cy5-conjugated AffiniPure goat antirabbit IgG (H + L; Jackson ImmunoResearch, West Grove, PA). Nuclei were counterstained with 4’6’-diamidino-2-phenylindole (DAPI) for 10 minutes. Images were acquired with confocal microscopy using Olympus IX81. For statistical analysis, categorical variables were evaluated using Fisher’s or Chi-square tests and continuous variables were evaluated with Student t tests or analysis of variance. All statistical analyses were performed using JMP 9.0 (Stata Corp LP, College Station, TX). RESULTS D-galactosamine can reproducibly induce lethal ALF. Twelve immunocompetent Lewis rats and 14 athymic nude rats were used to build the animal model of ALF. Serum ALT was used as the primary biomarker to monitor liver failure. A dose of 950–1,000 mg/kg resulted in a mean increase of ALT from 64 to 3,945 U/L 1 day after induction of ALF. Histologically, D-galactosamine caused massive hepatic necrosis (Fig 2). Among the 10- to 14-week-old athymic nude rats (n = 14) who achieved an initial increase in serum ALT of 3,000 U/L (9/14 rats), the 3-day mortality after induction of ALF with 975 mg/kg of D-galactosamine was 89% (8/9 rats). Those that had an ALT of 3,000 U/L on the first day after induction of ALF were used for experimentation. Stem cell xenotransplant resulted in increased survival and in vivo function. Rats transplanted with iPSC (n = 14) had 71% survival (10/14) to 14 days, those with iPSC + EC (n = 11) had 75% survival (8/11) to 14 days, and those with MSC (n = 8) had 73% survival (6/8) to 14 days (Table I). Controls (n = 6) had 14% survival (1/6) to 14 days. All the transplanted animals demonstrated a greater survival to 14 days (P < .05; Fig 4). Compared with controls, there was increased survival to 14 days in animals transplanted with iPSC-derived and MSC-derived hepatocyte-like
ARTICLE IN PRESS Surgery Volume j, Number j
Ramanathan et al 5
Fig 2. Representative liver section with hematoxylin and eosin staining displaying widespread hepatic necrosis noted 1 day after induction of acute liver failure with 975 mg/kg of D-galactosamine. Left panel displays 10 3 magnification and right panel 40 3 magnification. Box identifies magnified area. Scale bar represents 50 mm.
Fig 3. Kaplan–Meier survival curve of 10- to 14-week-old control animals. Those animals that incurred a liver injury with an alanine aminotransferase (ALT) level of >3,000 U/L at 1 day after D-galactosamine injection had a 3-day mortality of 8 of 9, compared with a 3-day mortality of 2 of 5 in those with an ALT of
Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure Rajesh Ramanathan, MD,a Giuseppe Pettinato, PhD,b John T. Beeston, RN,a David D. Lee, MD,c Xuejun Wen, MD, PhD,b Martin J. Mangino, PhD,a and Robert A. Fisher, MD,d Richmond, VA, Jacksonville, FL, and Boston, MA
Introduction. Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Methods. Intraperitoneal D-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. Results. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Conclusion. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. (Surgery 2015;j:j-j.) From the Departments of Surgerya and Chemical and Life Science Engineering,b Virginia Commonwealth University, Richmond, VA; the Department of Surgery,c Mayo Clinic Jacksonville, Jacksonville, FL; and the Transplant Institute,d Beth Israel Deaconess Medical Center, Boston, MA
CELL TRANSPLANTATION holds great promise as an alternative to whole-organ liver transplantation for patients suffering from some forms of acute,
R.R. and G.P. contributed equally to this report. Supported by the Virginia Commonwealth University HumeLee Transplant Center’s Irvin Grant for supplies and resident salary support. There was no industry funding for this study. Conflicts of interest: None of the authors have any personal conflict of interests to report. Presented at the 10th Annual Academic Surgical Congress in Las Vegas, Nevada, February 3–5, 2015. Accepted for publication April 22, 2015. Reprint requests: Robert A. Fisher, MD, FACS, Transplant Institute, Beth Israel Deaconess Medical Center, 110 Francis Street, 7th Floor, Boston, MA 02215. E-mail: [email protected]. edu. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.04.014
life-threatening liver disease. Despite its promise and preliminary success in human trials, widespread application of cell transplantation for liver failure has been hindered by the scarcity of human donor livers for primary cell isolation.1,2 Stem cell-derived hepatocytes are an alternate source for cell transplantation. Induced pluripotent stem cells (iPSC) and mesenchymal stem cells (MSC) are adult-derived cell sources that can be expanded to generate large numbers of cells. These cells display the potential for multilineage differentiation, are devoid of ethical controversy, may be less immunogenic, and are a potential autologous cell source enabling immunosuppression minimization or cessation.3-6 The iPSC and MSC have been differentiated successfully into hepatocyte-like cells. We recently developed a novel, suspension-based, embryoid body approach for differentiating multipotent stem cells from different sources into hepatocyte-like cells.7 SURGERY 1
ARTICLE IN PRESS 2 Ramanathan et al
Because the transplantation of stem cell-derived hepatocytes results in transiency in function, we hypothesized that incorporating endothelial cells (ECs) into clusters of stem cell-derived hepatocytes may improve engraftment and prolong function.8 In this study, we utilized an animal xenotransplant model of lethal acute liver failure (ALF) to investigate the efficacy of human stem cell-derived hepatocyte-like cells in reversing ALF. Using an identical differentiation protocol, we evaluated the in vivo function of hepatocyte-like cells derived from human iPSC, human MSC, and human iPSC + EC. METHODS Rat model of ALF. The Institutional Animal Care and Use Committee approved the use of rats for experimentation in this study. The goal for the model of ALF was reproducible mortality within 3 days of onset of liver failure. Sterile D-galactosamine (Sigma-Aldrich, St. Louis, MO) dissolved in Hank’s balanced salt solution (HBSS) was used to induce ALF. Anesthesia was induced by placement of the rats in an isofluorane induction chamber. Intraperitoneal injection of 800–1,000 mg/kg of D-galactosamine was evaluated during the developmental stage of this model of ALF to determine the dose responsiveness. The model was initially developed in male Lewis rats (LEW/SsNHsd; Harlan Laboratories, Indianapolis, IN) and repeated and translated in male nude athymic rats (Crl:NIH-Foxn1rnu [Charles River Laboratories, Wilmington, MA] and Hsd:RH-Foxn1rnu [Harlan Laboratories]). The Lewis rats were housed in our institutional vivarium with 12-hour light–dark cycles and received standard chow and water ad libitum; the athymic nude rats were housed in a specialized barrier facility and received irradiated chow and water. All rats were weighed daily and signs of hepatic encephalopathy scored using published neurologic criteria.9 Liver injury was quantified by measuring rat serum alanine transferase (ALT) by tail venipuncture (VetScan 2.0, Abaxis, Union City, CA). Animals were monitored for 14 days after transplantation, and when humanely killed, liver and spleen samples were retrieved. Animals were killed at earlier time points if they met the following humane criteria: moribund state, weight loss of $30% from before induction of ALF, or increase in ALT to >15 times baseline. The data were analyzed to determine the optimal dosage required to achieve a reproducible increase in ALT and mortality within 72–96 hours after induction of ALF.
Surgery j 2015
Xenotransplant model. To develop the model, primary hepatocytes isolated from mouse livers were transplanted into the spleens of athymic nude rats 16–18 hours after induction of ALF. ALF was induced in 270–350 g male, athymic nude rats with 950–975 mg/kg of sterile D-galactosamine dissolved in HBSS at a concentration of 97.5 mg/ mL. Isolation of hepatocytes. Primary mouse hepatocytes were isolated from 6- to 8-week-old male mice (C57BL/6NHsd, Harlan Laboratories) weighing 25–30 g. We used 22 mice to establish and optimize the technique. An additional 16 mice were used to obtain donor hepatocytes for culture and transplantation. Under ketamine anesthesia, the inferior vena cava was cannulated with a 24-gauge angiocath (Becton-Dickson, Franklin Lakes, NJ) and the liver flushed and digested retrograde. The flush and digestion solutions were oxygenated for 10 minutes before perfusion and warmed to 378C. The flush solution consisted of 6.67 g/L NaCl, 0.16 g/L KCl, 2.1 g/L NaHCO3, 0.34 g/L KH2PO4, 0.11 g/L MgCl2, and 0.14 g/L MgSO4. The digestion solution consisted of the flush solution with 1.2 mmol/L calcium chloride and 1 mg/mL Type 2 Collagenase (Worthington Biochemical Corp, Lakewood, ND). Both solutions were administered for 7 minutes each at a flow rate of 6 mL/min. After digestion, the peritoneal and diaphragmatic attachments of the liver were transected and the liver was removed in a 50-mL, conical tube with sterile Krebs-Henseleit buffer (25 mmol/L NaHCO3, 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L NaH2PO4, 10 mmol/L glucose and 1.3 mmol/L CaCl2) at 48C. Using a sterile, cotton-tipped applicator, the liver capsule was ruptured and cells released. The solution was strained through a sterile nylon mesh, and the resultant solution was washed twice at 50g for 2 minutes with cold Krebs-Henseleit buffer. The final pellet was re-suspended in 5 mL of cold, hepatocyte maintenance medium (HMM) consisting of enriched Iscove’s Modified Dulbecco’s Medium (Invitrogen, Grand Island, NY) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1 3 penicillin/streptomycin (100 U/mL penicillin + 0.1 mg/mL streptomycin), 2 g/L glucose, 1 g/L galactose, 0.5 g/L nicotinamide, 0.110 g/L sodium pyruvate, 10 mg linoleic acid, 0.5 mL of insulin–transferrin–selenium, 50 mg/L dexamethasone, and 10 mg/L epidermal growth factor (all from Sigma-Aldrich). The resulting sample was analyzed for yield and viability using Trypan blue exclusion. Using our isolation protocol, the mean yields were 1–5 3 107 hepatocytes per
ARTICLE IN PRESS Ramanathan et al 3
Surgery Volume j, Number j
Fig 1. Isolated mouse hepatocytes displaying strong green autofluorescence, Magnification = 20x (A) and close to 90% viability by Trypan blue staining (B).
Table I. Posttransplant survival and human albumin detection in the rat serum at 3 and 14 days after cell transplant Variable
n
iPSC iPSC + EC MSC Mouse hepatocytes Control
14 8 11 9 6
Survivors to 14 d 10 6 8 4 1
(71%)* (75%)* (73%)* (44%) (14%)
Mean survival (d) 11.2 11.2 11.6 8.1 5.4
± ± ± ± ±
Human albumin at 3 d
Human albumin at 14 d
58% (7/12)* 63% (5/8)* 22% (2/9) — (0/2)
0% (0/10) 50% (3/6)* 0% (0/8) — (0/1)
4.7* 4.5* 4.2* 5.6 4.5
*P < .05 versus control. EC, Endothelial cells; iPSC, induced pluripotent stem cells; MSC, mesenchymal stem cell.
mouse. The isolated hepatocytes showed strong autofluorescence and viability between 75 and 90% (Fig 1). The sample was diluted to approximately 1.2 3 106 cells/mL using warm HMM. For immediate transplantation, 2 3 106 cells in 500 mL of HMM were used per rat. Nine athymic nude rats were transplanted with 2 3 106 mouse hepatocytes per rat. Rats that were transplanted with mouse hepatocytes demonstrated survival to 14 days in 4 of 9 rats compared with 1 of 6 rats among the controls (Table I). Cell transplantation. Under isofluorane anesthesia, the caudal pole of the spleen in the recipient rat was eviscerated through a left subcostal incision. After ligating the caudal spleen hilum, cells and media were injected via the caudal spleen into the body of the spleen, and the caudal spleen parenchyma was ligated with 6-0 silk ties. The spleen was placed back in the abdomen, and the muscle and skin were closed with 4-0 Vicryl suture. The animals were monitored for #14 days after transplantation. They were weighed and examined daily. Animals were killed in accordance with predefined humane care criteria. Experimental groups. Cell transplantation was performed using hepatocyte-like cells derived from
3 cell types: iPSC, MSC, and iPSC + EC. The iPSC (WiCell Research Institute, Madison, WI), the MSC (ScienceCell, Carlsbad, CA) and the iPSC combined with human adipose-derived EC (ScienceCell) were differentiated into hepatocyte-like cells using a 4-stage, embryoid body-based, differential protocol in suspension developed based on the in vivo differentiation process of human hepatocytes.7 Cells differentiated using this protocol demonstrated in vitro hepatocyte morphology, albumin synthesis, urea metabolism, and sequential mRNA expression and protein expression of the hepatocyte markers SOX-17, FOXA2, Hhex, GATA4, HNF-4a, AFP, albumin, and CK-18. For transplantation, 100 clusters (approximately 1.2 3 106 cells) of iPSC-derived, MSC-derived and iPSC + EC-derived hepatocyte-like cells were injected into the body of the spleen via the caudal spleen pole through an 18-gauge needle. The control group consisted of transplantation with 500 mL of HMM vehicle. Endpoints. The primary endpoint used was mortality with complete survival defined as survival to 14 days after cell transplantation. Secondary endpoints included serum ALT, presence of human albumin in the rat serum, and histologic
ARTICLE IN PRESS 4 Ramanathan et al
evidence of human cells in the rat liver and spleen. Concentration of serum ALT in whole blood was measured using VetScan 2.0 (Abaxis). The tail vein was used to obtain blood samples before cell transplantation, at 48–72 hours after transplantation, and at killing when possible. The presence of human albumin in the rat serum was evaluated using a Human Albumin ELISA Quantitation Set that was non-cross reactive with rat albumin (Bethyl Laboratories, Montgomery, TX). Liver and spleen samples were recovered at time of death and fixed with 10% neutral buffered formalin. The cells were embedded in paraffin and sectioned with hematoxylin and eosin staining for histologic assessment. The paraffin-embedded slides were deparaffinized using xylene substitute and ethanol. Immunohistochemistry and immunofluorescence were performed on liver and spleen sections to identify the presence of human hepatocyte markers and human ECs. Immunohistochemistry. For detection of human albumin, after nonspecific binding was blocked with 2% donkey serum (Sigma-Aldrich), the slides were incubated with a non–cross-reactive goat antibody to human albumin primary antibody (Bethyl Laboratories; 1:500) for 60 minutes. The secondary antibody used was HRP-conjugated donkey antibody to goat immunoglobulin (Ig)G (Santa Cruz Biotechnology, Dallas, TX; 1:200) for 60 minutes. For detection of ECs, a non–crossreactive mouse antibody to human platelet EC adhesion molecule (PECAM)-1 (Santa Cruz) was used as the primary antibody. PECAM-1, also known as CD31, is expressed in multiple hematopoietic cell types and has high expression on human ECs. A goat antibody to mouse IgG1-HRP (AbD Serotec, Raleigh, NC) was used as the secondary antibody for 60 minutes. Metal-enhanced diaminobenzidine substrate was used to activate the horseradish peroxidase in both cases (ThermoScientific, Waltham, MA). The slides were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Immunofluorescence. After deparaffinization, antigen retrieval was achieved through boiling the slides at 1008C for 10 minutes. Permeabilization was performed with 0.3% (v/v) Triton-X 100 for 1 hour and nonspecific blocking was performed with 0.5% (v/v) goat serum (Sigma-Aldrich) for 1 hour. The slides were incubated with the following primary antibodies overnight at 48C: mouse anti-human HNF-3b, goat anti-human albumin, rabbit anti-human C-MET, and mouse antihuman PECAM-1 (Santa Cruz). The following secondary antibodies were used: Cy2-AffiniPure goat anti-mouse IgG (Fc Subclass 1 Specific),
Surgery j 2015
Cy2-AffiniPure goat anti-mouse IgG (Fc Subclass 2 Specific), Cy3-AffiniPure donkey anti-goat IgG (H + L), and Cy5-conjugated AffiniPure goat antirabbit IgG (H + L; Jackson ImmunoResearch, West Grove, PA). Nuclei were counterstained with 4’6’-diamidino-2-phenylindole (DAPI) for 10 minutes. Images were acquired with confocal microscopy using Olympus IX81. For statistical analysis, categorical variables were evaluated using Fisher’s or Chi-square tests and continuous variables were evaluated with Student t tests or analysis of variance. All statistical analyses were performed using JMP 9.0 (Stata Corp LP, College Station, TX). RESULTS D-galactosamine can reproducibly induce lethal ALF. Twelve immunocompetent Lewis rats and 14 athymic nude rats were used to build the animal model of ALF. Serum ALT was used as the primary biomarker to monitor liver failure. A dose of 950–1,000 mg/kg resulted in a mean increase of ALT from 64 to 3,945 U/L 1 day after induction of ALF. Histologically, D-galactosamine caused massive hepatic necrosis (Fig 2). Among the 10- to 14-week-old athymic nude rats (n = 14) who achieved an initial increase in serum ALT of 3,000 U/L (9/14 rats), the 3-day mortality after induction of ALF with 975 mg/kg of D-galactosamine was 89% (8/9 rats). Those that had an ALT of 3,000 U/L on the first day after induction of ALF were used for experimentation. Stem cell xenotransplant resulted in increased survival and in vivo function. Rats transplanted with iPSC (n = 14) had 71% survival (10/14) to 14 days, those with iPSC + EC (n = 11) had 75% survival (8/11) to 14 days, and those with MSC (n = 8) had 73% survival (6/8) to 14 days (Table I). Controls (n = 6) had 14% survival (1/6) to 14 days. All the transplanted animals demonstrated a greater survival to 14 days (P < .05; Fig 4). Compared with controls, there was increased survival to 14 days in animals transplanted with iPSC-derived and MSC-derived hepatocyte-like
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Fig 2. Representative liver section with hematoxylin and eosin staining displaying widespread hepatic necrosis noted 1 day after induction of acute liver failure with 975 mg/kg of D-galactosamine. Left panel displays 10 3 magnification and right panel 40 3 magnification. Box identifies magnified area. Scale bar represents 50 mm.
Fig 3. Kaplan–Meier survival curve of 10- to 14-week-old control animals. Those animals that incurred a liver injury with an alanine aminotransferase (ALT) level of >3,000 U/L at 1 day after D-galactosamine injection had a 3-day mortality of 8 of 9, compared with a 3-day mortality of 2 of 5 in those with an ALT of
