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Page 1: Non-Anastomotic Biliary Strictures After Liver Transplantationscripties.umcg.eldoc.ub.rug.nl/FILES/root/... · Non-anastomotic biliary strictures (NAS) are a feared complication causing

Non-Anastomotic Biliary Strictures After Liver Transplantation:

Injury, Regeneration and Preservation of the Biliary Tree

Final report for the M3 Research Clerkship of: Pepijn D. Weeder S1823264 Under supervision of: Prof. Dr. R.J. Porte, Chirurg Afdeling Hepatobiliaire Chirurgie & Levertransplantatie Universitair Medisch Centrum Groningen/ Rijksuniversiteit Groningen

Prof. Korkut Uygun, PhD Center of Engineering in Medicine Harvard Medical School/ Massachusetts General Hospital Boston, MA, USA

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Summary: Non-anastomotic biliary strictures (NAS) are a feared complication causing significant morbity and mortality in liver transplant recipients. The etiology of NAS is not fully understood, although ischemia is believed to be a major risk factor for biliary injury. In this study, injury of the peribiliary vascular plexus and deep peribiliary glands (dPBGs) in donor bile ducts at the end of cold storage were strongly associated with donor risk factors. Injury of the dPBGs is an independent risk factor for the development of NAS after transplantation. A method for the quantification of biliopancreatic progenitor cells in the PBGs was developed and a proof-of-concept was achieved in a pilot study. Secondly, a putative mouse model to study the role of biliopancreatic progenitor cells in biliary regeneration after injury was presented. Finally, a new bile duct model for the comparison of different static preservation solutions was designed. This model was applied to assess the effect of oxygen (carrier) enrichment of University of Wisconsin preservation solution. However, because of high variability and relatively low power, the results were inconclusive. Overall, the work presented in this thesis may contribute to a better understanding of injury, regeneration and preservation of the biliary tree in the liver transplant patient. Samenvatting: Het ontstaan van niet-anastomotische galwegstricturen (NAS) is een gevreesde complicatie bij levertransplantatiepatiënten die aanzienlijke morbiditeit en mortaliteit veroorzaakt. Het exacte onstaansmechanisme van NAS is niet bekend, maar ischemie wordt beschouwd als een belangrijke risicofactor voor galwegschade. Dit onderzoek toont aan dat schade aan de peribiliare vasculaire plexus en de diepe peribiliary klieren (dPBGs) in de extrahepatische donorgalweg aan het einde van de koude bewaartijd sterk is geassocieerd met al bekende risicofactoren. Schade aan de dPBGs is een onafhankelijke risicofactor voor het ontwikkelen van NAS na transplantatie. Verder wordt een methode beschreven voor de kwantificatie van biliopancreatische voorloper cellen in de PBGs, die succesvol werd getest in een pilot studie. Ten tweede wordt in deze scriptie een protocol voorgesteld voor de ontwikkeling van een nieuw muismodel waarmee de rol van biliopancreatische voorlopercellen in het herstel van de galweg na ischemische schade zou kunnen worden bestudeerd. Tot slot is er een nieuw galwegmodel ontwikkeld dat kan worden gebruikt om het effect van verschillende preservatievloeistoffen te vergelijken. Dit model is toegepast om het effect van oxygenatie van University of Wisconsin oplossing te onderzoeken. Helaas heeft dit door hoge variabiliteit en een kleine groepsgrootte nog geen eenduidig resultaat opgeleverd. In het algemeen kan dit verslag mogelijk een bijdrage leveren aan een beter begrip van schade, herstel en bescherming van de galwegen in de context van levertransplantatie.

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Contents

1. Preface..............................................................................................................

2. Chapter 1: Is the number of bipotent biliary progenitor cells associated with

the development of non-anastomotic biliary strictures after liver

transplantation?................................................................................................

i. Introduction

ii. Materials and methods……………………………………….

iii. Results……………………………..........................................

iv. Discussion and Conclusion......................................................

3. Chapter 2: Brief proposal of a novel mouse model for non-anastomotic

biliary strictures. .............................................................................................

4. Chapter 3: Does oxygenation during static cold storage enhance bile duct

preservation in liver transplantation?...............................................................

i. Introduction..............................................................................

ii. Materials and methods.............................................................

iii. Results .....................................................................................

iv. Discussion and Conclusion......................................................

5. Conclusion.......................................................................................................

6. References........................................................................................................

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Preface Orthotopic liver transplantation is currently the only available life-saving treatment for patients with end-stage liver disease. Unfortunately, the number of organs available for transplantation greatly surpasses the supply, prompting strict selection criteria for transplant candidates and long waiting lists for those patients that reach candidate status. As a result of this shortage about 15% of patients die while on the waiting list.1 More notably, according to studies of death certificates, about 60,000 patients die of liver disease annually in the United States2; Many of whom could (theoretically) have been treated with a liver transplant, causing gross underestimation of the magnitude of the problem when only waitlist mortality is considered. In fact, only about 1.5% of liver disease mortality concerns waitlisted patients.3 Because of the grave shortage of livers available for transplantation, the transplant community is continuously pushing to increase the availability of organs. Also, improved treatments for liver diseases are pursued aiming to decrease the demand for transplantation. As a part of this effort, the limits of the criteria used for donor selection have been progressively expanded, allowing more marginal grafts to be used. Donors that fall outside of standard criteria, termed ‘extended criteria donors (ECDs), have gained ground significantly worldwide in the past decades. For example, in the United Kingdom 42% of transplanted liver now come from donation after cardiac death (DCD) donors.4 The most important other criteria that are being extended include donor age, prolonged cold ischemia time, ABO incompatibility, steatosis and infection.5,6 It has been estimated that the use of ECDs can increase the organ pool by 10% to 20%.7,8 At first, the use of ECDs came at the cost of increased primary non-function, delayed graft function and decreased survival, but with meticulous donor selection the initial rise of these problems has been successfully reversed to a level comparable to grafts from donation after brain death10-13 However, a serious remaining problem is the rise of biliary complications in recipients of ECD grafts.14-17 The incidence of non-anastomotic biliary strictures (NAS) varies between 4 and 15% after transplantation of livers donated after brain death (DBD) but can be as high as 30% after transplantation of DCD grafts.18 Developing NAS critically impacts patients’ long-term survival, rate of retransplantation, quality of life and the cost of care.9,19 Patients that develop NAS typically present between one to twelve months after liver

Figure 1. Examples of the radiological findings associated with NAS. Left: “ERCP showing a long nonanastomotic stricture extending proximally from the site of the anastomosis.”(adopted from Sharma et al. 20089). Right: ERCP showing the hallmark sign of ‘Beads on a string’ resulting from alternating stenosis and dilatation (adopted from Buis et al. 2007 24).

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transplantation. Their symptoms are characterized by cholestasis, often accompanied by cholangitis and abdominal pain.20 The diagnosis is made by ultrasound examination followed by endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC).9 Examples of the radiological findings associated with NAS can be found in figure 1. Strictures are progressive in 60% of cases, ultimately leading to cirrhosis.21 The work reported in this thesis is centered on the problem of NAS. It focuses on the etiology of NAS (chapter 1) and proposes a novel animal model to study the role of progenitor cells in this process (chapter 2). Finally, an experiment was conducted to investigate whether oxygenation has a protective effect on the bile duct during cold storage (chapter 3). The research efforts presented here were made between November 1, 2013 and March 21, 2014 in the context of my research clerkship, which is an integral part of the Master’s of Medicine program that I attend at the University of Groningen. The work took place at the Center of Engineering in Medicine at Harvard Medical School in close collaboration with the transplant division of Massachusetts General Hospital.

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Chapter 1: Is the number of bipotent biliary progenitor cells associated with the development of non-anastomotic biliary strictures after liver transplantation? Introduction The formation of non-anastomotic biliary strictures (NAS) is a devastating complication that can occur after liver transplantation. It is associated with significant morbity and mortality and currently limits the use of extended criteria donors.9,19 The rise of the incidence of NAS in conjuction with extended warm and cold ischemia time, points towards a causal role of ischemia in the occurrence of biliary stricturing after transplantation.18 The liver has a dual inflow of blood from both the portal vein and the hepatic artery. However, the bile duct relies exclusively on the arterial vasculature for its supply of oxygen and nutrients.18 This is evidenced by the fact that thrombosis of the hepatic artery can lead to rapid development of massive biliary stenosis and/or necrosis.22 Furthermore, cholangiocytes, the specialized epithelial cells lining the biliary tree, are known to be highly susceptible to ischemia-reperfusion injury. 23 During the past two decades, the prevailing paradigm was that excessive cholangiocyte injury formed the most important determining factor prompting post-transplant scar formation in the biliary tree.24 Surprisingly however, recent studies have shown that 86-88% of grafts have sustained severe injury to the bile duct epithelium at the time of transplantation.25,26 This led Karimian et al. to hypothesize that the (failing) regenerative capacity of the bile duct may be more important than the initial amount of injury in the development of NAS.27 In a histological study, op den Dries et al. showed that injury of the deep peribiliary glands (PBGs) and the peribiliary vascular plexus (PVP) predict the formation of NAS after transplantation.28 The PBGs are a known niche for stem/progenitor cells.29-31 This result suggests that loss of the bilio-pancreatic progenitor cell niche may play a pivotal role in the etiology of NAS, possibly under the upstream influence of microvascular injury. However, with the H&E staining and scoring system that was used in this previous study the presence or absence of stem/progenitor cells cannot be shown. The biliopancreatic progenitor-like celltype has been relatively well localized and described. It is characterized by the co-expression of transcription factors “pancreatic and duodenal homebox 1” (PDX1) and “(sex determining region Y)-box 17” (SOX17).29 The progenitors are thought to be bi-potent, yielding cells that differentiate towards either a pancreatic or a cholangiocyte faith.30 The default path of differentiation is pancreatic, which is inhibited by “Hairy and enhancer of split-1” (HES1) to maintain the cholangiocyte lineage.29,32,33 Biliary progenitor cells have been implied to play a role in biliary regeneration in humans, which is supported by the observation that they proliferate upon injury in mice.31,34 Table 1 shows an overview of markers expressed by different relevant celtypes in the hepatopancreatobiliary region.

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The aims of this study are: i) To determine if donor-related risk factors for NAS predict the occurrence of injury to the PVP and to the deep PBGs; ii) To develop a technique for the quantification of the number of biliopancreatic progenitor cells in the PBGs of the extrahepatic bile duct. The hypothesis is that donor-related risk factors for NAS are associated with the occurrence of injury to the PVP and to the deep PBGs because the latter are mechanistic effectors in the etiology of this complication. Moreover, it is hypothesized that the fraction of progenitors cells in the PBGs will be higher in patients with NAS, because these cells are highly resilient and will therefore survive longer while the surrounding tissue ceases from ischemic injury. Materials and methods

Study subjects and tissue collection The multivariate analysis performed in this study forms an additional analysis of data that was previously studied univariately.28 Biopsies of the distal common bile duct were taken at the end of cold storage from 67 donor livers in Massachusetts General Hospital (MGH) and the University Medical Center Groningen (UMCG) combined. Biopsies were either snap frozen or formalin fixed and then sectioned and stained for H&E. The Institutional Review Board approved collection of tissue and follow up data at MGH. In the UMCG, acquisition of patient information and distal bile duct sections were in accordance with national legislation and the code for usage of human remnant material (Code Goed Gebruik, Federation of Medical Scientific Societies in the Netherlands).

Histological scoring

H&E slides of bile ducts were graded in a blinded fashion using a semi-quantitative scoring system that was initially published by Hansen et al. and later modified by op den Dries et al. 25,28 A grade of injury (grade 0, 1 or 2 for no injury, <50% injury or >50%injury) was assigned to different aspects of the tissue. An overview of the scoring system can be found in table 2.

Table 1. Overview of marker profiles expressed by different relevant celltypes in the hepatopancreatobiliary region. Cell type Marker Reference Definitive endoderm cell OCT4, FOXa2, CXCR4, SOX17, SOX9, FoXa2,

HNF6, Prox1, Sall4, CD326+/EpCAM, CD56+/NCAM, CD133

29,30

Bilio-pancreatic progenitor cell PDX1+/SOX17+, EPCAM, AFP- , K19+/C-kit+ 29,31,34,35

Pancreatic progenitor cell PDX1+/SOX17- 29 Biliary progenitor cell PDX1-/SOX17+, HES1 29,32

Intestinal stem cell LGR5 29,36 General stem cell markers SOX9, EpCAM, NCAM 29

Markers of mature liver/cholangiocytic cells

Albumin, HepPar-1, secretin receptor (SR), ck19, ck7, α-tubulin, α-foetoprotein, Gamma-GT

29-31

Markers of mature pancreatic cells

Insulin, Glucagon 29,30

Cholangiocyte lineage cells Nuclei+/EPCAM+/SR+ 29 Mucus secreting cells (goblet) PAS+/Alcian blue+ 29,31

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Table 2. Overview of the Hansen et al. histological grading system. (Table adopted from op den Dries et al 28)

Bile duct wall component

Injury Score

Grade 0

Grade 1

Grade 2

Grade 3

Biliary epithelium

No loss

≤50% loss

>50% loss

N/A

Mural stroma No injury ≤25% necrotic 25-50% necrotic >50% necrotic

Peribiliary vessels No injury ≤50% of vessels with changes

>50% of vessels with changes

Grade 2 + arteriolonecrosis

Thrombosis Absent Present N/A N/A Intramural bleeding None ≤50% of duct wall >50% of duct wall N/A Periluminal PBG No injury ≤50% loss of cells >50% loss of cells N/A

Deep PBG No injury ≤50% loss of cells >50% loss of cells N/A

Inflammation None At least 10 leukocytes / HPF

At least 50 leukocytes / HPF

N/A

Recorded variables and follow-up

67 patients transplanted between May 1, 2010 and January 1, 2013 were included in the study. The last date of follow-up was January 1, 2014. Patients were classified as NAS or non-NAS according to the definition described by op den Dries et al.28 Additionally, among other demographic variables (table 3, results section), the type of donor (brain death [DBD] vs cardiac death [DCD]), cold ischemia time (CIT), donor warm ischemia time (WIT), type of preservation solution (HTK or UW) and donor age were recorded.

Immunofluorescent staining Common bile duct tissue from one NAS graft, two non-NAS grafts (controls) and two biopsies taken at procurement right the after cold flush (baseline) were selected for immunofluorescent staining. Sections were mounted on glass slides coated with 0.1% poly-Llysine, hydrated in graded alcohol and rinsed in phosphate-buffered saline with Tween 20 (0.1%). 30 minute incubation with BSA 5% in PBS followed, succeeded by overnight incubation with primary antibodies (PDX1: Rabbit anti-human, Santa Cruz, catalog no. sc-25403. SOX17: Goat anti-human, R&D systems, catalog no. AF1924) overnight at 4°C. Slides were then washed three times with PBS-T and incubated with secondary antibodies (Donkey anti-rabbit, catalog no. A-31573 and donkey anti-goat, catalog no. A-1105 , both from Life Technologies) for one hour at room temperature in the dark. After three washings with PBS-T in the dark, tissue was mounted with mounting media containing DAPI nuclear background staining.

Slide scanning and progenitor cell quantification Fluorescently stained samples were scanned entirely using a TissueFaxs®, Zeiss AxioObserver Z1 Microscope System (Tissue-Gnostics GmbH, Vienna, Austria). TissueQuest® image analysis software (Tissue-Gnostics GmbH, Austria) was used to select all visible peribiliary glands. Data from all PBG’s in each slide were pooled. Additionally, a random 1mm2 section of tissue was selected for background calibration. For each detected cell, the surface area and signal intensity for DAPI, PDX1, SOX17 were recorded. Threshold

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values for the co-expression of PDX1 and SOX17 were determined by calculating the 99% confidence interval of the signal intensity for PDX1 and SOX17 in the background. Cells of the peribiliary glands with signals higher than the 99% confidence interval for both markers were considered progenitor cells. The area covered by progenitor cells divided by the total cell area of the peribiliary glands resulted in the progenitor cell fraction. Statistical analysis Continuous variables are shown as medians and interquartile range. Categorical variables are presented as the number of cases and percentage. For univariate analysis variables were compared using the students’ t-test if they met the test’ assumptions. For other variables the non-parametric Mann-Whitney-U test was used and Chi-square for categorical variables. Multivariate analysis was performed using a stepwise logistic regression model. Throughout, variables with a p-value of <0.05 were considered to be significant. All statistical analyses were performed using IBM SPSS version 21 for Mac. Results Determination of risk factors for histological injury and NAS Relevant demographic information about patients and recipients is shown in table 3. 23 (31%) patients received a DCD graft and 50 (69%) were transplanted with an organ from a DBD donor. The median donor age was 50 (35.3-61) and the median cold ischemia time was 421 (341-492) minutes. 12 (16%) of included cases were re-transplantations. Table 3. Demographic variables describing study subjects. Variable Number (%) or

Median (IQR)

Donor characteristics Age (years) 50 (35.3 – 61) Gender (male) 38 (52.1%) Body Mass Index 25.1 (22.8 – 27.3) Cause of Death

Cerebrovascular Accident Post-anoxia Trauma other

40 16 16

1

(55%) (22%) (22%) (1%)

Type of donor DBD DCD

Graft type Full sized graft Reduced graft

50 23

69 4

(69%) (31%) 95% 5%

Type of preservation fluid UW solution HTK solution

45 28

(62%) (38%)

Donor warm ischemia time (minutes) 16.5 (11.8-20.8) Recipient characteristics Age (years) 53 (35-60) Gender (male) 50 (68.5%) Indication for transplantation

Alcoholic liver disease Hepatocellular carcinoma Biliary cirrhosis (PSC, PBC or atresia) Acute liver failure Post-viral cirrhosis (hepatitis B or C)

11 10 10

7 6

(15%) (14%) (14%) (10%) (8%)

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Non-alcoholic steatohepatitis Retransplantation Miscellaneous

5 12 12

(7%) (16%) (16%)

MELD score 19 (11.0-30.5) Surgical variables Cold ischemia time (minute) 421 (341-492) Implantation warm ischemia time (minutes) 42 (38 – 49) Type of biliary anastomosis

Duct-to-duct Roux-en-Y hepatico-jejunostomy Duct-duodenum

Biopsy preservation Formalin (10%) Snap frozen

61 11

1

36 34

(84%) (15%) (1%) (51%) (49%)

Univariate analysis was performed to screen for relevant demographic co-variates (not shown). Variables with a p-value <0.20 were included in the logistic regression model. Table 4 summarizes the logistic regression for the association between the selected covariates and the three main endpoints. Table 4. Stepwise logistic regression for association of selected co-variates to i. Vascular lesions; ii. Deep PBG injury and; iii. NAS. #Odds ratio=1.*significant. Odds ratio (OR) Confidence

interval P-value

PVP injury: Cold ischemia time

(per half hour) 1.233

0.966 – 1.574

0.092

Preservation solution UW# vs. HTK

5.780

1.001 – 33.354

0.05*

Donor age <55# vs. >55

4.491

1.264 – 15.956

0.02*

Biopsy fixation method Formalin# vs. freezing

5.264

1.357 – 20.423

0.016*

Deep PBG injury:

Donor type DBD# vs. DCD

19.163

(1.542 – 238.120)

0.022*

Cold ischemia time (per half hour)

1.609

(1.107 – 2.338)

0.013*

Donor age <55# vs. >55

25.258

(2.120 – 300.948)

0.011*

NAS:

Deep PBG mucosal loss <50%# vs >50%

14.000

2.908 – 67.392

0.001

PVP injury The use of HTK preservation solution (OR 5.780) and donor age >55 (OR 4.491) were found to be independently associated with the histological observation of PVP injury. Cold ischemia time showed a trend, but this association was not significant. Furthermore, biopsy preservation by snap freezing was associated with a higher incidence of PVP injury. PBG injury DCD donor type (OR 19,.163), longer Cold ischemia time (OR 1.609 per half hour) and donor age >55 (OR 25.258) showed independent association with deep PBG injury.

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NAS When entering all relevant variables into the model, including the histological grading, only deep PBG mucosal loss was found to be independently associated (OR 14.0) with the outcome of NAS. Biliopancreantic progenitor cell quantification Bile duct sections showed specific staining of cells in the peribiliary glands for both PDX1 and SOX17 as expected. Visual intensity of staining was high at baseline and in the NAS subject but low in controls. Representative images of PBGs as observed by fluorescence microscopy are shown in figure 2.

Figure 2. Peribiliary glands of control patient (left) and NAS patient (right) stained for PDX1 (red), SOX17 (green) and DAPI nuclear background staining (blue). The difference between NAS and controls in the number of cells co-expressing PDX1 and SOX17 in the PBGs was confirmed by the quantitative analysis of microscopic images (figure 3). The median percentage of PBG area populated by progenitor-like cells in baseline samples was 30% (IQR 29%-32%), 3,8% (IQR 3.4%-4.3%) in controls and 43% in NAS (n=1).

Figure 3. Fraction of PBG area populated by progenitor-like cells shown per case.

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Discussion and conclusion The fact that PBG injury is the only variable that exhibits an independent association to NAS, taking into account a multitude of possible risk factors, suggests a critical role for deep PBGs in the etiology of NAS. The finding that classical risk factors for NAS such as DCD donation, donor age and CIT are independently associated with injury to the PVP and PGBs further supports this hypothesis. A more in-depth understanding of the processes that lead to NAS will open new alleys for directed prevention and treatment. The association between the use of HTK preservation solution and vascular lesions adds to controversy surrounding its use in liver transplantation. 37 The question which solution is superior has been around for a long time. A high-powered retrospective study based on data from the United Network for Organ Sharing (UNOS) showed that use of HTK leads to inferior outcomes. 38 Additionally, HTK has been implicated as a cause of increased NAS incidence. 39 But other studies disagree. 40 In some regions, HTK is preferably used over UW in DCD donors, which may cause a false association with inferior outcome. One of the reasons for this is the fact that HTK comes in 3-liter bags (as opposed to 1 liter for UW), which is convenient when rapid flushing is desirable. To the best of my knowledge, this is the first study to show an association between histological injury and the use of HTK solution compared to UW. This study showed a highly significant association between dPGB injury and the development of NAS, independent from many major risk factors that were taken into account. Consequentially the role of PBGs deserves further scrutiny. The establishment of a method for the quantification of progenitor cells in the wall of the bile duct forms an important advance in this process. In a pilot study, the fraction of progenitor cells in the PBGs seemed to be higher in NAS, compared to controls. Using the techniques described in this chapter, progenitor cell quantification will be performed in the coming months comparing NAS patients to a group of matched controls. This study has several limitations. Analysis of observational data can only yield associations and does not prove any causal relations. The confidence intervals associated with the risk factors that were identified in this study are very wide. This implies that although the associations that were found are significant, the effect size remains unknown. A possible confounding factor is the preservation method of bile duct biopsies. Tissue was initially collected by snap freezing, which was replaced by formalin fixation at a later phase of tissue collection. The method of fixation proved to be a significant risk factor for vascular lesions; because of this artefact, outcomes with regard to vascular lesions in this study should be interpreted with caution. The immunofluorescent staining lacked negative and positive controls (which will be added to the experiment in the near future). Moreover, although there is a significant body of literature describing the transcription factor expression profile of biliary progenitor cells. Progenitor cell status (e.g. pluripotency) of marker positive cells can be debated and inversely loss of expression of these markers does not necessarily equate to loss of pluripotency. This study shows that injury of the PVP and DPBG in donor bile ducts at the end of cold storage is associated with donor risk factors such as DCD, age, type of preservation fluid, and CIT. Injury of the DPBG’s is an independent risk factor for the development of NAS after transplantation. Moreover, a proof-of-concept has been demonstrated for the quantification of progenitor cells in the PBGs.

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Chapter 2: Brief proposal or the development of a novel mouse model for non-anastomotic biliary strictures. Research efforts to investigate the etiology of NAS are currently limited by the lack of any experimental model that can be used to study this problematic complication. In addition, to address the question whether progenitor cells play a causal role in the development of NAS, observational studies cannot suffice. The development of an animal model that expresses a NAS-like phenotype holds promise to address these issues. A putative model to study the role of progenitor cells in biliary regeneration after ischemic injury in mice is presented here. Biliopancreatic progenitor cells are characterized by the co-expression of PDX1 and SOX1. Constitutional knockout of these genes leads to pancreatic agenesis (PDX1-/-) and disruption of definitive endoderm development including biliary atresia (SOX17-/-) respectively.41-43 Because the conditions induced by these null mutations cause malformations and are lethal, constitutional knockout of progenitor cells cannot be used to study the specific function of SOX17/PDX1 co-expressing cells in the normal or injured adult bile duct. Cre-Lox recombination provides the opportunity to circumvent these problems by means of inducible knockout in adult animals. When a gene locus is transduced with a Cre (“Causes REcombination”) label, Cre-recombinase causes a recombination event on the LoxP-flanked (floxed) site that has been placed elsewhere on the genome. This recombination results in removal of a stop codon causing the expression of a reporter gene, such as green fluorescent protein, together with the expression of the floxed site. The end result of this mechanism is that only those cells expressing the Cre-labeled gene express the reporter. ROSA26 is a gene that is permanently constitutively expressed in mice.44 Expression of the diptheria toxin receptor (DTR) makes cells highly sensitive to low-dose diphtheria toxin (DT). Floxing the ROSA26 site with a DTR gene induces continuous expression of DTR in Cre positive cells. DTR expressing cells can then be selectively eliminated by injection of DT. Cre-labeled mice strains for both PDX1 and SOX17 exist and have been used for lineage tracing studies. 45-47 Rosa26-DTR and PDX1-cre mice are commercially available (JAX® mice, stock no. 007900 (ROSA26-DTR) and 014647 (PDX1-cre)). A SOX17-cre strain exists but can currently only be acquired through academic collaboration from Germany.46 Cross breeding of these three strains would lead to a PDX1-cre/SOX17-cre/ROX26-DTR genotype that allows for the inducible obliteration of PDX1/SOX17 positive (i.e. biliopancreatic progenitor) cells. Figure 4 shows a schematic overview of this system.

Figure 4. Inducible knockout of PDX1+/SOX17+ cells by injection of DT. (Figure adapted from Clausen and Kel.48) Because PDX1 and SOX17 individually are also widely expressed in other tissues such as the vascular endothelium and the pancreas dosing studies of DT need to be performed to

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determine the optimal dose that achieves sufficient toxicity to cells with two sites of Cre expression (PDX1 and SOX17) but not those cells expressing only one of the Cre-labeled genes.41,45 Finally, to examine the role of the PDX1+/SOX17+ progenitor cells in biliary regeneration, proficiency in the use of a model for total hepatic ischemia needs to be acquired by the investigator. This can be achieved by clamping the hilum of the liver in the presence of a portosystemic shunt that prevents (lethal) congestion of the intestines.49 The best method currently available to achieve portosystemic shunt is subdermal transposition of the spleen.50 In this procedure, the spleen is mobilized through a small incision and placed under the skin. After several weeks collaterals form connecting the spleen to the epigastric vein, creating the desired shunt (figure 5).

Figure 5. Splenic transposition into the extra-abdominal cavity in the mouse leads to the formation of a portosystemic shunt. During hilar occlusion visceral blood drain into the epigastric vein (EV) through collateral vessels (CLV). The dotted line indicates the peritoneum, the solid line reperesents the skin. (Image adopted from Matsumoto et al. 50) Relevant endpoints to study the biliary regeneration after ischemic injury in this model are short- and long-term survival, histology of the liver and bile acids, bilirubin, ALP, AST, ALT and Gamma-GT in serum. Additionally, CT imaging could be applied to detect the formation of biliary strictures in vivo, if they occur.51 Successful development of the model presented in this chapter would possess exceptional potential to experimentally clarify the role of biliopancreatic progenitor cells in the process of bile duct regeneration and perhaps contribute to the understanding of the etiology of NAS.

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Chapter 3: Does oxygenation during static cold storage enhance bile duct preservation in liver transplantation? Introduction To allow for successful transplantation of the liver, the organ has to be kept alive outside the human body for a period of several hours to make the transfer from donor to recipient possible. Currently, the standard method of preservation is based on cooling the organ to approximately 4ºC.52 At this temperature, the cellular metabolism is slowed to a very low rate, enabling tissue survival without the opportunity of oxygen or nutrient exchange. This low-tech mode of preservation only requires a plastic bag with preservation solution, ice cubes and a Styrofoam box, making it inexpensive and highly resistant to technical failure.53 A major disadvantage of static cold storage (SCS) is that although the devastating effects of hypoxia and ischemia are blunted at low temperatures, significant injury still occurs.54,55 This is probably due to incomplete shutdown of the hepatic metabolism at 4°C.   Even at temperatures between 0°C and 4°C the liver still requires 0.27 µmol/min/g liver of oxygen.56 The biliary tree, which is often referred to as the “Achilles heel” of the transplant liver, is especially sensitive to ischemic injury.23,57,58 Ischemia and subsequent reperfusion injury, are thought to be the most important contributors to the development or non-anastomotic biliary strictures after liver transplantation.18,20 Reduction of ischemia could therefore potentially contribute to the prevention of NAS in liver transplantation. A potential approach to alleviate ischemic stress during cold storage is oxygen enrichment of static the preservation solution. Because the solubility of oxygen in water is relatively low, the addition of an oxygen carrier to considerably increase the oxygen storing capacity of the solution is desirable.59 perfluorocarbons (PFC) are a class of compounds with high oxygen storing capacity that have been used for this purpose in pancreas transplantation.60 Perfluorodecalin (C10F18, PFD) is a bicyclic perfluorinated alkane that can dissolve approximately 16 times more oxygen than water.61 Because PFD is hydrophobic and has a density about double that of water, two discrete phases form when it is added to University of Winsconsin preservation solution (UW). Pancreatic tissue that is preserved in this biphasic mixture floats on the interface of the two layers because its density is higher than UW but lower than PFD.62 This “two-layer method” (TLM) has been shown to extend the maximum organ preservation time in pancreas transplantation and support the production adenosine triphosphate (ATP) , an important marker of viability.63 Hemopure™ (OPK biotech, Cambridge, MA) may be used as an alternative to PFD for enhancing the oxygen carrying capacity of UW solution. The product is synthesized by polymerization of purified bovine hemoglobin.64 It is water soluble, stable at a wide range of temperatures and has excellent oxygen binding and dissociation qualities.65 The fact that it is a hydrophilic protein is a possible advantage over lipophilic PFC, which may (theoretically) leave a residue on tissue that cannot be washed out. Moreover, the intimate contact between oxygenated Hemopure dissolved in UW and tissue may potentially aid oxygen delivery. The aim of this experiment is to investigate the effect of cold storage in three different oxygen (carrier) enriched UW solutions compared to UW alone, on the histological quality, energy content and amount of cholangiocyte injury of human common bile duct derived from organ donors. It is hypothesized that pre-oxygenation of UW solution has a protective effect on the

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bile duct during cold storage, and that the presence of an oxygen carrier is essential in achieving adequate oxygen delivery. Materials and methods The study protocol was approved by the Institutional Review Board of Massachusetts General Hospital (MGH) and by the Executive Committee of the of the Clinical Policy Board of the New England Organ bank, the local Organ Procurement Organization (OPO). Written informed consent for the harvest and research use of specified tissues was always acquired from the donors’ next of kin prior to procurement. During the period from December 1, 2013 until April 20, 2014 the author was permanently available to attend all organ procurements performed by the MGH transplant division. During organ procurements, in cases when the pancreas is left in situ, a section of common bile duct (CBD) is routinely discarded. For this study, 4-5 centimeter sections of this discarded tissue were collected (figure 6).

Figure 6. Anatomical clarification. During liver procurement, the bile duct is divided at its junction with the pancreas. The intrapancreatic portion of the duct, that is usually left in situ (colored green), was collected for this study. The age, gender, body mass index (BMI) and type (DCD or DBD) of all donors were recorded. Additionally the time of withdrawal of life support (in the case of DCD donors), the time of cold flush and the start time and end time of cold storage were registered. The warm ischemia time and the time from flush to the start of oxygen treatment were calculated from this. During procurement, the bile duct was routinely flushed with UW preservation solution through the gallbladder. After the liver had been harvested for either transplant or research, the intrapancreatic bile duct was dissected free by the procuring surgeon and removed from the donor. The duct was then atraumatically cut into ten equally sized rings on the back table by the investigator using either Metzenbaum scissors or a scalpel blade. Two rings were immediately preserved as baseline samples in 10% formaldehyde and by freezing on dry ice respectively. The other eight sections were randomly assigned to one of four preservation solutions: i. UW solution. ii. Oxygenated UW solution. iii. Oxygenated UW solution with 20% oxygenated PFD. iv. Oxygenated UW with 25% Hemopure™ (Hemopure solution). Oxygenated preservation formulations were bubbled with pure oxygen for 20 minutes at a rate of 300 ml/min by submersion of perforated tubing that was connected to a

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portable oxygen tank (Walk-O2-Bout™, Airgas Inc., USA). The total volume of preservation solution was 5ml in all groups. Samples were kept on ice in a tightly sealed 15ml falcon tube for 7 hours. At the end of cold storage, a 0.3 ml sample was drawn from each tube for analysis in a blood gas analyzer (RAPIDpoint 500, Siemens AG, Germany) and pO2, pCO2 and pH were recorded. The two rings of tissue per group were weighed and preserved by fixation in 10% formaldehyde and by snap freezing in liquid nitrogen. Preservation solutions were weighed and 3 ml was sampled and frozen on dry ice. Frozen samples were stored at -80ºC until they were analyzed. Formalin fixed tissue was embedded in paraffin, cut and prepared for microscopic assessment using Haemotoxilin & Eosin staining (H&E). A schematic overview of fresh common bile duct collection, processing and analysis is presented in figure 7.

Figure 7. Schematic overview of intrapancreatic bile duct collection, processing and analysis. Frozen samples were stored at -80ºC for 15-55 days before analysis. ATP was measured using a modified protocol based on a commercially available kit (Biovision, catalog no. K254). In brief, 50 sections of 16µM thickness were cut at -20ºC using cryostat and stored in an eppendorf tube at -80ºC overnight. Samples were measured in carrousels of four at a time.

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500µL of Nucelotide Releasing Buffer was added to each sample. After vortexing for three seconds cells were lysated by pipetting the contents up and down 3 times with a 1ml pipette. Next, samples were centrifuged for 2 minutes at 10,000G at 4ºC. After centrifuging, 105 µL of supernatant was transferred to a luminometer tube and 10µL of ATP monitoring enzyme was added. After swinging the tube for 3 seconds luminescence was measured for one minute and the highest reading was recorded. Lysates were frozen on dry ice immediately after each carrousel of four had been measured. Following, protein concentrations in all lysates were determined for ATP normalization utilizing a calorimetric assay (Biovision, catalog no. K813). Lactate dehydrogenase (LDH) was detected using a calorimetric assay (Biovision, catalog no. K726). Samples of preservation solution where thawed at room temperature and then put on ice. 10µL of each sample was transferred into a 96-well plate and 90 µL of UW solution was added to create a ten-fold dilution. Next, pre-treated samples were pipetted into a second 96-well plate (30 µL from UW and PFD groups, 5 µL of Hemopure solution group) in duplex. Additionally, a standard sequence of NADH, a positive control and two 5 µL ten-fold diluted samples of 25% Hemopure™ in UW (for background subtraction) were added. All wells were supplemented with assay buffer to bring the total volume to 50µL. Finally, 50 µL of reaction mix was added to all wells. The optical density was at 450nm was then measured immediately and every ten minutes for fifty minutes while the sample was incubated at 37ºC in the plate reader. The LDH activity was calculated from the standard curve using the averages of duplex samples and normalized to tissue mass. Values were expressed as fold increase or decrease of mU LDH/mL/gram tissue relative to the non-oxygenated UW group. H&E sections of all bile duct rings were scored in a blinded fashion using the modified Hansen et al. scoring system as described in chapter 1.28 The scores were analyzed both separately and cumulatively to create an overall quality score. Statistial analysis Continuous variables are shown as medians and interquartile range. Categorical variables are presented as the number of cases and percentage. Internally controlled parameters are shown as fold increase or decrease compared to a baseline reference from the same donor. Multiple group comparisons were made with the Kruskal-Wallis test unless specifically stated otherwise. Post-hoc, the non-parametric Mann-Whitney U test was used. Throughout, variables with a p-value of <0.05 were considered to be significant. All statistical analyses were performed using IBM SPSS version 21 for Mac. Results Tissue was collected from eight organ donors. Three donors were excluded from the analysis. The first two cases were excluded because the protocol was changed based on lessons-learned from these cases. Another was excluded because the biopsy was found to be arterial upon histological examination. 2 donors (40%) were DBD type and 3 (60%) were DCD.

Table 5. Demograhpic variables of included tissue donors (n=5). Donor variable Number (%) or

Median (IQR) Age 43 (39-53)

Gender (male) 3 -60%

DBD type (vs. DCD) 2 -40%

Body Mass Index 34.6 (31.3-39.3)

Warm Ischemia Time (min) 10 (0-22)

Time from cold flush to start preservation treatment (min)

91

(85-116)

Liver accepted for transplant 1 -20%

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The median age was 43 (IQR 39-53) and the median time from cold flush to start of storage in the investigatory solutions was 91 minutes (IQR 85-116). An overview of demographic variables can be found in table 5. Because of technical issues, the blood gas analyzer was only available in two cases. Although the differences in pO2 and pH did not reach statistical significance, baseline studies showed that pO2 was significantly elevated in oxygenated groups compared non-oxygenated UW throughout the 7-hour period of cold storage (Figure 8). Furthermore, pH seemed to be higher in the Hemopure solution group (pH 7.45) compared to the other groups (pH 7.35). Median tissue mass was equal between groups. Histological scoring analyzed both separately and as a cumulative score normalized to baseline, showed no association with treatment group (figure 9). An overview of the results is presented in table 6. Table 6. Variables of tissue (Tissue mass, ATP) and preservation solutions (pO2,pH and LDH) measured at the end of cold storage. # pO2 and pH were only measured in two cases. Preservation group P-value

Standard UW UW + O2 UW + PFD (20%) UW + Hemopure (25%)

Variable: Tissue mass (mg) 104 (74-121) 83.5 (51.7-119) 122 (94.9-139) 144 (116-147) 0.436 pO2 (mmHg) 247 (222-250) 475 (467-482) 370 (343-434) 498 (464-532) 0.265# pH 7.361 (7.35-7.37) 7.36 (7.36-7.37) 7.36 (7.36-7.36) 7.44 (7.43-7.45) 0.256# ATP (mg/µg protein)

3.15E-7

(1.7E-7-5.70 E-7) 4.12E-7

(2.67 E-7- 12.8 E-7) 2.87E-7

(1.88E-7- 11.0 E-7) 1.79E-7

(0.86 E-7-7.81 E-7) 0.395

LDH (mU/mL/gr) 344 (200-711) 222 (152-308) 816 (397-1064) 647 (541-824) 0.386

Figure 8. Baseline partial oxygen pressure (pO2) over time in preservation solutions not containing tissue. n=2 per group.

Figure 9. Cumulative Hansen score per group.

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The level of ATP in al groups was significantly decreased compared to baseline levels (P=0.008). ATP levels between treatment groups were not significantly different (P=0.395). A graphic depiction of normalized ATP levels is shown in figure 10.

Figure 10. Box plot of ATP expressed as a fraction of the within-case baseline (tissue frozen at procurement). n=5 per group. No statistically significant differences in LDH release were seen in the preservation solution after 7 hours of cold storage (P=0.322). However, a trend was detected pointing towards increased release of LDH from the tissue preserved in oxygenated Hemopure solution (P=0.066, Mood's median test). Figure 11 shows the amount of LDH released by tissue stored in an oxygenated solution compared to ‘gold standard’ UW as fold decrease/increase.

Figure 11. Box-plot of LDH released in the preservation solution compared tot level detected in non-oxygenated UW group from the same donor (fold decrease/increase). n=5 per group.

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Discussion and conclusion In this chapter, a new internally controlled model for the study of bile duct preservation is presented. It was shown that in a sealed container, high partial oxygen pressures can be maintained for up to 10 hours. Unfortunately, no clear effects of oxygen (carrier) enriched UW solutions on histology, tissue ATP or release of injury markers were found. This is most likely due to the combination of high variability associated with the use of human tissue and relatively small group sizes (n=5). Nevertheless, the feasibility of the protocol has been established. It still holds potential for finding more comprehensive results when the study gains power with ongoing tissue collection. Remarkably, the amount of LDH released by ±100 mg of bile duct in 5ml of preservation solution of the course of 7 hours can be easily detected. The cell lysate In the Hemopure solution group was colored red, almost certainly due to the presence of Hemopure. Because the amount of ATP was normalized to protein concentrations, and Hemopure is a protein, it is likely that the ATP normalized to protein for this group is unreliable. Normalization of ATP to the concentration of DNA in the solution should solve this problem in future analysis. Although admittedly the effect is not significant, the three-fold increase of LDH release from tissue stored in Hemopure solution compared to non-oxygenated UW raises a red flag. The hypothesis in this study was that oxygen should prevent injury rather than aggravate it. A possible explanation for this observation may be found in the offsetting effect of mixing Hemopure into UW. UW preservation solution has been developed to mimic the intracellular milieu of the cell, which reduces the energy required for ion pumping across the cell membrane. The addition of Hemopure, for which the solvent is Lactated Ringer’s solution, disturbs the carefully titrated intracellular-like ion concentrations. In general, this study has several limitations. The finding that the histological quality of basline samples (n=8) compared to the end of cold storage in any group (n=31) showed no difference (p=0.79) suggests that the major injury occurred even before the baseline sample was fixated. The median time between the cold flush and the start of preservation in oxygenated media was one and a half hours. It is possible that the ischemia during this period causes injury to the bile duct to a degree that it is already beyond rescuing when oxygenated storage is applied. Moreover, on theoretical grounds it may be questioned whether sufficient cellular respiration can be achieved through diffusion by submersion of solid tissue in an oxygen rich fluid. Based on these arguments, an endovascular approach in which the vascular system of the liver is flushed with oxygenated fluid may be more beneficial. This method is currently being investigated in a rat model by the author. In conclusion, the experiment reported here is currently underpowered to detect trends in the data that is characterized by a high natural variability. Ongoing tissue collection will hopefully resolve this problem. Nevertheless, the protocol for this model has been established and all endpoints were successfully measured.

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Conclusion The experimental results that were attained during my 5-month research clerkship are presented in this thesis. In a multivariate analysis, significant associations between classical donor-related risk factors for NAS and histological injury to the PVP and dPBGs were uncovered. Moreover, dPBG injury was strongly and independently associated with the outcome of NAS. These associations come to support the hypothesis that the dPBGs play a role in the etiology of NAS. Additionally, a method for the quantification of biliopancreatic progenitor cells in the PBGs was developed and a proof-of-concept was achieved in a pilot study. In chapter two, a putative mouse model to study the role of biliopancreatic progenitor cells in biliary regeneration after injury was presented. All the building blocks for the creation of this model are available; at present it’s just a matter of putting them together. The first steps in this process will be taken in the months to come. Finally, a new bile duct model for the comparison of different static preservation solutions was designed. This model was applied to assess the effect of oxygen (carrier) enrichment of UW preservation solution. Because of high variability and relatively low power, the results were inconclusive. However, the experiment is still ongoing and hopefully, with continued tissue collection, will yield a more unambiguous outcome in the future. The projects that were discussed here could not be entirely completed within the scope of the research clerkship and are still ongoing. After the end date of the research clerkship, I will stay at Harvard Medical School (HMS) for an additional five months. To date, the experience of working with a very high level of independence at institutions of excellence such as HMS and Massachusetts General Hospital (MGH) has already been invaluable. I am thrilled to have the opportunity to extend my stay and attempt to finish the work that I have started. Finally, doing research is not a one-man job. I would like to thank prof. Uygun and prof. Porte for their continuing supervision and guidance. Other key mentors are dr. Markmann and dr. Yeh from the MGH transplant division and dr. Martins from UMass Memorial Medical Center. Also, I would like to thank my colleagues Bote Bruinsma, Nima Saeidi, Gautham Sridharan and James Avruch for welcoming me into their group.

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