Wessam Hassanein

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

ABSTRACT Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model

Wild‐type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody‐mediated xenograft injury. One such carbohydrate is N‐glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model.

Standard Induction Immunosuppression Interferes with the Role of Vascularized Bone Marrow in Vascularized Composite Allograft Protection

Introduction Clinical induction therapies have been demonstrated to interfere with the development of immunologic tolerance in experimental models. Vascularized bone marrow (VBM) has been demonstrated to provide immunologic protection to vascularized composite allografts (VCA). We utilized a non-human primate (NHP) model of facial transplantation to investigate the effects of recipient T cell depletion and steroid therapy on VBM-mediated graft protection. Materials and Methods We utilized a well-established model of heterotopic facial subunit VCA in a NHP model. Recipients (N=3) were administered 50 mg/kg of anti-thymocyte globulin (ATGAM) on Day -2, -1, and 0 and transplanted Day 0. Steroids were administered with an initial 20 mg/kg dose and tapered over 21 days and maintained on tacrolimus (Tac) and mycophenylate mofetil (MMF). Chimerism was determined using flow cytometry from peripheral blood samples and final tissues. Comparison was made to historical cohorts, VCA + VBM (n=4) and VCA (no VBM, n=3) treated with Tac/MMF. Results and Discussion Mean survival in the ATGAM with steroid taper group was 28.3 ±8.02 days, as compared to VCA + VBM group 348.2±85.9 days, and VCA (no VBM) 24.7±16.6 days (p=0.008). NHP receiving induction therapies developed early Banff 4 rejection, as well as histopathologic evidence of thrombophlebitis and muscle necrosis. In contrast to the VCA + VBM group, chimerism was undetected in peripheral blood and graft bone marrow was nearly acellular, with an average of 91.5%±2.4 (range 89.8-93.2%) of the lymphocytes present recipient in origin (n=2). Also in contrast to VCA + VBM but comparable to VCA (no VBM), the development of IgM and IgG donor specific antibodies was noted in the ATGAM with steroid taper group, with a mean of 357.5 and 180.5 MFI respectively. Figure. No caption available. Conclusion Presumptive VBM cell populations that regulate immune responses are susceptible to T cell depleting and steroid therapies. The observed interference of standard induction therapies on the protective value of VBM highlights relationships between pharmacologic and biologic mechanisms of VCA graft protection. Henry Jackson Foundation, Department of Defense Award # W81XWH-13-2-0056. Astellas Pharma US, Inc.

Re-Endothelialization of Liver Xenografts Utilizing Human Endothelial Cells.

Introduction Acute antibody mediated rejection due to preformed antibodies present in human serum remains a hurdle to successful xenotransplantation. Xenograft vascular endothelium represents the initial site of recipient immune exposure to xenoantigens. We hypothesized that biologically engineered liver xenografts after re-endothelialization of the vascular tree with allogenic endothelial cells has the potential to evade the recipient’s immune response against xenografts. Methods Adult rat livers were procured using aseptic technique (n=6). A novel protocol of selective de-endothelialization was developed utilizing 0.1% SDS perfusion for 15 min using our established bioreactor perfusion system. Treated grafts were examined by H&E staining and under immunofluorescent microscopy using rat specific anti-CD34 as endothelial cell marker. Human umbilical vein endothelial cells (HUVECs) were then introduced into the de-endothelialized livers, and perfused with culture media suplemented with appropriate growth factors. Grafts were kept for 3 days at 37C. Constructs were subsequently examined immunohistochemically for HUVECs using human specific anti-CD31. Results Livers treated with 0.1% SDS showed selective yet complete de-endothelialization compared to native liver (Fig 1A, 1B). The liver architecture remained intact and the majority of liver cells were viable following treatment. HUVECs were successfully engrafted onto the de-endothelialized rat liver vessels. This was confirmed by H&E and immunohistochemistry staining (Fig 1C, 1D). Figure. No caption available. Conclusions Selective de-endothelialization of rat livers was successful, and human cells could re-establish the xenograft vascular bed. This method demonstrates the ability to manipulate a key component of the immune response to xenogeneic antigen. Future experiments are directed at ex-vivo perfusion of the re-endothelilaized grafts with human blood.

Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model

Background Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway. Methods Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized &agr;CD154, n = 9), 5C8H1 (mouse-human chimeric &agr;CD154, n = 5), or 2C10R4 (mouse-rhesus chimeric &agr;CD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant. Results Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20+ lymphocytes from baseline at day 14 after transplant (−457 ± 152 cells/&mgr;L) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype. Conclusions In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

2582: Bone marrow regulated local protective mechanisms of vascularized composite allografts in non-human primates

2582: Bone marrow regulated local protective mechanisms of vascularized composite allografts in non-human primates Mehmet C. Uluer, MD, ScM, Arthur J. Nam, Jhade D. Woodall, MD, Wessam Hassanein, MD, David A. Bruno, MD, Dawn Parsell, BS, Urmil Drhu, BS, Branko Bojovic, Stephen T. Bartlett, MD, and Rolf N. Barth, MD University of Maryland School of Medicine, Baltimore, MD, USA; University of Maryland Medical Center, Baltimore, MD, USA Background Vascularized composite allografts (VCA) containing vascularized bone marrow (VBM) prolongs graft survival Cell populations within the co-transplanted VBM are believed to protect the skin and muscle graft components We performed experiments to define these mechanisms in our established non-human primate model of facial transplantation. Methods Three experimental groups were performed of facial VCA Group 1: VCACVBM segments to MHCmismatched cynomolgus macaques after donor irradiation (n D 4) Group 2: VCA without bone C heterotopic VBM without skin (n D 4) Group 3: VCACVBM after donor lymphodepletion with ATGAM (n D 2) Immunosuppression consisted of tacrolimus and mycophenolate mofetil End points were graft rejection or PTLD Chimerism was assayed using donor specific anti-MHC antibodies. Results Group 1 recipients underwent early graft loss (mean 32 § 21 d) from rejection (Banff IV) Chimerism was undetectable in 3 animals Donor VBM demonstrated replacement with recipient cells (50%, 70%, 100%, and 100%) Group 2 recipients also experienced early skin rejection (mean 37§18d), while one animal had PTLD Chimerism was likewise undetectable and heterotopic VBM demonstrated fibrosis and recipient replacement Of the 2 preliminary group 3 recipients one rejected early at day 12, while the other developed PTLD Complete replacement of the bone marrow was seen in the early rejecting animal These data compared to historical VCACVBM grafts with mean survival of 348§ 86 days (rejection only after immunosuppression withdrawal), 75% chimerism, and viable donor VBM. Conclusions Facial VCACVBM contain cell populations that protect the graft from early rejection and graft loss Our data support that these cell populations are radiosensitive, and do not confer systemic protective effects These results support a hypothesis that regulatory cell populations within VBM have local down-regulatory functions toward graft infiltrating alloreactive lymphocytes The finding that cells are noted to be susceptible to depletion or downregulation of their protective effect by irradiation should be kept in consideration when choosing pre-operative conditioning regimens and treating rejection in VCA. CONTACT Mehmet C. Uluer, MD, ScM mculuer@gmail.com