Mehmet C. Uluer

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.

a Rare Cervical Dystonia Mimic in adults: Congenital Muscular torticollis (Fibromatosis colli), a Follow-up

Neglected or undiagnosed congenital muscular torticollis in adults is quite rare, although it is the third most common congenital deformity in the newborn (1). When left untreated at an early age, deficits in lateral and rotational range of motion can occur along with irreversible facial and skeletal deformities that develop over time. Subtle cases can go unnoticed until early adulthood, with predominant fibrotic replacement in the sternocleidomastoid (SCM) making physical therapy and chemodenervation mostly ineffective. Surgical intervention, in these cases, can prove effective in alleviating pain, improving function and cosmesis (2). We report an update on a previously reported case, misdiagnosed as cervical dystonia, which had undergone partial myectomy of the anterior belly of the SCM with some relief of symptoms but without total resolution after the correct diagnosis of fibromatosis colli (3).

Early Microchimerism After Face Transplantation Detected by Quantitative Real-time Polymerase Chain Reaction of Insertion/Deletion Polymorphisms.

V composite allografts (VCA), such as hand and face transplants, offer an alternative approach to complex reconstructive scenarios that otherwise require multiple complex operations. Multiple face transplants with various components of skin, muscle, and bone have been performed worldwide, with the most extensive including mandible and maxilla, tongue, and skin performed in 2012 at the University of Maryland Medical Center. Microchimerism, defined as the presence of low levels of donor-derived cells (<1%), has not been definitively associated with improved immunologic outcomes. We investigated the presence of chimerism in our clinical full-face VCA with upper and lower jaw vascularized bone marrow components using established techniques of flow cytometry and short tandem repeat analysis without detecting any evidence of macrochimerism. We subsequently analyzed postoperative whole blood samples from our face transplant recipient using a commercial assay (AlleleSEQR) that screens and quantifies DNA by quantitative polymerase chain reaction (PCR) using insertion/deletion (InDel) polymorphisms as genetic markers sensitive to 0.001%.

2586: Next generation quantitative sequencing for detecting chimerism in a NHP model

2586: Next generation quantitative sequencing for detecting chimerism in a NHP model Jhade D. Woodall, MD, Mehmet C. Uluer, MD, ScM, Matthew Chrencik, Arthur J. Nam, MD, Stephen T. Bartlett, MD, and Rolf N. Barth, MD University of Maryland Medical Center; University of Maryland School of Medicine, Baltimore, MD, USA Background The association between chimerism and immunologic tolerance in transplantation has shown a path toward eliminating the need for lifelong immunosuppression We have been investigating vascularized composite allograft (VCA) facial transplantation in a well-established non-human primate (NHP) model and have observed transient chimerism without the use of preconditioning therapies when these grafts include vascularized bone marrow The detection of chimerism in the Mauritian cynomolgus macaque (MCM) model relies on flow cytometry with limited ability to detect microchimerism We explored the use of next generation quantitative sequencing as a method of chimerism detection in our VCA model. Methods: Non-specific PCR primers were designed in a conserved region to amplify all Mafa-B major alleles for each of the 7 haplotypes (M1-M7) in MCMs Two animals DNA were initially used: with M3/M3 and M6/M6 haplotypes After confirming PCR efficiency and that products were of appropriate length the primers were coupled with barcodes for use with the ion torrent sequencing system The barcoded primers were added to mixed DNA from M3 and M6 animals and amplified for 25 cycles These products were purified and then sequenced by ion torrent. Results Initial efficiency was determined with the non-barcoded primers on 2 different animals (M3/M3, M6/ M6) to ensure our conditions and primers were at an optimal range These haplotypes had efficiencies in a reasonable range between 90–105% The addition of the barcode to these primers resulted in a drop in the PCR efficiencies to 77% and 79% respectively both with R values of 099 Despite similar but decreased efficiencies, sequencing was subsequently performed Sequencing results indicated that both haplotypes were present at inexact ratios, thus not permitting quantitative assessments of chimerism. Conclusions Real time PCR and next generation quantitative sequencing technologies are feasible techniques to detect chimerism in our MCM model Optimization of PCR reaction conditions for barcoded primers are necessary for quantitative interpretations Furthermore, more specific primers for sets of haplotypes may be necessary given the genetically close nature of MCM’s CONTACT Mehmet C. Uluer, MD, ScM mculuer@gmail.com

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

Vascularized Composite Allotransplantation: Medical Complications

The objective of this review is to summarize the collective knowledge regarding the risks and complications in vascularized composite tissue allotransplantation (VCA), focusing on upper extremity and facial transplantation. The field of VCA has entered its second decade with an increasing experience in both the impressive good outcomes, as well as defining challenges, risks, and experienced poor results. The limited and selective publishing of negative outcomes in this relatively new field makes it difficult to conclusively evaluate outcomes of graft and patient survival and morbidities. Therefore, published data, conference proceedings, and communications were summarized in an attempt to provide a current outline of complications. These data on the medical complications of VCA should allow for precautions to avoid poor outcomes, data to better provide informed consent to potential recipients, and result in improvements in graft and patient outcomes as VCA finds a place as a therapeutic option for selected patients.

Lost in translation? Microchimersim detection in experimental and clinical transplantation

Abstract The importance of further elucidating the properties surrounding microchimerism in various experi- mental models and clinical transplantation are limited by current techniques and the sensitivity of available platforms. Development of reliable methods and use routine use of microchimerism detection in clinical practice could guide clinical decision making regarding rejection, stable function, and tolerance.