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Scientific Forum Abstracts
RESULTS: Confocal fluorescence microscopy at 1 to 3 days
showed that incremental reversal of the S1P blood-tissue gradient
across in-vitro endothelialized microvessels and monolayers, increases permeability, and induces cell migration and lumen formation, in increasing magnitude to the gradient. Incrementally
restoring the gradient attenuates migration and restores monolayer
stability in proportion to the gradient. Shear magnifies these effects.
CONCLUSIONS: The S1P concentration gradient across the endothelium regulates vascular stability and provides a basis for dissection of this differential response and mediation by its family of S1P
G-protein coupled receptors. We hypothesize that this differential
response can be used to fine-tune angiogenesis to drive healthy
vascular-extensions within engineered tissues.
Directed Differentiation of Human Pluripotent
Stem Cells Generates Tissue-Engineered Liver
Populated by PROM1 and EpCAM Hepatic
Stem/Progenitor Cells
Christopher R Schlieve, MD, Candida Toribio, Kathryn L Fowler,
Johnny L Castillo, Laura-Marie Nucho, MBA,
Tracy C Grikscheit, MD, FACS
Children’s Hospital Los Angeles, Los Angeles, CA
INTRODUCTION: Liver disease affects roughly 30 million patients
worldwide, with liver transplantation being the only definitive therapy for end-stage liver failure. Directed differentiation of human
pluripotent stem cells (hPSC) can produce multiple tissues of the
gastrointestinal tract, including immature hepatocytes and cholangiocytes. Therefore, we explored whether directing hPSC into naı̈ve
hepatoblasts would develop tissue-engineered liver (TELi) after in
vivo implantation.
METHODS: H9 hESC or WTC hiPSC underwent directed differentiation to day 11 hepatoblasts, as previously published. Hepatoblasts were seeded onto a PGA/PLA biodegradable scaffold and
implanted into the subcutaneous space or omentum of immunocompromised mice and harvested after 1 month. Samples were
analyzed by hematoxylin and eosin (H&E) and immunofluorescence staining of HNF4a, albumin, PROM1, EpCAM, Desmin,
and Ki67.
RESULTS: Directed differentiation of hPSC into immature
hepatoblasts implanted into the omentum or subcutaneous space
generated hPSC-TELi after 1 month of in vivo growth. Clusters
of hepatocytes were identified by H&E staining and demonstrated extensive proliferation by nuclear Ki67 immunostaining.
TELi derived from hPSC contained numerous albumin-expressing hepatocytes with nuclear staining of HNF4a, a known
regulator of lipid and glucose metabolism, differentiation, and
morphogenesis. PROM1- and EpCAM-positive human hepatic
stem/progenitor cells were identified within developing hPSCTELi. Additionally, hPSC-TELi repopulated Desmin-positive
perisinusoidal cells.
CONCLUSIONS: TELi derived from directed differentiation of
hESC or hiPSC is a feasible approach for regenerating hepatic
J Am Coll Surg
tissue. hPSC-TELi contained numerous hepatic cell populations,
including hepatocytes, stellate cells, and stem/progenitor cells.
Further exploration of functionality and methods to promote
maturation of hPSC-TELI could provide a useful strategy for the
treatment of patients with liver failure.
Effects of Polyethylene Glycol 20,000 on the
Management of Brain Dead Organ Donors in a
Canine Model
Niluka Wickramaratne, MD, Loren Liebrecht, MD,
Heather Reichstetter, Ria Fyffe-Freil, Charles Blocher,
Michel B Aboutanos, MD, MPH, FACS, Martin Mangino, PhD
Virginia Commonwealth University, Richmond, VA
INTRODUCTION: The purpose of this study was to compare the
efficacy of polyethylene glycol 20,000 kDa (PEG-20k), a novel
intravenous fluid with hybrid cell-impermeant and oncotic properties, to crystalloid for hemodynamic management of brain dead organ donors in a pre-clinical canine model, and to evaluate
subsequent renal graft function after cold storage using an isolated
perfusion (IP) model of transplantation.
METHODS: Anesthetized Beagle dogs underwent brain death.
Mean arterial pressure was maintained using epinephrine, vasopressin, and physiologic crystalloid infusion (n ¼ 4) or PEG-20k
(n ¼ 4). After 16 hours, 1 kidney was recovered, flushed (UW),
cold stored for 24 hours, and reperfused on an ex-vivo IP device
for 60 minutes. The other kidney was immediately reperfused on
the IP device. Outcomes for donor management were hemodynamic variables, total IV fluid and medications, and urine output.
Groups were compared by ANOVA.
RESULTS: The total IV fluid administered, urine output, and total epinephrine were decreased in the PEG-20k group vs control.
There were no differences in blood pressure, heart rate, total vasopressin, or lactate. During IP, the cold-stored PEG-20k kidneys
produced less lactate dehydrogenase (LDH) and produced a
glomerular filtration rate (GFR) closer to that of fresh kidneys;
the control kidneys had a decreased GFR.
CONCLUSIONS: The reduction in IV fluid and urine output suggest that PEG-20k is able to nonpharmacologically prevent diabetes insipidus while minimizing electrolyte shifts and pressor
requirements. Kidneys from donors managed with PEG-20k
released less LDH during IP studies, suggesting less cell injury.
The GFR data indicate that using PEG-20k during donor management may attenuate preservation injury.
Epigenetic Analysis of Scar Forming
Fibroblasts Reveals Key Differences in Genes
Associated with Fibrosis
Alessandra L Moore, MD, Clement D Marshall, MD,
Ulrike Litzenburger, PhD, Leandra Barnes, Ryan Chase Ransom,
Michael Hu, MD, Tripp Leavitt, Howard Y Chang,
Michael T Longaker, MD, MBA, FACS
Stanford University, Stanford, CA
Vol. 225, No. 4S1, October 2017
INTRODUCTION: Skin scarring, though present in the adult, does
not occur in fetal wounds. Rather, fetal wounds heal by regeneration.
Understanding the fetal wound healing mechanism could allow for
scarless healing in human patients. Recently, our group showed that
a group of fibroblasts in murine dorsal skin are responsible for all scar
tissue deposition in adults, and they appear around the time of
phenotypic change from scarless to scarring. These cells, known as
engrailed positive fibroblasts (EPFs) are hypothesized to be responsible for the transition from scarless to scar-forming phenotype in the
late gestational fetus, via numerous epigenetic changes.
METHODS: Dorsal dermal fibroblasts from En1Cre, R26mTmG
mice were isolated at gestational ages E10, E16, E18, P1, and P30.
EPFs and ENFs (engrailed negative fibroblasts) from these time
points were sorted using fluorescence-activated cell sorting
(FACS) and were analyzed using assay for transposase-accessible
chromatin using sequencing (ATAC-seq). This assay compared
the accessibility of segments of genomic DNA.
RESULTS: Preliminary ATAC-seq data reveals a strong association between epigenetic changes and time course between scar
forming and other fibroblasts. Additionally, epigenetic changes
are highly preserved among cells of the same time course. Also,
ubiquitously expressed fibroblast genes have marked variation in
genomic accessibility.
CONCLUSIONS: ATAC-seq of EPFs and other fibroblasts proves
they are functionally distinct, not only physiologically, but in their
epigenetic regulation. In future experiments, we aim to achieve
scarless skin healing using CRISPR-Cas9 editing to target genes
identified using ATAC-seq.
Evaluation of a System for Normothermic and
Subnormothermic Machine Perfusion of the
Rat Liver
Nathanael Raschzok, MD, Joseph Gassner, Maximilian Noesser,
Lara Wegener, Simon Moosburner, Ruza Arsenic,
Johann Pratschke, MD, PhD, FACS, Igor M Sauer, MD
Charite-Universitaetsmedizin Berlin, Berlin, Germany
INTRODUCTION: Poor patient outcomes of extended criteria
donor liver grafts after static cold storage call for new methods of
ex vivo organ preservation. In recent years, ex vivo machine perfusion at different temperatures, ranging from hypothermic (4 C) to
normothermic (37 C), re-emerged as possible alternative to static
cold storage. Our aim was to establish and evaluate an ex vivo
perfusion system for rat livers that mimics the clinical condition
of machine perfusion.
METHODS: We investigated perfusion conditions at both subnormothermic (21 C) and normothermic (37 C) temperatures, with
varying setups. The perfusion system consists of a pressurecontrolled roller pump, an oxygenator, a custom-made perfusion
chamber, and a dialysis membrane for plasma expansion. Male
Wistar rat livers were perfused via the portal vein for up to 12 hours
using solely oxygenated culture medium, or oxygenated medium
supplemented with rat erythrocytes with and without dialysis.
Scientific Forum Abstracts
RESULTS: Perfusion without erythrocytes led to excessive transaminase secretion and unphysiological system pressure beyond 6
hours of perfusion. The addition of erythrocytes led to significantly
lower transaminase secretion, but to potassium excess in the
perfusate. Inclusion of the dialysis system resulted in a physiological
perfusion pressure and normal potassium levels. Subnormothermic
perfusion led to lower transaminase levels compared to normothermic perfusion, but also to significantly lower urea and bile
CONCLUSIONS: As observed in normothermic human liver machine perfusion, plasma expansion was the key for maintaining
physiological perfusion conditions during perfusion. Our system
might be suitable for small animal studies of machine perfusion
of the liver at both temperatures.
Ex Vivo Hyperbaric Normothermic Perfusion
for up to 14 Hours Mitigates Reperfusion
Injury in Porcine Vascular Composite
Kevin Y Wu, MD, Sharon D Lawson, MD, Lin C Wang, MD,
Samuel Tahk, MD, Nicholas L Robbins, DO,
Matthew Wordsworth, BM BCh, Bijaya Parida, PhD,
George E Wolf, MD, Michael R Davis, MD, FACS
59th Medical Dental Wing, Science and Technology, San
Antonio, TX
INTRODUCTION: Modern body armor, rapid evacuation, and
advanced combat casualty care have improved survival after catastrophic extremity and maxillofacial trauma. Vascularized composite allotransplantation (VCA) is a superior restorative option
compared to traditional reconstruction in these complex injuries.
To mitigate obligate reperfusion injury in VCA, we evaluated the
efficacy of a novel normothermic hyperbaric oxygen warm ex
vivo perfusion strategy in a porcine VCA model.
METHODS: Semitendinosus myocutaneous flap autotransplants
were performed heterotopically in the cervical area of Yorkshire
swine. Group 1 (controls, n ¼ 5) flaps were perfused with cold
static preservation solution (SPS) at 4 C for 3 hours prior to transplant. Group 2 (experimental, n ¼ 8) flaps were perfused with
hyper-oxygenated kidney perfusion solution (KPS) for 7 hours at
37 C in a hyperbaric chamber at 3 atm before transplantation.
Flaps were monitored daily for clinical viability and biopsied per
protocol, with an endpoint of 21 days.
RESULTS: For group 1 control flaps at cold static preservation,
clinical and histological evaluation revealed extensive diffuse evidence of necrosis by the endpoint. Group 2 autotransplanted flaps
remained viable by the endpoint and showed decreased histologic
evidence of ischemic injury or necrosis. Preliminary data are
currently being collected for our 14-hour window.
CONCLUSIONS: Hyperbaric normothermic perfusion dramatically extends the viability of composite tissues ex vivo. Injuries secondary to ischemia and cold preservation can potentially be
mitigated in 14 hours of treatment. This technology has the
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