Jason A. Wertheim

Major Challenges Limiting Liver Transplantation in the United States

Liver transplantation is the gold standard of care in patients with end‐stage liver disease and those with tumors of hepatic origin in the setting of liver dysfunction. From 1988 to 2009, liver transplantation in the United States grew 3.7‐fold from 1713 to 6320 transplants annually. The expansion of liver transplantation is chiefly driven by scientific breakthroughs that have extended patient and graft survival well beyond those expected 50 years ago. The success of liver transplantation is now its primary obstacle, as the pool of donor livers fails to keep pace with the growing number of patients added to the national liver transplant waiting list. This review focuses on three major challenges facing liver transplantation in the United States and discusses new areas of investigation that address each issue: (1) the need for an expanded number of useable donor organs, (2) the need for improved therapies to treat recurrent hepatitis C after transplantation and (3) the need for improved detection, risk stratification based upon tumor biology and molecular inhibitors to combat hepatocellular carcinoma.

Optimization and Critical Evaluation of Decellularization Strategies to Develop Renal Extracellular Matrix Scaffolds as Biological Templates for Organ Engineering and Transplantation

The ability to generate patient‐specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three‐dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long‐range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention—indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X‐100, 1% Triton X‐100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin‐0.05% EGTA/1% Triton X‐100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix‐bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS‐decellularized scaffolds maintained 3 h of leak‐free blood flow in a rodent transplantation model and supported repopulation with human iPSC‐derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS‐based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering.

Perspectives on whole-organ assembly: moving toward transplantation on demand

There is an ever-growing demand for transplantable organs to replace acute and chronically damaged tissues. This demand cannot be met by the currently available donor organs. Efforts to provide an alternative source have led to the development of organ engineering, a discipline that combines cell biology, tissue engineering, and cell/organ transplantation. Over the last several years, engineered organs have been implanted into rodent recipients and have shown modest function. In this article, we summarize the most recent advances in this field and provide a perspective on the challenges of translating this promising new technology into a proven regenerative therapy.

Bcr-abl-positive cells secrete angiogenic factors including matrix metalloproteinases and stimulate angiogenesis in vivo in Matrigel implants

To further elucidate the role of angiogenesis in the pathogenesis of chronic myelogenous leukemia (CML) we evaluated the effects of the bcr-abl translocation on the secretion of the angiogenic factors VEGF, FGF-2, HGF, IL-8 and matrix metalloproteinases (MMPs) as well as on the angiogenic potential in vivo of bcr-abl+ cells. First, we examined murine FL5.12 cells transfected with the bcr-abl constructs p185, p210 and p230 and found that the transfected cells secreted as much as four-fold more VEGF (p185 > p210 >p230) than wild-type (wt) cells, as well as MMP-9 and MMP-2. When Matrigel fragments containing these bcr-abl+ cells were implanted subcutaneously in SCID or Balb-C mice they became significantly more vascularized and hemoglobinized than implants containing normal or wt cells (p185 > p210 > p230). Similarly, we found that myeloblasts expanded from bone marrow (BM) CD34+ cells derived from Philadelphia-positive CML patients secreted up to 10 times more VEGF, FGF-2, HGF and IL-8 compared to myeloblasts derived from normal donors’ BM CD34+ cells and that BM mononuclear cells (MNC) isolated from CML patients induced vascularization of Matrigel implants in mice. Moreover, we found that peripheral blood MNC expressed MMP-2 and membrane-type (MT)1-MMP in about 50% of CML patients studied, and MMP-9 in all of them. Furthermore, VEGF stimulated the secretion of MMP-9 in these primary CML cells. We conclude that stimulation of angiogenesis by angiogenic factors, including MMPs, could play an important role in the pathogenesis of CML, suggesting that therapies targeting the newly formed endothelium could be developed for CML.

Challenging Regeneration to Transform Medicine

The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public‐private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White Houses Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of “universal donor tissues” should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies.

The promise of organ and tissue preservation to transform medicine

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

Assessment of hepatic steatosis by transplant surgeon and expert pathologist: A prospective, double‐blind evaluation of 201 donor livers

An accurate clinical assessment of hepatic steatosis before transplantation is critical for successful outcomes after liver transplantation, especially if a pathologist is not available at the time of procurement. This prospective study investigated the surgeons accuracy in predicting hepatic steatosis and organ quality in 201 adult donor livers. A steatosis assessment by a blinded expert pathologist served as the reference gold standard. The surgeons steatosis estimate correlated more strongly with large‐droplet macrovesicular steatosis [ld‐MaS; nonparametric Spearman correlation coefficient (rS) = 0.504] versus small‐droplet macrovesicular steatosis (sd‐MaS; rS = 0.398). True microvesicular steatosis was present in only 2 donors (1%). Liver texture criteria (yellowness, absence of scratch marks, and round edges) were mainly associated with ld‐MaS (variance = 0.619) and were less associated with sd‐MaS (variance = 0.264). The prediction of ≥30% ld‐MaS versus <30% ld‐MaS was excellent when liver texture criteria were used (accuracy = 86.2%), but it was less accurate when the surgeons direct estimation of the steatosis percentage was used (accuracy = 75.5%). The surgeons quality grading correlated with the degree of ld‐MaS and the surgeons steatosis estimate as well as the incidence of poor initial function and primary nonfunction. In conclusion, the precise estimation of steatosis remains challenging even in experienced hands. Liver texture characteristics are more helpful in identifying macrosteatotic organs than the surgeons actual perception of steatosis. These findings are especially important when histological assessment is not available at the donors hospital. Liver Transpl 19:437–449, 2013.

Polymer-drug conjugates: manipulating drug delivery kinetics using model LCST systems

Model studies of the kinetics of hydrolysis of side chain substituted polymers exhibiting a lower critical solution temperature (LCST) were undertaken. Copolymers of N-isopropyl acrylamide (NiPAm) and N-acryloxy succinimide (AS) were prepared by radical polymerization of the monomers or by reaction of isopropylamine with poly(N-acryloxy succinimide). The LCSTs of the copolymers were measured by DSC and shown to depend on both the NiPAm/AS ratio and the copolymer microstructure. The kinetics of hydrolysis of the succinimide side chain were measured at temperatures below, at, and above the LCST of the copolymers. The LCST increased on conversion of the acryloxy succinimide groups to acrylic acid groups during the hydrolysis process. At temperatures below the LCST, the polymers were water soluble throughout the hydrolysis and exhibited first order kinetics. Above the LCST, the polymers were initially insoluble but became soluble as hydrolysis caused a progressive increase in the LCST. The rate of hydrolysis accelerated on dissolution. As a result, the kinetics exhibited either a zero order or an S-shaped profile. In the latter case the initial slow phase was dependent on the difference between the reaction temperature and the initial LCST.

Cellular therapy and bioartificial approaches to liver replacement.

Purpose of reviewThe success of liver transplantation has increased over the past 20 years due to improved immunosuppressive medications, surgical technique and donor-recipient selection. To date, the number of patients waiting for a liver transplant exceeds the number of transplants performed yearly by over a 2 : 1 ratio. Despite efforts to expand the donor pool, mortality of patients waiting for a liver remains high due to the shortage of donor organs. Herein, we discuss options for liver replacement that are currently under development. Recent findingsExtracorporeal bioactive liver perfusion devices were investigated in the late 1990s and preliminarily demonstrated safety but failed to show clinical efficacy. Current research is ongoing, but the focus has shifted to xenotransplantation of whole organs, organ engineering and cell transplantation. These new modalities are limited to small and large animal studies and each present unique advantages and limitations. SummaryDiscovery of new sources of organs or cells to replace a damaged liver may be the only long-term solution to provide definitive therapy to all patients who require transplantation. The past 2 years have seen notable achievements in xenotransplantation, tissue engineering and cell transplantation. Though challenges remain, now identified, they may be readily solved.

Ascites after liver transplantation—A mystery

Ascites after liver transplantation, although uncommon, presents a serious clinical dilemma. The hemodynamic changes that support the development of ascites before liver transplantation are resolved after transplant; therefore, persistent ascites (PA) after liver transplantation is unexpected and poorly characterized. The aim of this study was to define the clinical factors associated with PA after liver transplantation. This was a retrospective case–control analysis of patients who underwent liver transplantation at the University of Pennsylvania. PA occurring for more than 3 months after liver transplantation was confirmed by imaging studies. PA was correlated with multiple recipient and donor variables, including etiology of liver disease, preoperative ascites, prior portosystemic shunt (PS), donor age, and cold ischemic (CI) time. There were 2 groups: group 1, cases with PA transplanted from November 1990 to July 2001, and group 2, consecutive, control subjects who underwent liver transplantation between September 1999 and December 2001. Both groups were followed to censoring, May 2002, or death. Twenty‐five from group 1 had ascites after liver transplantation after a median follow‐up of 2.6 years. In group 1 vs group 2 (n = 106), there was a male predominance 80% vs 61% (P = .10) with similar age 52 years; chronic hepatitis C virus (HCV) was diagnosed in 88% vs 44% (P < .0001); preoperative ascites and ascites refractory to treatment were more prevalent in group 1 (P = .0004 and P =.02, respectively), and CI was higher in group 1, (8.5 hours vs 6.3 hours, P = .002). Eight of the 25 (group 1) had portal hypertension with median portosystemic gradient 16.5 mm Hg (range, 16–24). PS was performed in 7 of 25 cases, which resulted in partial resolution of ascites. The development of PA after liver transplantation is multifactorial; HCV, refractory ascites before liver transplantation, and prolonged CI contribute to PA after liver transplantation. (Liver Transpl 2004;10:654–660.)