Nripen Sharma

Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation

BACKGROUND Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C. METHODS Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls. RESULTS Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values. CONCLUSIONS MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.

Recovery of Warm Ischemic Rat Liver Grafts by Normothermic Extracorporeal Perfusion

Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.

Impact of co-culture on pancreatic differentiation of embryonic stem cells.

Promise of cellular therapy for type 1 diabetes has inspired the search for transplantable cell sources, and embryonic stem cells (ESCs) have emerged as strong candidates. We have developed a directed differentiation protocol to obtain insulin‐producing cells from ESCs. The ESCs are first induced towards a homogeneous monolayer of definitive endoderm‐like cells by co‐culture with primary hepatocytes. Pancreatic commitment is induced by plating the ESC‐derived endoderms on Matrigel, along with Sonic hedgehog inhibition and retinoid induction. More than 70% of differentiated cells positively upregulated Pdx‐1, along with pro‐endocrine transcription factors Ngn3, β2/neroD1, Nkx2.2 and Nkx6.1. Final maturation to islet‐specific cells is achieved by co‐culturing the ESC‐derived pancreatic endocrine cells with endothelial cells, which resulted in Insulin 1 upregulation in 60% of the cell population, along with high levels of IAPP and Glut2. The differentiated cell population also secreted high levels of insulin. Our findings illustrate the significant effect of co‐culture in different stages of differentiation and maturation of ESCs in vitro. Such a high yield of pancreatic islet cells has not yet been reported. Our findings establish a robust protocol for islet differentiation. Copyright

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post‐burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady‐state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham‐burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic‐like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose‐dependent metabolic response, but rather leads to qualitatively different responses. Biotechnol. Bioeng. 2011; 108:839–852.

Adipocyte-derived basement membrane extract with biological activity: applications in hepatocyte functional augmentation in vitro

Natural and synthetic biomaterials utilized in tissue engineering applications require a dynamic interplay of complex macromolecular compositions of hydrated extracellular matrices (ECMs) and soluble growth factors. The challenges in utilizing synthetic ECMs is the effective control of temporal and spatial complexity of multiple signal presentation, as compared to natural ECMs that possess the inherent properties of biological recognition, including presentation of receptor‐binding ligands, susceptibility to cell‐triggered proteolytic degradation, and remodeling. We have developed a murine preadipocyte differentiation system for generating a natural basement membrane extract (Adipogel) comprising ECM proteins (collagen IV, laminin, hyaluronan, and fibronectin) and including relevant growth factors (hepatocyte growth factor, vascular endothelial growth factor, and leukemia inhibitory factor). We have shown the effective utilization of the growth factor‐enriched extracellular matrix for enhanced albumin synthesis rate of primary hepatocyte cultures for a period of 10 d as compared to collagen sandwich cultures and comparable or higher function as compared to Matrigel cultures. We have also demonstrated comparable cytochrome P450 1A1 activity for the collagen‐Adipogel condition to the collagen doublegel and Matrigel culture conditions. A metabolic analysis revealed that utilization of Adipogel in primary hepatocyte cultures increased serine, glycine, threonine, alanine, tyrosine, valine, methionine, lysine, isoleucine, leucine, phenylalanine, taurine, cysteine, and glucose uptake rates to enhance hepatocyte protein synthesis as compared to collagen double‐gel cultures. The demonstrated synthesis, isolation, characterization, and application of Adipogel provide immense potential for tissue engineering and regenerative medicine applications.—Sharma, N. S., Nagrath, D., Yarmush, M. L. Adipocyte‐derived basement membrane extract with biological activity: applications in hepatocyte functional augmentation in vitro. FASEB J. 24, 2364–2374 (2010). www.fasebj.org

Enrichment of hepatocyte-like cells with upregulated metabolic and differentiated function derived from embryonic stem cells using S-NitrosoAcetylPenicillamine.

The generation of a large number of fully functional hepatocytes from a renewable cell source can provide an unlimited resource for bioartificial liver devices and cell replacement therapies. We have established a directed differentiation system using sodium butyrate treatment to generate an enriched population of hepatocyte-like cells from embryonic stem cells. A metabolic analysis of the hepatocyte populations revealed glycolytic and mitochondrial phenotypes similar to mouse hepatoma cells, implying that these cells represent an immature hepatocyte phenotype. To mediate further differentiation, S-NitrosoAcetylPenicillamine (SNAP), a nitric oxide donor, was utilized to induce mitochondrial development in the precursor populations. A comparative analysis of the different treated populations showed that 500microM SNAP treatment resulted in the generation of an enriched population of metabolically mature hepatocyte-like cells with increased differentiated function. Specifically, 500microM SNAP treatment increased glucose consumption, lactate production rates, mitochondrial mass, and potential as compared to untreated populations. In addition, functional analysis revealed that intracellular albumin content, urea secretion rates, and cytochrome P450 7a1 promoter activity were increased in the treated population. The methodology described here to generate an enriched population of metabolically and functionally mature hepatocyte-like cells may have potential implications in drug discovery and regenerative medicine.

Diluted Blood Reperfusion as a Model for Transplantation of Ischemic Rat Livers: Alanine Aminotransferase Is a Direct Indicator of Viability

Donors after cardiac death present a significant pool of untapped organs for transplantation, and use of machine perfusion strategies has been an active focus area in experimental transplantation. However, despite 2 decades of research, a gold standard has yet to emerge for machine perfusion systems and protocols. Whole blood reperfusion has been used as a surrogate for organ transplantation, especially as a model for the short-term response posttransplantation, and for optimization of perfusion systems. Although it is known that there is a strong correlation between liver function in whole-blood reperfusion and survival, the exact nature of these correlations, and to what extent they can be considered as an indicator of viability for transplantation/recipient survival, remain unclear. In this work, we demonstrate that diluted whole-blood reperfusion can be used as a direct model for transplantation of ischemic rat liver grafts. Specifically, we show that recipient survival can be predicted based simply on the value of alanine aminotransferase during perfusion, providing quantitative criteria of viability for use in this animal model. These results indicate that in the rat model graft survival is highly correlated with hepatocellular damage.

Metabolic Profiling Based Quantitative Evaluation of Hepatocellular Metabolism in Presence of Adipocyte Derived Extracellular Matrix

The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.

Augmentation of EB-Directed Hepatocyte-Specific Function via Collagen Sandwich and SNAP

The development of implantable engineered liver tissue constructs and ex vivo hepatocyte‐based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)‐mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin‐18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin‐M (OSM) or S‐nitroso‐N‐acetylpenicillamine (SNAP) supplementation of EB‐derived hepatocyte‐lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long‐term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver‐specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte‐like function. Maintenance and augmentation of hepatocyte‐like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.

Microdevice integrating innate and adaptive immune responses associated with antigen presentation by dendritic cells

Dendritic cells are the principal antigen presenting cells that are responsible for acquiring and transporting antigen from the peripheral tissue to the secondary lymphoid tissue. There they present it to T cells which ultimately initiate an antigen specific immune response. In vivo, the migration of dendritic cells (DCs) and T cell activation are intimately linked. However, ex vivo systems that facilitate integrated evaluation of DC chemotaxis and resulting T cell activation by migrated DCs are lacking. In this work, we have developed a microfabricated platform that integrates DC chemotaxis with T cell activation. The basic design of the microdevice includes two layers of PDMS, with the top layer comprising the chemotaxis compartment and the bottom layer containing a T cell compartment. In the chemotaxis compartment, the DCs are subjected to a chemokine gradient, and their migratory response is evaluated. In the T cell compartment, rapid DC-induced activation of T cells is evaluated by measuring the level of calcium in T cells. We demonstrate the efficacy of our approach by evaluating the integrated response of mature DCs, whereby the overall T cell activation response is governed both by the chemotaxis and the T cell activation potential of mature DCs relative to immature DCs. Our system provides a powerful platform for systematically probing various aspects of antigen induced immune responses - DC maturation, migration and T cell activation - in an integrated fashion.