Alejandro Soto-Gutierrez

Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix

Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.

Mesenchymal Stem Cells: Mechanisms of Immunomodulation and Homing

Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissue. A growing corpus of studies have highlighted two important aspects of MSC therapy: 1) MSCs can modulate T-cell-mediated immunological responses, and (2) systemically administered MSCs home to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. First, we examine the low immunogenicity of MSCs and their antigen presentation capabilities. Next, we discuss the paracrine interactions between MSCs and innate [dendritic cells (DC)] and adaptive immune cells (T lymphocytes) with a focus on prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and toll-like receptor (TLR) signaling pathways. We transition to outline the steps of activation, rolling/adhesion, and transmigration of MSCs into target tissues during inflammatory or ischemic conditions. These aspects of MSC grafts—immunomodulation and homing—are contextualized to understand a reported side effect of MSC therapy, cancer development.

Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes

BACKGROUND & AIMS The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. METHODS To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. RESULTS Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. CONCLUSIONS Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

A Whole-Organ Regenerative Medicine Approach for Liver Replacement

BACKGROUND & AIMS The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue. METHODS A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion. RESULTS The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction. CONCLUSIONS Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter–based cell sorting. ES cell–derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell–derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell–derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

Human-Scale Whole-Organ Bioengineering for Liver Transplantation: A Regenerative Medicine Approach

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

Barriers to the successful treatment of liver disease by hepatocyte transplantation

Management of patients with hepatic failure and liver-based metabolic disorders is complex and expensive. Hepatic failure results in impaired coagulation, altered consciousness and cerebral function, a heightened risk of multiple organ system failure, and sepsis [1]. Such manifold problems are only treatable today and for the foreseeable future by transplantation. In fact, whole or auxiliary partial liver transplantation is often the only available treatment option for severe, even if transient, hepatic failure. Patients with life-threatening liver-based metabolic disorders similarly require organ transplantation even though their metabolic diseases are typically the result of a single enzyme deficiency, and the liver otherwise functions normally. For all of the benefits it may confer, liver transplantation is not an ideal therapy, even for severe hepatic failure. More than 17,000 patients currently await liver transplantation in the United States, a number that seriously underestimates the number of patients that need treatment [2], as it has been estimated that more than a million patients could benefit from transplantation [3]. Unfortunately, use of whole liver transplantation to treat these disorders is limited by a severe shortage of donors and by the risks to the recipient associated with major surgery [4].

Reactive bone marrow stromal cells attenuate systemic inflammation via sTNFR1

Excessive systemic inflammation following trauma, sepsis, or burn could lead to distant organ damage. The transplantation of bone marrow stromal cells or mesenchymal stem cells (MSCs) has been reported to be an effective treatment for several immune disorders by modulating the inflammatory response to injury. We hypothesized that MSCs can dynamically secrete systemic factors that can neutralize the activity of inflammatory cytokines. In this study, we showed that cocultured MSCs are able to decrease nuclear factor κ-B (NFκB) activation in target epithelial cells incubated in inflammatory serum conditions. Proteomic screening revealed a responsive secretion of soluble tumor necrosis factor (TNF) receptor 1 (sTNFR1) when MSCs were exposed to lipopolysaccharide (LPS)-stimulated rat serum. The responsive effect was eliminated when NFκB activation was blocked in MSCs. Intramuscular transplantation of MSCs in LPS-endotoxic rats decreased a panel of inflammatory cytokines and inflammatory infiltration of macrophages and neutrophils in lung, kidney, and liver when compared to controls. These results suggest that improvements of inflammatory responses in animal models after local transplantation of MSCs are at least, in part, explained by the NFκB-dependent secretion of sTNFR1 by MSCs.

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.