Arno W. Tilles

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 Cell-Derived Molecules Reverse Fulminant Hepatic Failure

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo.

Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D‐galactosamine–induced rat model of acute liver injury, we show that systemic infusion of MSC‐conditioned medium (MSC‐CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC‐CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three‐fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up‐regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC‐CM on hepatocytes were demonstrated using in vitro assays. Conclusion: These data provide the first clear evidence that MSC‐CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF. (HEPATOLOGY 2008.)

Bone Marrow‐Derived Mesenchymal Stem Cells Ameliorate Autoimmune Enteropathy Independently of Regulatory T Cells

Cell‐based tolerogenic therapy is a relatively new approach for the treatment of autoimmune diseases. Mesenchymal stem cells (MSCs) have been shown to be potent immunomodulatory agents in a number of experimental and clinical scenarios; however, their use in various autoimmune diseases is undefined. Herein, we report the efficacy of MSC transplantation in a multiorgan autoimmunity model. Mice with defective peripheral tolerance caused by a deficiency in regulatory T cells were used as a testbed for therapy. After screening multiple target tissues of autoimmune attack, we observed an MSC‐specific improvement in the histopathology of the distal ileum of treated mice. We then showed that MSCs can reduce mesenteric lymph node (MLN) cellularity in autoimmune mice during active disease and decrease activated T‐cell populations in the MLN. Trafficking studies using enhanced green fluorescent protein (eGFP)‐reporter MSCs revealed no appreciable engraftment in the intestine, but it did reveal the presence of eGFP+ cells organized in clusters within the MLN, as well as ancillary nodes. Semiquantitative analysis showed no difference in the number of clusters; however, eGFP+ cells in MLNs compared with ancillary nodes had distinct fibroblastoid morphology and formed a network with neighboring eGFP+ cells. Finally, we show evidence that transplantation of MSCs caused global immunosuppression, as measured by increased CD4+ CD8+ thymocyte production and serum interleukin‐10 and decreased serum interferon‐γ. These data implicate the intestine as a new site of MSC tolerance induction and should motivate additional studies evaluating the use of MSCs as a treatment for autoimmune enteropathies.

Hepatic tissue engineering for adjunct and temporary liver support: Critical technologies

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end‐stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte‐based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the “proof of principle” test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist. (Liver Transpl 2004;10:1331–1342.)

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.

Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure

One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm‐like cells with 95% purity. Mouse ES cells cultured on top of collagen‐sandwiched hepa‐tocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm‐like cells. The endoderm‐like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte‐like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte‐like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.—Cho, C. H., Parashurama, N., Park, E. Y. H., Suganuma, K., Nahmias, Y., Park, J., Tilles, A. W., Berthiaume, F., Yarmush, M. L. Homogeneous differentiation of hep‐atocyte‐like cells from embryonic stem cells: applications for the treatment of liver failure. FASEB J. 22, 898–909 (2008)

Analysis of oxygen transport to hepatocytes in a flat-plate microchannel bioreactor.

AbstractOxygen transfer to cultured hepatocytes in microchannel parallel-plate bioreactors with and without an internal membrane oxygenator was investigated with a mathematical model and the results were corroborated with fluorescence imaging experiments. The consumption of oxygen by hepatocytes was assumed to follow Michaelis–Menten kinetics. Our simulations indicate that under conditions of low Péclet (Pe) number (<80) and fixed Damkohler number (=0.14, corresponding to rat hepatocytes) the cells are hypoxic in the bioreactor without an internal membrane oxygenator. Under the same conditions, the bioreactor with an internal membrane oxygenator can avoid cell hypoxia by appropriate selection of membrane Sherwood number and/or the gas phase oxygen partial pressure, thus providing greater control of cell oxygenation. At high Pe, both bioreactors are well oxygenated. Experimentally determined oxygen concentrations within the bioreactors were in good qualitative agreement with model predictions. At low Pe, cell surface oxygen depletion was predicted from analytically derived criteria. Hepatocytes with oxygen dependent functional heterogeneity may exhibit optimal function in the bioreactor with the internal membrane oxygenator.

Bone Marrow Mesenchymal Stromal Cells Attenuate Organ Injury Induced by LPS and Burn

Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.

Layered patterning of hepatocytes in co-culture systems using microfabricated stencils

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 microm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.