Christina Philippeos

Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure.

Background and Aim Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF) providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use. Methods Human hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality. Results The optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×106 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function. Conclusion An optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.

Expression of a chimeric antigen receptor specific for donor HLA class I enhances the potency of human regulatory T cells in preventing human skin transplant rejection

Regulatory T cell (Treg) therapy using recipient‐derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor‐MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA‐A2‐specific CARs were engineered: one comprising a CD28‐CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA‐A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA‐A2‐expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune‐mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.

Introduction to Cell Culture

The basics of cell culture as applied to human cells are discussed. Biosafety when working with human tissue, which is often pathogenic, is important. The requirements for a tissue culture laboratory are described, particularly the range of equipment needed to carry out cell isolation, purification, and culture. Steps must be taken to maintain aseptic conditions to prevent contamination of cultures with micro-organisms. Basic cell-handling techniques are discussed, including choice of media, primary culture, and cryopreservation of cells so they can be stored for future use. Common assays which are used to determine cell viability and activity are considered.

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.

Use of indocyanine green for functional assessment of human hepatocytes for transplantation

BACKGROUND/OBJECTIVE Hepatocyte transplantation is a promising alternative to liver transplantation in children with liver metabolic disorders and acute liver failure. Currently, it is difficult to assess rapidly hepatocyte function before transplantation. The aim of this study was to investigate whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used. METHODS Human hepatocytes (10(6) cells) isolated from unused donor livers were incubated at 37°C for 30 minutes with ICG (0-2mg/mL) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 hours with supernatants collected at 1, 2 and 3 hours for measurement of ICG release. Cell viability was determined by trypan blue exclusion, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (mitochondrial dehydrogenase activity) and sulforhodamine B (SRB) assay (cell attachment). HepG2 cells were also used. RESULTS ICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG > 1.0mg/mL had toxic effects on hepatocytes. Hepatocytes incubated with 1.0mg/mL ICG had higher mitochondrial dehydrogenase activity compared to 0.5mg/mL ICG or control cells (0.025 ± 0.0004 OD unit vs. 0.019 ± 0.0008 OD unit or 0.020 ± 0.002 OD unit, p<0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9 ± 0.02 ng/mL, 66.6 ± 0.05 ng/mL and 39.1 ± 0.4 ng/mL for control cells, and 0.5mg/mL and 1.0mg/mL ICG, respectively), and decreased [(3)H]-thymidine incorporation in a dose-dependent manner. Addition of taurine (20mM) to plated hepatocytes gave greater release of ICG and hepatocyte attachment compared to controls, at all ICG concentrations (SRB 1.360 ± 0.083 optical density units vs. 0.908 ± 0.159 optical density units, p=0.011 at 1.0mg/mL). CONCLUSION With further refinement, ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes.

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.

Fibroblast state switching orchestrates dermal maturation and wound healing

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.