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Introduction: The past few years saw an increasing trend in liver disease prevalence [1]. Orthotopic transplantation is the current gold standard treatment. However, its practice is affected by strong complications represented by the paucity of available organs and the necessity of long-term immunosuppressive therapies. Tissue engineering strategies represent suitable alternatives by combining scaffolds matrices and patients’ autologous cells. In this setting, CRISPR/Cas9 genome edited HLA knockout human induced pluripotent stem cells (hiPSCs) represent a promising cell candidate for a universal strategy, conjugating high cell availability and wide tissue-specific differentiation capacity [2]. The present work aims to rebuild a functional liver substitute seeking for the definition of a promising strategy to evade T cell surveillance upon in vivo transplantation.
Methodology: hiPSCs underwent CRISPR/Cas9 genome-editing to prevent expression of HLA class I and II. HLA Class I/II-/- hiPSCs were differentiated according to standard protocols to obtain definitive endoderm (DE), hepatic endoderm (HE) and immature hepatocytes (IH) cells in a 2D monolayer [3]. hiPSCs-IH were transferred to Matrigel® 3D culture in hepatic organoid expansion medium to generate self-replicating hepatic organoids [3]. To obtain decellularized scaffolds, mouse livers were cannulated via portal vein and decellularized via already established detergent-enzymatic treatment [4]. 17 M cells were obtained from enzyme mediated organoid disaggregation and were further seeded in the decellularized scaffold via the portal vein. Repopulated livers were cultured in a custom-designed bioreactor in a dynamic condition provided by peristaltic pump (flowrate=0.5 ml/min). Constructs were cultured for 7 days in hepatic organoid expansion medium and further supplemented with Oncostatin-M and Dexamethasone to induce terminal hepatocyte differentiation for the following 7 days [5]. Histological and immunohistochemistry analysis were performed to study scaffold repopulation and expression of hepatic maturation and biliary polarity markers. qRT-PCR analysis was performed to analyse the expression of mature hepatic markers including cytochrome 3A4,1A2 (CYP3A4, CYP1A2), hepatocyte nuclear factor 4 alpha (HNF4α).
Results: Complete HLA double knockout was confirmed by genome-sequencing analysis. H&E staining showed successful scaffold repopulation supported by the bioreactor. Immunofluorescence analysis revealed the expression of mature hepatocytes markers (HNF4α, Alpha1-anti-trypsin, human albumin) together with biliary polarity markers such as zonula occludens 1 (ZO1) and multi-drug resistance protein 2 (MRP2), proving a nearly complete mature state. HLA class I and II expression were not detected in immunofluorescence staining, supporting sequencing analysis. qRT-PCR results showed enhanced expression of HNF4α, CYP3A4 and CYP1A2 in the bioreactor culture compared to the static in vitro control, highlighting the effect of the scaffold environment and the dynamic culture on hepatic maturation.
Conclusion: Universal therapeutic strategies are mostly unavailable for the majority of end-stage diseases, including liver diseases. The present work demonstrated the development of a universal functional liver graft by combining organotypic acellular scaffolds and universal hepatocytes obtained from HLA class I/II-/- hiPSCs. The decellularized 3D liver microenvironment efficiently supported hiPSCs-derived hepatocytes engraftment and proliferation. This experimental evidence proves that universal hiPSCs represent a valid candidate to be employed in “ready-to-use” tissue engineering and regenerative therapies, with the promise to overcome immune rejection upon graft transplantation.
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