Yongjie Zhou

Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts

Whole‐organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole‐organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human‐sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale.

Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization.

Whole-liver perfusion-decellularization is an attractive scaffold–preparation technique for producing clinical transplantable liver tissue. However, the scaffold’s poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin. Heparin was immobilized on decellularized liver scaffolds (DLSs) by electrostatic binding using a layer-by-layer self-assembly technique (/h-LBL scaffold), covalent binding via multi-point attachment (/h-MPA scaffold), or end-point attachment (/h-EPA scaffold). The effect of heparinization on anticoagulant ability and cytocompatibility were investigated. The result of heparin content and release tests revealed EPA technique performed higher efficiency of heparin immobilization than other two methods. Then, systematic in vitro investigation of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), platelet adhesion and human platelet factor 4 (PF4, indicates platelet activation) confirmed the heparinized scaffolds, especially the /h-EPA counterparts, exhibited ultralow blood component activations and excellent hemocompatibility. Furthermore, heparin treatments prevented thrombosis successfully in DLSs with blood perfusion after implanted in vivo. Meanwhile, after heparin processes, both primary hepatocyte and endothelial cell viability were also well-maintained, which indicated that heparin treatments with improved biocompatibility might extend to various hemoperfusable whole-organ scaffolds’ preparation.

Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

Aim. To refine the decellularization protocol of whole porcine liver, which holds great promise for liver tissue engineering. Methods. Three decellularization methods for porcine livers (1% sodium dodecyl sulfate (SDS), 1% Triton X-100 + 1% sodium dodecyl sulfate, and 1% sodium deoxycholate + 1% sodium dodecyl sulfate) were studied. The obtained liver scaffolds were processed for histology, residual cellular content analysis, and extracellular matrix (ECM) components evaluation to investigate decellularization efficiency and ECM preservation. Rat primary hepatocytes were seeded into three kinds of scaffold to detect the biocompatibility. Results. The whole liver decellularization was successfully achieved following all three kinds of treatment. SDS combined with Triton had a high efficacy of cellular removal and caused minimal disruption of essential ECM components; it was also the most biocompatible procedure for primary hepatocytes. Conclusion. We have refined a novel, standardized, time-efficient, and reproducible protocol for the decellularization of whole liver which can be further adapted to liver tissue engineering.

Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host’s immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.

Loss of histone deacetylases 1 and 2 in hepatocytes impairs murine liver regeneration through Ki67 depletion

Histone deacetylases 1 and 2 (HDAC1 and HDAC2) are ubiquitously expressed in tissues, including the liver, and play critical roles in numerous physiopathological processes. Little is known regarding the role of HDAC1 and HDAC2 in liver regeneration. In this study we generated mice in which Hdac1, Hdac2 or both genes were selectively knocked out in hepatocytes to investigate the role of these genes in liver regeneration following hepatic injury induced by partial hepatectomy or carbon tetrachloride administration. The loss of HDAC1 and/or HDAC2 (HDAC1/2) protein resulted in impaired liver regeneration. HDAC1/2 inactivation did not decrease hepatocytic 5‐bromo‐2‐deoxyuridine uptake or the expression of proliferating cell nuclear antigen, cyclins, or cyclin‐dependent kinases. However, the levels of Ki67, a mitotic marker that is expressed from the mid‐G1 phase to the end of mitosis and is closely involved in the regulation of mitotic progression, were greatly decreased, and abnormal mitosis lacking Ki67 expression was frequently observed in HDAC1/2‐deficient livers. The down‐regulation of either HDAC1/2 or Ki67 in the mouse liver cancer cell line Hepa1‐6 resulted in similar mitotic defects. Finally, both HDAC1 and HDAC2 proteins were associated with the Ki67 gene mediated by CCAAT/enhancer‐binding protein β. Conclusion: Both HDAC1 and HDAC2 play crucial roles in the regulation of liver regeneration. The loss of HDAC1/2 inhibits Ki67 expression and results in defective hepatocyte mitosis and impaired liver regeneration. (Hepatology 2013; 58:2089–2098)

Octreotide prevents liver failure through upregulating 5'-methylthioadenosine in extended hepatectomized rats.

Insufficient liver regeneration and hepatocyte injury caused by excessive portal perfusion are considered to be responsible for post‐hepatectomy liver failure (PLF) or small‐for‐size syndrome in living‐donor liver transplantation. Somatostatin can decrease portal vein pressure (PVP) but simultaneously inhibits liver regeneration. This interesting paradox motivated us to investigate the outcome of PLF in response to somatostatin treatment.

MiR-17~92 ablation impairs liver regeneration in an estrogen-dependent manner.

As one of the most important post‐transcriptional regulators, microRNAs (miRNAs) participate in diverse biological processes, including the regulation of cell proliferation. MiR‐17~92 has been found to act as an oncogene, and it is closely associated with cell proliferation. However, its role in liver regeneration is still unclear. We generated a hepatocyte‐specific miR‐17~92‐deficient mouse and used a mouse model with 70% partial hepatectomy (PH) or intraperitoneal injection of carbon tetrachloride to demonstrate the role of MiR‐17~92 in liver regeneration. In quiescent livers, the expression of the miR‐17~92 cluster showed a gender disparity, with much higher expression in female mice. The expression of four members of this cluster was found to be markedly reduced after 70% PH. The ablation of miR‐17~92 led to obvious regeneration impairment during the early‐stage regeneration in the female mice. Ovariectomy greatly reduced miR‐17~92 expression but significantly promoted liver regeneration in wild‐type mice. In addition, early regeneration impairment in miR‐17~92‐deficient livers could be largely restored following ovariectomy. The proliferation suppressors p21 and Pten were found to be the target effectors of miR‐17~92. MiR‐17~92 disruption resulted in elevated protein levels of p21 and Pten in regenerating livers. MiR‐17~92 functions as a proliferation stimulator and acts in an oestrogen‐dependent manner. The loss of this miRNA results in increases in p21 and Pten expression and therefore impairs liver regeneration in female mice.

Loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation

AIM To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1. METHODS We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver. Immunohistochemistry and Western blotting were performed to detect Dicer1 expression. We performed hematoxylin and eosin staining, Periodic acid-Schiff staining, Oil Red O staining, and Massons trichrome staining to detect histological changes in Dicer1-deficient livers. Ki67 immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and Western blotting were used to determine hepatocyte proliferation and apoptosis. Serum biochemistry, cytokine assays, and flow cytometric analysis were performed to quantity liver necrosis and inflammation. Fibrogenic markers were determined by Western blotting and qPCR. CK19, CD133, and OV6 immunofluorescence were used to observe liver progenitor cells. Immunofluorescence and qPCR were performed to reveal embryonic gene expression. We also performed histological staining and Western blotting to analyze hepatocellular carcinoma (HCC) development. RESULTS Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruption in the liver. Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis. In contrast to previous reports, the mutant liver exhibited chronic inflammation and progressive fibrosis, and could not be repopulated by Dicer1-positive cells. In addition, extensive activation of hepatic progenitor cells was observed. Primary HCC was observed as early as 4 mo after birth. CONCLUSION Hepatic loss of Dicer1 results in complex chronic pathological processes, including hepatocyte death, inflammatory infiltration, chronic fibrosis, compensatory proliferation, progenitor activation, and spontaneous hepatocarcinogenesis.

Loss of Gsα impairs liver regeneration through a defect in the crosstalk between cAMP and growth factor signaling.

BACKGROUND & AIMS The stimulatory G protein α subunit (Gsα) activates the cAMP-dependent pathway by stimulating the production of cAMP and participates in diverse cell processes. Aberrant expression of Gsα results in various pathophysiological disorders, including tumorigenesis, but little is known about its role in liver regeneration. METHODS We generated a hepatocyte-specific Gsα gene knockout mouse to demonstrate the essential role of Gsα in liver regeneration using a mouse model with 70% partial hepatectomy (PH) or an intraperitoneal injection of carbon tetrachloride (CCl4). RESULTS Gsα inactivation dramatically impaired liver regeneration and blocked proliferating hepatocytes in G1/S transition due to the simultaneous depression of cyclin-dependent kinase 2 (CDK2) and cyclin E1. Loss of Gsα led to a fundamental alteration in gene profiles. Among the altered signaling cascades, the MAPK/Erk pathway, which is downstream of growth factor signaling, was disrupted secondary to a defect in phosphorylated Raf1 (pRaf1), resulting in a deficiency in phosphorylated CREB (pCREB) and CDK2 ablation. The lack of pRaf1 also resulted in a failure to phosphorylate retinoblastoma, which releases and activates E2F1, and a decrease in cyclin E1. Although these factors could be phosphorylated through both Gsα and growth factor signaling, the unique function of Raf1 in the growth factor cascade collapsed in response to the lack of Gsα. CONCLUSION The growth factor signaling pathway that promotes hepatocyte proliferation is dependent on Gsα signaling. Loss of Gsα leads to a breakdown of the crosstalk between cAMP and growth factor signaling and dramatically impairs liver regeneration.

Proportions of acetyl-histone-positive hepatocytes indicate the functional status and prognosis of cirrhotic patients

AIM To investigate whether the proportions of acetyl-histone-positive hepatocytes could be used as markers of deteriorating liver function. METHODS In total, 611 cirrhotic cases from 3701 patients who were diagnosed during the past 15 years were screened, and 152 follow-up cases were selected. Paraffin tissue microarray was prepared for immunohistochemistry to examine acetyl-histone expression. The proportions of positive hepatocytes were recorded, and their correlations to clinical and laboratory indicators were analyzed statistically. RESULTS The proportions of H2AK5ac(+), H3K9/K14ac(+) and H3K27ac(+) hepatocytes gradually increased with deteriorating liver function and with increasing levels of serum markers of liver injury. In the follow-up cases, patients with > 70% H2AK5ac(+), H3K9/K14ac(+) or H3K27ac(+) hepatocytes had statistically lower survival rates (P < 0.05). Furthermore, > 70% H2AK5ac(+) or H3K27ac(+) hepatocytes were strong independent predictors of overall survival (P < 0.05). CONCLUSION The proportions of acetyl-histone-positive hepatocytes are closely associated with the liver function and prognosis of cirrhotic patients.