Lisha Mou

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Circulating Organ-Specific MicroRNAs Serve as Biomarkers in Organ-Specific Diseases: Implications for Organ Allo- and Xeno-Transplantation

Different cell types possess different miRNA expression profiles, and cell/tissue/organ-specific miRNAs (or profiles) indicate different diseases. Circulating miRNA is either actively secreted by living cells or passively released during cell death. Circulating cell/tissue/organ-specific miRNA may serve as a non-invasive biomarker for allo- or xeno-transplantation to monitor organ survival and immune rejection. In this review, we summarize the proof of concept that circulating organ-specific miRNAs serve as non-invasive biomarkers for a wide spectrum of clinical organ-specific manifestations such as liver-related disease, heart-related disease, kidney-related disease, and lung-related disease. Furthermore, we summarize how circulating organ-specific miRNAs may have advantages over conventional methods for monitoring immune rejection in organ transplantation. Finally, we discuss the implications and challenges of applying miRNA to monitor organ survival and immune rejection in allo- or xeno-transplantation.

Pig-to-Primate Islet Xenotransplantation: Past, Present, and Future:

Islet allotransplantation results in increasing success in treating type 1 diabetes, but the shortage of deceased human donor pancreata limits progress. Islet xenotransplantation, using pigs as a source of islets, is a promising approach to overcome this limitation. The greatest obstacle is the primate immune/inflammatory response to the porcine (pig) islets, which may take the form of rapid early graft rejection (the instant blood-mediated inflammatory reaction) or T-cell-mediated rejection. These problems are being resolved by the genetic engineering of the source pigs combined with improved immunosuppressive therapy. The results of pig-to-diabetic nonhuman primate islet xenotransplantation are steadily improving, with insulin independence being achieved for periods >1 year. An alternative approach is to isolate islets within a micro- or macroencapsulation device aimed at protecting them from the human recipients immune response. Clinical trials using this approach are currently underway. This review focuses on the major aspects of pig-to-primate islet xenotransplantation and its potential for treatment of type 1 diabetes.

Potential alternative approaches to xenotransplantation

There is an increasing worldwide shortage of organs and cells for transplantation in patients with end-stage organ failure or cellular dysfunction. This shortage could be resolved by the transplantation of organs or cells from pigs into humans. What competing approaches might provide support for the patient with end-stage organ or cell failure? Four main approaches are receiving increasing attention - (i) implantable mechanical devices, although these are currently limited almost entirely to devices aimed at supporting or replacing the heart, (ii) stem cell technology, at present directed mainly to replace absent or failing cells, but which is also fundamental to progress in (iii) tissue engineering and regenerative medicine, in which the ultimate aim is to replace an entire organ. A final novel potential approach is (iv) blastocyst complementation. These potential alternative approaches are briefly reviewed, and comments added on their current status and whether they are now (or will soon become) realistic alternative therapies to xenotransplantation.

Human IL‐6, IL‐17, IL‐1β, and TNF‐α differently regulate the expression of pro‐inflammatory related genes, tissue factor, and swine leukocyte antigen class I in porcine aortic endothelial cells

Pro‐inflammatory cytokines play important pathological effects in various diseases and allotransplantation; however, their pathological role in xenotransplantation remains elusive. In pig‐to‐human cell or organ transplantation, whether porcine cells or organs are activated by human cytokines or not as an important question needs to be investigated.

Angiopoietin‐1 and angiopoietin‐2 protect porcine iliac endothelial cells from human antibody‐mediated complement‐dependent cytotoxicity through phosphatidylinositide 3‐kinase/AKT pathway activation

Cytokines play crucial roles in inflammation, but their role in xenotransplantation remains elusive. We assessed the role of several cytokines using an in vitro model of human antibody‐mediated complement‐dependent cytotoxicity (CDC). Recombinant human angiopoietin‐1 (Ang‐1) protected porcine iliac endothelial cells (PIECs) from human antibody‐mediated CDC. Interestingly, human angiopoietin‐2 (Ang‐2) had a similar protective effect on PIECs. By flow cytometry analysis, the extent of human IgM and IgG binding to PIECs did not decrease when PIECs were exposed to Ang‐1/Ang‐2. The mRNA level of complement regulators (CD46, CD55, CD59) was not upregulated in PIECs treated with Ang‐1/Ang‐2, both of which activated the PI3K/AKT pathway in PIECs. Wortmannin, which inhibits phosphatidylinositide 3‐kinase (PI3K), suppressed Ang‐1/Ang‐2‐induced AKT phosphorylation and consequent Ang‐1/Ang‐2‐mediated protection of PIECs in human antibody‐mediated CDC model. Moreover, dominant negative AKT also suppressed Ang‐1/Ang‐2‐mediated protection of PIECs in this model. In conclusion, our data suggest that human Ang‐1/Ang‐2 induces the protection of PIECs from human antibody‐mediated CDC by activating the PI3K/AKT pathway. Ang‐1/Ang‐2 is likely to protect porcine endothelial cells and may be beneficial in xenotransplantation research.

Porcine IL-6, IL-1β, and TNF-α regulate the expression of pro-inflammatory-related genes and tissue factor in human umbilical vein endothelial cells

Whether porcine cytokines are induced after pig‐to‐primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro‐inflammatory cytokines (IL‐6, IFN‐γ, and IL‐1β) and chemokines (IL‐8, MCP‐1, and CXCL2) were upregulated in the both cell types. TNF‐α was induced 10‐fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using western blotting and real‐time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL‐1β and TNF‐α significantly activated NF‐κB and P38, and STAT3 was activated by porcine IL‐6 in HUVECs. The adhesion genes (E‐selectin, VCAM‐1, and ICAM‐1), inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α), chemokines (MCP‐1 and IL‐8), and the pro‐coagulation gene (tissue factor) were upregulated by porcine IL‐1β and TNF‐α. Porcine IL‐6 increased the expression of ICAM‐1, IL‐6, MCP‐1, and tissue factor, but decreased IL‐8 expression slightly. Surprisingly, porcine IFN‐γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL‐6, IL‐1β, and TNF‐α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.

Circulating miRNA or circulating DNA-Potential biomarkers for organ transplant rejection

Accurate and timely diagnosis of transplant rejection is essential for the long‐term survival of solid‐organ transplant recipients. Biopsy is the “gold standard” for the diagnosis of rejection, but this approach is a time‐consuming, expensive, and invasive process. An efficient, cheap, and noninvasive tool for monitoring of immune rejection is urgently needed. Circulating miRNA and circulating DNA can be easily amplified and quantified by PCR which could be potential biomarkers for monitoring organ survival and/or immune rejection. In this review, we briefly introduce the origins and characteristics of circulating miRNA and circulating DNA. Furthermore, we review their clinical application and potential as noninvasive biomarkers for rejection in organ transplantation. Finally, we sum up their potential as biomarkers, which may help scientists and surgeons choose the proper biomarker for routine assays.

Molecular hydrogen protects against ischemia-reperfusion injury in a mouse fatty liver model via regulating HO-1 and Sirt1 expression

Fatty liver has lower tolerance against ischemia-reperfusion (I/R) injury in liver operations, including liver transplantation. Seeking to ameliorate liver injury following I/R in fatty liver, we examined the protective effect of hydrogen (H2) saline on I/R liver injury in a methionine and choline-deficient plus high fat (MCDHF) diet-induced fatty liver mouse model. Saline containing 7 ppm H2 was administrated during the process of I/R. Livers were obtained and analyzed. Primary hepatocytes and Kupffer cells (KCs) were obtained from fatty liver and subjected to hypoxia/reoxygenation. Apoptosis-related proteins and components of the signaling pathway were analyzed after treatment with hydrogen gas. The MCDHF I/R group showed higher levels of AST and ALT in serum, TUNEL-positive apoptotic cells, F4/80 immunopositive cells, mRNA levels of inflammatory cytokines, constituents of the signaling pathway, pro-apoptotic molecules in liver, and KCs and/or primary hepatocytes, compared to the control group. In contrast, H2 treatment significantly suppressed the signs of I/R injury in fatty liver. Moreover, the expression of Bcl-2, HO-1, and Sirt1 in liver, KCs, and hepatocytes by hydrogen gas were increased, whereas caspase activation, Bax, and acetylation of p53 were suppressed by hydrogen gas. These results demonstrated that H2 treatment ameliorated I/R liver injury in a fatty liver model by reducing hepatocyte apoptosis, inhibiting macrophage activation and inflammatory cytokines, and inducing HO-1 and Sirt1 expression. Taken togather, treatment with H2 saline may have a protective effect and safe therapeutic activity during I/R events, such as in liver transplantation with fatty liver.

Drug repurposing: Ibrutinib exhibits immunosuppressive potential in organ transplantation

Long-term administration of classic immunosuppressants can induce severe adverse effects. The development of novel immunosuppressants confronts great challenges and opportunities. Ibrutinib, an approved drug for B-cell lineages and chronic graft versus host disease (cGVHD), exhibits immunosuppressive efficacy in autoimmune diseases. Ibrutinibs potential as an immunosuppressant in organ transplantation has not been investigated to date. In a xeno-artery patch model ex vivo, ibrutinib inhibited the proliferation of PBMCs (POD 14-42), mainly CD3+CD4+ and CD3+CD8+ T cells ex vivo. The secretion of cytokines (IL-6, IL-2 and IFN-γ) was suppressed in response to ibrutinib. In allo-skin transplantation models, ibrutinib delayed the rejection of grafted skins. Ibrutinib decreased the amount of T/B cells and lymphocyte infiltration. Altogether, ibrutinib exhibited immunosuppressive potential through cytokine regulation and T cell inhibition ex vivo and in vitro. Repositioning of ibrutinib as an immunosuppressant will greatly facilitate novel immunosuppressant development.