Elimination of GGTA1,CMAH,β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Background:Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic,while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs.Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection(HAR)that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure,in which process the MHCⅡmolecule plays critical roles.Methods:Thus,we generate a 4-gene(GGTA1,CMAH,β4GalNT2,and CIITA)knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.Results:We successfully obtained 4KO piglets with deficiency in all alleles of genes,and at cellular and tissue levels.Additionally,the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping.Piglets have survived for more than one year in the barrier,and also survived for more than 3 months in the conventional environment,suggesting that the piglets without MHCⅡcan be raised in the barrier and then gradually mated in the conventional environment.Conclusions:4KO piglets have lower immunogenicity,are safe in genomic level,and are easier to breed than the model with both MHCⅠandⅡdeletion.

Effects of salidroside on glioma formation and growth inhibition together with improvement of tumor microenvironment

Objective：To test the effects of salidroside on formation and growth of glioma together with tumor microenvironment.Methods：Salidroside extracted from Rhodiola rosea was purified and treated on human glioma cells U251 at the concentration of 20 μg/mL.3-（4,5-dimethylthiazol-2-yl）-2,5-dephenyltetrazolium bromide （MTT） assay for cytotoxicity and flow cytometry （FCM） for cell cycle analysis were performed.Then for in vivo study,xenotransplantation tumor model in nude mice was generated and treated with salidroside at the concentration of 50 mg/kg.d for totally 20 d.Body weight and tumor size were detected every 2 d after the treatment.The levels of 8-isoprostane,superoxide dismutase （SOD） and malondialdehyde （MDA）,special markers for oxidative stress,were detected while immunofluoresence staining was performed for astrocyte detection.Results：For in vitro study,salidroside could decrease the viability of human glioma cells U251 and the growth of U251 cells at G0/G1 checkpoint during the cell cycle.For in vivo study,salidroside could also inhibit the growth of human glioma tissue in nude mice.The body weight of these nude mice treated with salidroside did not decrease as quickly as control group.In the tumor xenotransplantation nude mice model,mice were found of inhibition of oxidative stress by detection of biomarkers.Furthermore,overgrowth of astrocytes due to the stimulation of oxidative stress in the cortex of brain was inhibited after the treatment of salidroside.Conclusions：Salidroside could inhibit the formation and growth of glioma both in vivo and in vitro and improve the tumor microenvironment via inhibition of oxidative stress and astrocytes.

Human benign prostatic hyperplasia heterotransplants as an experimental model

Benign prostatic hyperplasia is a nonmalignant adenomatous enlargement of the pefiurethral prostate gland. It is a common disease in older men. In addition to man,spontaneous benign prostatic hyperplasia occurs in chimpanzee and the dog. Alternatives to these spontaneous models are induced benign prostatic hyperplasia,xenografts and in vitro models. Xenografts may be induced by cells cultured in vitro or by the heterotransplantation of primary surgical specimens into immunosuppressed mice. The purpose of this review is to integrate data from more than 30 years of heterotransplantation research in the study of benign hyperplasia of the prostate. Heterotransplantation has provided data regarding the histopathology,morphology,tissue markers,androgen receptor expression,tissue kinetics,take rate and tissue vasculature for this prostate disease.There are advantages,as well as limitations,that have been identified for human prostate disease heterotransplants versus xenotransplantation of cultured cells.Overall,heterotransplanted tissue is better at retaining tissue morphology,pathology,secretory activity,expression of tissue markers and human vasculature of the patient＇s original specimen. Furthermore,heterotransplanted tissue preserves the three-dimensional tissular architecture of the prostate to maintain critical stromal-epithelial cell interactions.

A comparison of three methods of decellularization of pig corneas to reduce immunogenicity

·AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells(CECs).Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts.·METHODS: Six-month-old wild-type pig corneas were cut into 100-200 μm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate(SDS); 2) hypoxic nitrogen(N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin(H&E), and for the presence of galactose-α1,3-galactose(Gal) and N-glycolylneuraminic acid(NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining.· RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by 】80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue.· CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.

Cloning and cDNA Sequence Analysis of SLA-DQA Gene from Hainan Wuzhishan Miniature Pig

[ Objective] The study aimed to lay a foundation for studying immune recognition mechanism in xenotransplantation. [ Method] SLA- DQA gene of Hainan Wuzhishan miniature pig was cloned by RT-PCR and sequenced. J-Result] The SLA-DQA gene sequence of Wuzhishan minia- ture pig contained 768 nucleotides, among which 1 -765 were the complete open reading frame, encoding 255 amino acids. SLA-DQA cDNA se- quence and its deduced amino acid sequence of Wuzhishan miniature pig were most similar to those of the other miniature pigs in China. When compared with corresponding cDNA sequences and the amino acid sequences in other species, the homology with human was obviously higher than that with mouse. [ Conclusion] As the Chinese pig breed suitable for xenotransplantation, Wuzhishan miniature pig has significant importance to ser- ial studies on immunogenetics characteristics.

Future of bioartifi cial liver support

Many different artificial liver support systems(biological and non-biological) have been developed,tested pre-clinically and some have been applied in clinical trials.Based on theoretical considerations a biological artificial liver(BAL) should be preferred above the non-biological ones.However,clinical application of the BAL is still experimental.Here we try to analyze which hurdles have to be taken before the BAL will become standard equipment in the intensive care unit for patients with acute liver failure or acute deterioration of chronic liver disease.

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strategies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their accession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A*0201) are better than those between pigs and mice (H-2Db/H-2Kb). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.

The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation

There is a critical shortage of organs, cells, and corneas from deceased human donors worldwide. There are also shortages of human blood for transfusion. A potential solution to all of these problems is the transplantation of organs, cells, and corneas from a readily available animal species, such as the pig, and the transfusion of red blood cells from pigs into humans. However, to achieve these ends, major immunologic and other barriers have to be overcome. Considerable progress has been made in this respect by the genetic modification of pigs to protect their tissues from the primate immune response and to correct several molecular incompatibilities that exist between pig and primate. These have included knockout of genes responsible for the expression of major antigenic targets for primate natural anti-pig antibodies, insertion of human complement- and coagulation-regulatory transgenes, and knockdown of swine leukocyte antigens that stimulate the primate＇s adaptive immune response. As a result of these manipulations, the administration of novel immunosuppressive agents, and other innovations, pig hearts have now functioned in baboons for 6-8 months, pig islets have maintained normoglycemia in diabetic monkeys for 〉 1 year, and pig corneas have maintained transparency for several months. Clinical trials of pig islet trans- plantation are already in progress. Future developments will involve further genetic manipulations of the organ- source pig, with most of the genes that are likely to be beneficial already identified.

Antigenicity of tissues and organs from GGTA1/CMAH/β4GalNT2 triple gene knockout pigs

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.

The role of donor hepatocytes and/or splenocytes pre-injection in reducing islet xenotransplantation rejection

OBJECTIVE: To observe the effect of tail vein injection with donor hepatocyte and/or splenocyte on islets xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islets xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: Streptozotocin (STZ) succeeded in inducing diabetes mellitus models of mice. Pre-injection of donor hepatocytes, and splenocytes or their mixture via tail vein was effective in preventing donor islets transplantation from rejection, which was demonstrated by the mentioned immunological marks. And each group of transplantation could decrease the blood glucose of recipients and prolong the survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection than pre-injection of donor hepatocytes or splenocytes separately. CONCLUSION: We propose that pre-injection of donor hepatocytes, splenocytes separately or their mixture before donor islets transplantation is a good way to prevent rejection.

Small-cell neuroendocrine carcinoma of the prostate： are heterotransplants a better experimental model？

Small-cell neuroendocrine carcinoma of the prostate （SCNCP） is an uncommon type of prostate cancer. However, it is of clinical importance because it is one of the most aggressive tumors of the prostate with a very poor prognosis. There exist few artificially cultured tumor cell lines to study SCNCE Then, another approach to that study consists in the use of fresh tumor tissue obtained from patients and its heterotransplantation into host mice. The purpose of this review is to integrate data from more than 20 years of heterotransplantation research in the study of small-cell neuroendocrine carcinoma of the prostate （SCNCP）. Heterotransplantation has provided data regarding the histopathology, karyotype, DNA content, cell cycle frequency, tumor markers, androgen receptor expression, metastasis and take rate of this prostate disease. When possible, comparisons between original in situ specimens removed from patients and heterotransplanted tissue from host mice have been made. There are advantages, as well as limitations, that have been identified for SCNCP heterotransplants versus xenotransplantation of cultured cells. Overall, heterotransplanted tumors are better than conventional tumor xenografts at retaining tumor morphology, pathology, secretory activity and expression of tumor markers of the patient＇s original specimen. Furthermore, heterotransplanted tissue preserves the three-dimensional tumor architecture of the prostate to maintain critical stromal-epithelial cell interactions.

HS-4,a highly potent inhibitor of cell proliferation of human cancer cell

Objective:To investigate the antitumor activity of the compound HS-4 and the action mechanism.Methods:MTT method was used to test in vitro antitumor activity of the compound HS-4.Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice,and.in vivo antitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system.Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics,using high-throughput sequencing technology.Results:HS-4 was found to have relatively high in-vitro antitumor activity against liver cancer cells,gastric cancer cells,renal cancer cells,lung cancer cells,breast cancer cells and colon cancer cells.The IC_(50) values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively,while the IC_(50) values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L,respectively.It was demonstrated that HS- 4 possessed a betler therapeutic effect in liver cancer.Conclusions:A new reliable orthotopicxenotransplantation tumor model of liver cancer in nude mice is established.The new compounds HS-4 was found to possess relatively high in vivo and in vitro antitumor activity against liver cancer cells.

Establishment of Reproducible Xenotransplantation Model of T Cell Acute Lymphoblastic Leukemia in NOD/SCID Mice

T cell acute lymphoblastic leukemia(T-ALL) is an aggressive leukemia.However the poor prognosis and low morbidity restrict further analysis of the disease.Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches.In this study,we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein.Four of the 9 patients and the cell line were successfully engrafted.Flow cytometry detected high percentage of human CD45 + cells in recipient mice.Immunohistochemistry showed infiltration of human CD45 + cells in different organs.Serial transplantation was also achieved.In vivo drug treatment showed that dexamethasone could extend survival,which was consistent with clinical observation.These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice,which recapitulated the characteristics of original disease.

Intracerebral xenotransplantation of semipermeable membrane- encapsuled pancreatic islets

AIM： To identify the decreasing effect of xenotransplantion in combination with privileged sites on rejection and death of biological semipermeable membrane-（BSM） encapsulated implanted islets. METHODS： After the BSM experiment in vitro, BSM- encapsulated SD rat＇s islet-like cell clusters （ICCs） were xenotransplanted into normal dog＇s brain. Morphological changes were observed under light and transmission electron microscope. The islets and apoptosis of implanted B cells were identified by insulin-TONEL double staining. RESULTS： The BSM used in our study had a favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts consisted of epithelioid cells and loose connective tissue. Severe infiltration of inflammatory cells was not observed. The implanted ICCs were identified 2 mo later and showed typical apoptosis. CONCLUSION： BSM xenotransplantation in combination with the privileged site can inhibit the rejection of implanted heterogeneous ICCs, and death of implanted heterogeneous B cells is associated with apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Establishment of Xenotransplantation Model of Human CN-AML with FLT3-ITD^(mut)/NPM1 in NOD/SCID Mice

Summary： Patients with FLT3-ITD^mmutt/NPM1- cytogenetically normal acute myeloid leukemia （CN-AML）, as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1- CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1- CN-AML primary cells. The FLT3-ITD^mut/NPM1- CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary genera- tion models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully estab- lished xenotransplantation model of human FLT3-ITD^mut/NPM1- CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

The expression and distribution of α-Gal gene in various species ocular surface tissue

AIM: To examine the α-Gal gene expression and distribution in the different species/genus and developing phase animal ocular surface tissue.METHODS: α-Gal binding assay were carried out on various animal eye sections. Photograph, slit-lamp observation on various eye showed normal corneal transparence.RESULTS: A strong α-Gal expression in invertebrates and some vertebrates ocular tissue, but no α-Gal binding in birds, fish and mammal. α-Gal expression change in the development of mice ocular surface tissue (except sclera) and display genus dependency in the different murine ocular surface tissue.CONCLUSION: This study identified specific α-Gal epitopes binding area in the ocular surface of several species and may solve the problem that naive ocular surface may be used as natural α-Gal gene knockout model/high risk immunologic rejection model or ocular surface scaffold material.

Update on acute myeloid leukemia stem cells:New discoveries and therapeutic opportunities

The existence of cancer stem cells has been wellestablished in acute myeloid leukemia. Initial proof of the existence of leukemia stem cells(LSCs) was accomplished by functional studies in xenograft models making use of the key features shared with normal hematopoietic stem cells(HSCs) such as the capacity of self-renewal and the ability to initiate and sustain growth of progenitors in vivo. Significant progress has also been made in identifying the phenotype and signaling pathways specific for LSCs. Therapeutically, a multitude of drugs targeting LSCs are in different phases of preclinical and clinical development. This review focuses on recent discoveries which have advanced our understanding of LSC biology and provided rational targets for development of novel therapeutic agents. One of the major challenges is how to target the selfrenewal pathways of LSCs without affecting normal HSCs significantly therefore providing an acceptable therapeutic window. Important issues pertinent to the successful design and conduct of clinical trials evaluating drugs targeting LSCs will be discussed as well.

How regenerative medicine and tissue engineering may complement the available armamentarium in gastroenterology?

The increasing shortage of donors and the adverse effects of immunosuppression have restricted the impact of solid organ transplantation.Despite the initial promising developments in xenotransplantation,roadblocks still need to be overcome and this form of organ support remains a long way from clinical practice.While hepatocyte transplantation may be effectively correct metabolic defects,it is far less effective in restoring liver function than liver transplantation.Tissue engineering,using extracellular matrix scaffolds with an intact but decellularized vascular network that is repopulated with autologous or allogeneic stem cells and/or adult cells,holds great promise for the treatment of failure of organs within gastrointestinal tract,such as endstage liver disease,pancreatic insufficiency,bowel failure and type 1 diabetes.Particularly in the liver field,where there is a significant mortality of patients awaiting transplant,human bioengineering may offer a source of readily available organs for transplantation.The use of autologous cells will mitigate the need for long term immunosuppression thus removing a major hurdle in transplantation.

The Effects of PDTC plus Leflunomide and Cyclosporine on the NF-КB Signaling Pathway in Mouse-to-rat Cardiac Xenografts

In this study, the effects of pirrolidine dithiocarbamate （PDTC） plus leflunomide （Lef） and cyclosporine （CsA） on the NF-κB signaling pathway in mouse-to-rat cardiac xeno-transplantation models were investigated. NIH mice and Wistar rats served as donors and recipients respectively. Mouse-to-rat cardiac xenotransplantation was performed. The recipients were divided into 5 groups： group A （the control group）, group B （PDTC group）, group C （PDTC plus CsA group）, group D （PDTC plus Lef group） and group E （PDTC plus Lef and CsA group）. The expressions of IKKa/[3, NF-κB-P65, IκBct, ICAM-1 and NF-κB DNA binding activity in xenograft tissues were determined by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay （EMSA）. The median survival time of cardiac xenografts in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef group and PDTC plus Lef and CsA group was （2.17±0.41）, （2.33±0.52）, （4.67±1.21）, （7.00±1.79） and （9.00±1.41） days respectively. The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups （P〈0.05 for all）, that in the PDTC plus Lef group longer than that in the control group, PDTC group and PDTC plus CsA group （P〈0.05 for all）, that in PDTC plus CsA group longer than the control group and PDTC group （P〈0.05 for all）. The expressions of IKKα/β, NF-κB-P65, IκBα and ICAM-1 and NF-κ3 DNA binding activity were notably increased in mouse-to-rat cardiac xenografts. The expressions were decreased in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef and PDTC plus Lef and CsA group in turn. It was concluded that PDTC plus Lef and CsA can significantly suppress the expressions of IKKα/β, NF-κB-P65, IκBα, ICAM-1 and NF-κB DNA binding activity, thereby prolonging the survival of the cardiac xenografts.

Over-expression of Heme Oxygenase-1 Does not Protect Porcine Endothelial Cells from Human Xenoantibodies and Complement-mediated Lysis

Accommodated organs can survive in the presence of anti-organ antibodies and complement. Heme oxygenase-1 （HO-1） is essential to ensure accommodation in concordant xenotransplant models. However, whether induction of HO-1 over-expression could protect porcine endothelial cells （PECs） against human xenoantibodies and complement-mediated lysis and induce an in vitro accommodation is still unknown. The SV40-immortalized porcine aorta-derived endothelial cell line （iPEC） was pre-incubated with 20, 50, or 80 μmol/L of cobalt-protoporphyrins IX （CoPPIX） for 24 h, and the HO-1 expression in iPECs was analyzed by using Western blotting. CoPPIX-treated or untreated iPECs were incubated with normal human AB sera, and complement-dependent cytotoxicity （CDC） was measured by both flow cytometry and lactate dehydrogenase （LDH） release assay. In vitro treatment with CoPPIX significantly increased the expression of HO-1 in iPECs in a dose-dependent manner. Over-expression of HO-1 was successfully achieved by incubation of iPECs with either 50 or 80 μmol/L of CoPPIX. However, HO-1 over-expression did not show any protective effects on iPECs against normal human sera-mediated cell lysis. In conclusion, induction of HO-1 over-expression alone is not enough to pro- tect PECs from human xenoantibodies and complement-mediated humoral injury. Additionally, use of other protective strategies is needed to achieve accommodation in pig-to-primate xenotransplantation.