The development of xenotransplantation is expected to alleviate the supply and demand gap of donors' organs. Currently,gene-edited pigs are considered as ideal organ donor source for clinical xenotransplantation. Driven by relevant technologies,substantial progress have been achieved in preclinical studies of xenotransplantation,which creates good conditions for the opening of early clinical trials. Especially in recent two years,the foreign clinical research in this field has made a breakthrough. Here,the progress in xenotransplantation of clinical trials is briefly reviewed home and abroad,the key issues in clinical trials of xenotransplantation are discussed from the perspectives of gene editing of donor pigs,principles of whole-course management of subjects,ethics and social psychology issue. It is believed that under the background of multidisciplinary cross-fusion,xenotransplantation will be gradually transferred to clinical application in the future,and better benefit human beings.
Animals ; Humans ; Swine ; Tissue Donors ; Transplantation, Heterologous

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.
Animals ; Animals, Genetically Modified ; Gene Knockout Techniques ; HLA Antigens ; Humans ; Nuclear Transfer Techniques ; Swine ; Transplantation, Heterologous

OBJECTIVE: To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft. METHODS: Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen. RESULTS: We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts. CONCLUSIONS β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.
Animals ; Disease Progression ; Heterografts ; Humans ; Mice ; T-Lymphocytes ; Transplantation, Heterologous ; beta-Arrestin 1

OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) is an important molecule in the early stress response of DNA damage, which is involved in DNA damage repair and cellular senescence. Olaparib, as PARP inhibitor, has an anti-tumor effect on high grade serous ovarian cancer, but its effects on cellular senescence have not been reported. This study intends to explore the role of olaparib in the regulation of senescence in ovarian cancer cells. METHODS: The effects of olaparib on the senescence of ovarian cancer cells were detected by using the senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated heterochromatin aggregation (SAHF). Quantitative real-time polymerase chain reaction was used to analyze the senescence-associated secretory phenotype (SASP). Cell cycle and apoptosis were detected by flow cytometry. The effect of olaparib on tumor growth was analyzed in a nude mouse xenograft transplantation model. RESULTS: Long-term (6 days) treatment with olaparib (5 μM) significantly inhibited the growth of ovarian cancer cells, leading to arrest the cell cycle at G0/G1 phase, significant increase the number of positive SA-β-Gal stained cells and positive SAHF cells. The expression of P16 and retinoblastoma protein (p-RB) were significantly enhanced in SKOV3 cells under olaparib treated, meanwhile, the expression of P53 and p-RB were upregulated in A2780 cells. In OVCAR-3 cells, the expression of P53 was downregulated and p-RB was upregulated. Mice with SKOV3 xenograft transplantation was given olaparib (10 mg/kg/day) via abdominal cavity administration, the tumor volume was reduced (p < 0.01). CONCLUSION: Continuous low dosage administration of olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.
Abdominal Cavity ; Aging ; Animals ; Apoptosis ; Cell Aging ; Cell Cycle ; DNA Damage ; Flow Cytometry ; Heterochromatin ; Mice ; Mice, Nude ; Ovarian Neoplasms ; Phenotype ; Real-Time Polymerase Chain Reaction ; Retinoblastoma Protein ; Transplantation, Heterologous ; Tumor Burden

BACKGROUND: Small cell lung cancer (SCLC) is characterized by poor differentiation, high malignancy and rapid growth fast, short double time, early and extensive metastatic malignancy. In clinical, chemotherapy is the main treatment method, while resistance to multiple chemotherapy drugs in six to nine months has been a major clinical challenge in SCLC treatment. Therefore, It has important clinical value to building SCLC aninimal model which is similar to patients with SCLC. Animal model of xenotransplantation (PDX) from the patients with small cell lung cancer can well retain the characteristics of primary tumor and is an ideal preclinical animal model. The study is aimed to establish SCLC PDX animal model and induce the chemoresistance model to help to study the mechanism of chemoresistance and individual treatment. METHODS: Fresh surgical excision or puncture specimens from SCLC patients were transplanted into B-NSGTM mice subcutaneous tissues with severe immunodeficiency in one hour after operation the B-NSGTM mice subcutaneous in 1 hour, and inject chemotherapy drugs intraperitoneally after its tumor growed to 400 mm³ with EP which is cisplatin 8 mg/kg eight days and etoposide 5 mg/kg every two days until 8 cycles. Measure the tumor volum and mice weights regularly, then re-engrafted the largest tumor and continue chemotherapy. RESULTS: Nine cases were conducted for B-NSG mice modeling. Three of nine cases could be engrafted to new B-NSG mice at least two generation. The SCLC PDX animal models have been established successfully. After adopting chemotherapy drugs, the chemoresistance PDX models have been established. High homogeneity was found between xenograft tumor and patient's tumor in histopathology, immunohistochemical phenotype (Syn, CD56, Ki67). CONCLUSIONS The SCLC PDX animal model and the chemoresistance PDX animal model have been successfully constructed, the success rate is 33%, which provides a platform for the clinical research, seeking for biological markers and choosing individual treatment methods of SCLC.
Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cisplatin ; administration & dosage ; Disease Models, Animal ; Drug Resistance, Neoplasm ; Etoposide ; administration & dosage ; Female ; Humans ; Interleukin Receptor Common gamma Subunit ; deficiency ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Small Cell Lung Carcinoma ; drug therapy ; metabolism ; pathology ; Transplantation, Heterologous ; methods ; Xenograft Model Antitumor Assays

Abstract　　Tumor xenograft model (PDTX) derived from leukemia patients is an animal model in which the leukemia cells or primary cell lines of patients are transplanted directly into immunodeficient mice.The emergence of nude mice and SCID mice opened early xenotransplantation, then the NSG, NOG mice and the improved model and humanized mice based on there mice significantly improves the success rate of transplantation. The late presented transplantation of leukemia LSC and transplantation of patient-derived and induced pluripotent stem cells obtained based on iPSC technology provide new insight for the anderstanding leukemia genesis and development, and the new type humanized mouse model with normal lymphatic hematopoietic reconstruction provides a new platform of leukemia cell therapy and immunotherapy for leukemia therapy. PDTX is an important platform for the study of the pathogenesis and drug resistance mechanism of leukemia, as well as the development of new drugs and individualized treatment. In this paper, the recent progress in the construction and application of models of immunodeficient mice and their models is reviewed.
Animals ; Disease Models, Animal ; Humans ; Leukemia ; Mice ; Transplantation, Heterologous

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.
Alleles ; Animals ; Endogenous Retroviruses ; Genes, MHC Class I ; Genes, MHC Class II ; Genome ; Heterografts ; Leukocytes ; Mass Screening ; Recombination, Genetic ; Retroviridae ; RNA ; Swine ; Transgenes ; Transplantation, Heterologous ; Viremia

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.
Adult ; Cell Line ; Cytidine ; DNA ; Ear ; Embryonic Development ; Female ; Fibroblasts ; Flow Cytometry ; Fluorescent Antibody Technique ; Genetics ; Heterografts ; Humans ; Kidney ; Korea ; Physiology ; Pregnancy ; Skin ; Swine ; Swine, Miniature ; Transplantation, Heterologous

Phosphoglycerate mutase 1 (PGAM1) is upregulated in many cancer types and involved in cell proliferation, migration, invasion, and apoptosis. However, the relationship between PGAM1 and prostate cancer is poorly understood. The present study investigated the changes in PGAM1 expression in prostate cancer tissues compared with normal prostate tissues and examined the cellular function of PGAM1 and its relationship with clinicopathological variables. Immunohistochemistry and Western blotting revealed that PGAM1 expression was upregulated in prostate cancer tissues and cell lines. PGAM1 expression was associated with Gleason score (P = 0.01) and T-stage (P = 0.009). Knockdown of PGAM1 by siRNA in PC-3 and 22Rv1 prostate cancer cell lines inhibited cell proliferation, migration, and invasion and enhanced cancer cell apoptosis. In a nude mouse xenograft model, PGAM1 knockdown markedly suppressed tumor growth. Deletion of PGAM1 resulted in decreased expression of Bcl-2, enhanced expression of Bax, caspases-3 and inhibition of MMP-2 and MMP-9 expression. Our results indicate that PGAM1 may play an important role in prostate cancer progression and aggressiveness, and that it might be a valuable marker of poor prognosis and a potential therapeutic target for prostate cancer.
Animals ; Apoptosis/genetics* ; Caspase 3/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Gene Deletion ; Gene Knockdown Techniques ; Humans ; Male ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Mice ; Mice, Nude ; Neoplasm Invasiveness/genetics* ; Neoplasm Transplantation ; PC-3 Cells ; Phosphoglycerate Mutase/genetics* ; Prostatic Neoplasms/pathology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Small Interfering ; Transplantation, Heterologous ; bcl-2-Associated X Protein/metabolism*

BACKGROUND: The shortage of human hearts for allotransplantation makes xenotransplantation a possible option for controllable organ providers. To detect acute xenograft rejection, invasive biopsy seems inevitable; however, this occasionally results in poor incision wound healing or infection. To date, no method of noninvasive imaging for early detection of xenograft rejection has been established. We hypothesized that ultrasound speckle tracking would better detect xenograft failure than routine left ventricular ejection fractions (EF). METHODS: From August 2013 to July 2015, a total of six cardiac heterotopic xenotransplants (α 1, 3-galactosyltransferase gene-knockout porcine heart) into cynomolgus monkeys were monitored with echocardiography every 3 to 7 days. M-mode and two-dimensional (2D)-EF measurements and myocardial strain analyses were performed. Cardiac xenograft pathology was reviewed from the immediate postoperative biopsy, as well as the necropsy. RESULTS: Myocardial speckle tracking analysis was feasible in all six cases. The longest survival was 43 days. Only one pathology-proven immunologic rejection occurred. Cardiac xenograft failure appeared as two types: a dilated pattern with decreased EF or a myocardial-thickening pattern with preserved EF. Both antibody-mediated rejection (n=1) and sepsis-induced myocardial dysfunction (n=2) revealed decreased radial or circumferential strains, but normal-range EF. Xenograft functional decline was significant with respect to radial or circumferential strain (P=0.028), but not to conventional M-mode or 2D-EFs (P=0.600, P=0.340, respectively). CONCLUSIONS: Radial and circumferential strains were significantly decreased in both types of xenograft failure, regardless of EF. Further studies are warranted to correlate the strain analysis and immunopathological details.
Biopsy ; Echocardiography ; Heart ; Heart Transplantation ; Heterografts ; Humans ; Macaca fascicularis ; Methods ; Pathology ; Stroke Volume ; Transplantation, Heterologous* ; Transplants* ; Ultrasonography ; Wound Healing