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1.
Transplantation ; 104(8): 1566-1573, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32732833

RESUMO

BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. METHODS: We simultaneously depleted GGTA1, ß2-microglobulin (ß2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1ß2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-ß2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, ß2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.


Assuntos
Rejeição de Enxerto/prevenção & controle , Xenoenxertos/imunologia , Transplante de Órgãos/métodos , Transplante Heterólogo/métodos , Aloenxertos/provisão & distribução , Animais , Animais Geneticamente Modificados/imunologia , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Feminino , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Técnicas de Inativação de Genes/métodos , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Xenoenxertos/transplante , Humanos , Masculino , Camundongos , Transplante de Órgãos/efeitos adversos , Suínos/genética , Suínos/imunologia , Transplante Heterólogo/efeitos adversos , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia
2.
Curr Opin Organ Transplant ; 25(3): 261-267, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32374577

RESUMO

PURPOSE OF REVIEW: Considerable advancements have been made in the field of cardiac xenotransplantation in the recent years, achieving prolonged survival of the life-supporting cardiac xenograft and paving the way toward first clinical implications. RECENT FINDINGS: The combination of genetic modifications and novel immunosuppression with costimulation blockade, as well as supporting therapy with antiinflammatory treatment, growth prevention, and adaptation of the heart procurement system to reduce myocardial ischemia and reperfusion injury improves the overall cardiac xenograft function and overall survival in nonhuman primates. Through the newly identified xenoantigens and novel gene-editing techniques, further genetic modification of the porcine xenografts should be explored, to ensure clinical safety. SUMMARY: With continuous progress in all fields of cardiac xenotransplantation, first clinical use in humans seems accomplishable. To ensure the clinical safety and to conform to the ethical regulations, further investigation of the infectious and immunological implications on humans should be explored prior to first clinical use. The first clinical use of cardiac xenotransplantation will be limited to only highly selected patients.


Assuntos
Transplante de Coração/métodos , Transplante Heterólogo/métodos , Animais , Humanos , Suínos
3.
Proc Natl Acad Sci U S A ; 117(16): 8845-8849, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253306

RESUMO

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.


Assuntos
Aminoácidos/genética , Diferenciação Celular/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/fisiologia , Engenharia de Proteínas/métodos , Animais , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Código Genético , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Plasmídeos/genética , Cultura Primária de Células/métodos , Transfecção/métodos , Quimeras de Transplante , Transplante Heterólogo/métodos
4.
J Immunol ; 204(7): 1998-2005, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32144163

RESUMO

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.


Assuntos
Anticorpos/imunologia , Epitopos/imunologia , Galactosiltransferases/imunologia , alfa-Galactosidase/imunologia , Animais , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante Heterólogo/métodos
5.
FASEB J ; 34(1): 1901-1911, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914605

RESUMO

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.


Assuntos
Células Endócrinas/fisiologia , Ilhotas Pancreáticas/fisiologia , Transcriptoma/fisiologia , Adulto , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Células Endócrinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Sobrevivência de Enxerto/fisiologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Camundongos , Transplante Heterólogo/métodos , Adulto Jovem
6.
Int J Cancer ; 146(8): 2078-2088, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31479514

RESUMO

Patient-derived xenograft (PDX) models are widely used as preclinical cancer models and are considered better than cell culture models in recapitulating the histological features, molecular characteristics and intratumoral heterogeneity (ITH) of human tumors. While the PDX model is commonly accepted for use in drug discovery and other translational studies, a growing body of evidence has suggested its limitations. Recently, the fidelity of cancer cells within a PDX has been questioned, which may impede the future application of these models. In this review, we will focus the variable phenotypes of xenograft tumors and the genomic instability and molecular inconsistency of PDX tumors after serial transplantation. Next, we will discuss the underlying mechanism of ITH and its clinical relevance. Stochastic selection bias in the sampling process and/or deterministic clonal dynamics due to murine selective pressure may have detrimental effects on the results of personalized medicine and drug screening studies. In addition, we aim to identify a possible solution for the issue of fidelity in current PDX models and to discuss emerging next-generation preclinical models.


Assuntos
Neoplasias/patologia , Transplante Heterólogo/métodos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Xenoenxertos/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-Zebra
7.
Nat Commun ; 10(1): 5776, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852888

RESUMO

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.


Assuntos
Moléculas de Adesão Celular/fisiologia , Lectinas Tipo C/fisiologia , Músculo Esquelético/fisiologia , Fator de Transcrição PAX7/genética , Regeneração , Células Satélites de Músculo Esquelético/fisiologia , Síndrome de Emaciação/genética , Animais , Biópsia , Pré-Escolar , Consanguinidade , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Mutação , Fator de Transcrição PAX7/metabolismo , Cultura Primária de Células , Células Satélites de Músculo Esquelético/transplante , Análise de Célula Única , Transplante Heterólogo/métodos , Síndrome de Emaciação/terapia , Sequenciamento Completo do Exoma
8.
JCI Insight ; 4(24)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31852842

RESUMO

Massive tears of the rotator cuff (RC) are associated with chronic muscle degeneration due to fibrosis, fatty infiltration, and muscle atrophy. The microenvironment of diseased muscle often impairs efficient engraftment and regenerative activity of transplanted myogenic precursors. Accumulating myofibroblasts and fat cells disrupt the muscle stem cell niche and myogenic cell signaling and deposit excess disorganized connective tissue. Therefore, restoration of the damaged stromal niche with non-fibro-adipogenic cells is a prerequisite to successful repair of an injured RC. We generated from human embryonic stem cells (hES) a potentially novel subset of PDGFR-ß+CD146+CD34-CD56- pericytes that lack expression of the fibro-adipogenic cell marker PDGFR-α. Accordingly, the PDGFR-ß+PDGFR-α- phenotype typified non-fibro-adipogenic, non-myogenic, pericyte-like derivatives that maintained non-fibro-adipogenic properties when transplanted into chronically injured murine RCs. Although administered hES pericytes inhibited developing fibrosis at early and late stages of progressive muscle degeneration, transplanted PDGFR-ß+PDGFR-α+ human muscle-derived fibro-adipogenic progenitors contributed to adipogenesis and greater fibrosis. Additionally, transplanted hES pericytes substantially attenuated muscle atrophy at all tested injection time points after injury. Coinciding with this observation, conditioned medium from cultured hES pericytes rescued atrophic myotubes in vitro. These findings imply that non-fibro-adipogenic hES pericytes recapitulate the myogenic stromal niche and may be used to improve cell-based treatments for chronic muscle disorders.


Assuntos
Células-Tronco Embrionárias Humanas/fisiologia , Transtornos Musculares Atróficos/terapia , Pericitos/transplante , Lesões do Manguito Rotador/complicações , Manguito Rotador/patologia , Animais , Diferenciação Celular , Linhagem Celular , Doença Crônica/terapia , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Injeções Intralesionais , Camundongos , Desenvolvimento Muscular/fisiologia , Transtornos Musculares Atróficos/etiologia , Transtornos Musculares Atróficos/patologia , Transtornos Musculares Atróficos/fisiopatologia , Pericitos/fisiologia , Manguito Rotador/fisiopatologia , Transplante Heterólogo/métodos
9.
BMB Rep ; 52(11): 625-634, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31722780

RESUMO

Severe combined immunodeficiency (SCID) is a group of inherited disorders characterized by compromised T lymphocyte differentiation related to abnormal development of other lymphocytes [i.e., B and/or natural killer (NK) cells], leading to death early in life unless treated immediately with hematopoietic stem cell transplant. Functional NK cells may impact engraftment success of life-saving procedures such as bone marrow transplantation in human SCID patients. Therefore, in animal models, a T cell-/B cell-/NK cell+ environment provides a valuable tool for understanding the function of the innate immune system and for developing targeted NK therapies against human immune diseases. In this review, we focus on underlying mechanisms of human SCID, recent progress in the development of SCID animal models, and utilization of SCID pig model in biomedical sciences. Numerous physiologies in pig are comparable to those in human such as immune system, X-linked heritability, typical T-B+NK- cellular phenotype, and anatomy. Due to analogous features of pig to those of human, studies have found that immunodeficient pig is the most appropriate model for human SCID. [BMB Reports 2019; 52(11): 625-634].


Assuntos
Modelos Animais de Doenças , Imunodeficiência Combinada Severa/imunologia , Suínos/imunologia , Animais , Humanos , Medicina Regenerativa , Transplante Heterólogo/métodos
10.
J Pak Med Assoc ; 69(11): 1617-1622, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31740866

RESUMO

OBJECTIVE: To compare the performance of collagenated bone graft substances with different collagen ratios after sinus floor augmentation. METHODS: The cross-sectional study was conducted at Hacettepe University, Ankara, Turkey, from September 2011 to September 2013. Sinus floor augmentation was done with two different equinederived xenografts in patients before dental implant application. Of the two randomised groups, one was treated with 100% collagenated bone mix (Group A), and the other half with 90% collagenated bone mix + 10% collagen gel (Group B).Six months after sinus augmentation, prior to dental implant surgery, a specimen was taken from the implant socket with trephine drill for histopathological evaluation of new bone, connective tissue and residual graft material at each augmented site. SPSS 19 was used for data analysis. RESULTS: Of the 19 patients, 12(63%) were females and 7(37%) were males. The overall mean age was 51.68±11,96 years (range: 24-69 years). A total of 30 sinus floor augmentations were done. New bone formation was significantly better in Group A(15 sinus floor augmentation) than in Group B (the other 15 sinus floor augmentation) (p<0.05), but there was no significant difference in connective tissue formation and residual graft materials between the groups (p>0.05). CONCLUSIONS: Collagenated bone mix was found to be a suitable graft material for sinus floor augmentation, but increased collagen ratio did not improve new bone formation over the 6-month healing process.


Assuntos
Substitutos Ósseos , Osso e Ossos , Levantamento do Assoalho do Seio Maxilar/métodos , Transplante Heterólogo/métodos , Adulto , Idoso , Animais , Substitutos Ósseos/química , Substitutos Ósseos/uso terapêutico , Osso e Ossos/química , Osso e Ossos/patologia , Estudos Transversais , Feminino , Cavalos , Humanos , Masculino , Pessoa de Meia-Idade , Osseointegração/fisiologia , Turquia , Adulto Jovem
11.
Med Sci Monit ; 25: 8694-8703, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31736477

RESUMO

BACKGROUND This study aimed to develop a nude mouse model of orthotopic liver transplantation of HCCLM3 human hepatocellular carcinoma (HCC) cell xenografts and the use of imaging and histology to evaluate tumor development and progression. MATERIAL AND METHODS HCCLM3 cells were injected subcutaneously into 25 healthy male athymic BALB/c (nu/nu) nude mice. The tumors that developed were transplanted into the liver of a new set of nude mice. After four weeks and six weeks, the mice were imaged using ultrasound (US), software-assisted contrast-enhanced ultrasound (CEUS), fluorodeoxyglucose-positron emission tomography (FDG-PET). Histology was performed on the liver and liver tumors, and included immunohistochemistry for vascular endothelial growth factor (VEGF), CD31, CD34, and alpha-smooth muscle actin (alpha-SMA). RESULTS The success rate for orthotopic tumor transplantation in the mouse liver was 90% (18/20). Liver tumors measured 11.8±2.6 mm in diameter and 525.9±250.8 mm3 in volume on the sixth week. CEUS showed rapid wash-in and washout in the liver tumors, and PET showed low tumor cell metabolism. Bone metastases were present in 45% (9/20) of mice in the sixth week. Immunohistochemistry showed positive expression for VEGF, CD31, CD34, and alpha-SMA. CONCLUSIONS The nude mouse orthotopic liver transplantation model of human HCC was shown to be a reliable model that has the potential for future research on the pathogenesis and progression of HCC and studies on drug development.


Assuntos
Transplante de Fígado/métodos , Transplante Heterólogo/métodos , Actinas/metabolismo , Animais , Antígenos CD34/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Xenoenxertos , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
PLoS One ; 14(11): e0224576, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697695

RESUMO

Intra-bone marrow transplantation (IBMT) has been adapted for mouse models to improve the seeding efficiency of transplanted hematopoietic stem and progenitor cells. Commonly used injection volumes for IBMT into the tibia differ between 10 and 40 µL even though considerable amounts of injected cells leak into the blood circulation immediately after injection. Injection of 3 µL trypan blue into the tibia of dead BALB/c mice showed staining in large vessels of hind limbs, even without supporting circulation. We therefore tested the effective capacity of the medullary cavity of dissected tibiae and femora of different mouse strains by bioluminescence imaging after injection of luciferase expressing cells. Cell leakage was already observed at 3 µL of injection volume and the measured emission rate increased significantly when 5 and 10 µL of volume with the same cell concentration were injected. Surprisingly, increasing injection volumes containing constant cell amounts resulted in comparable emission rates, suggesting a similar amount of leaked and absorbed cells independent of the injection volume. However, the absorption of a specific amount of injected cells could not be confirmed, as the ratio of leaked to absorbed cells was similar between IBMT that were performed with a constant injection volume containing either low or high cell amounts. In summary, for optimal cell transplantation via IBMT in mice we suggest to inject a high concentrated cell suspension with a maximum injection volume of 3 µL.


Assuntos
Transplante de Medula Óssea/métodos , Fêmur/transplante , Transplante de Células-Tronco Hematopoéticas/métodos , Tíbia/transplante , Animais , Movimento Celular/fisiologia , Fêmur/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Tíbia/fisiopatologia , Transplante Heterólogo/métodos
13.
Elife ; 82019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710289

RESUMO

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). Here, we report the in vitro reprogramming of fibroblasts to human induced Sertoli-like cells (hiSCs). Initially, five transcriptional factors and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles and cellular properties that are similar to those of primary human Sertoli cells. Moreover, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. hiSCs suppress the proliferation of human T lymphocytes and protect xenotransplanted human cells in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli cell only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.


Assuntos
Reprogramação Celular , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Síndrome de Células de Sertoli/genética , Células de Sertoli/metabolismo , Fator Esteroidogênico 1/genética , Animais , Diferenciação Celular/genética , Proliferação de Células , Técnicas de Cocultura , Fibroblastos/citologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Túbulos Seminíferos/citologia , Túbulos Seminíferos/crescimento & desenvolvimento , Túbulos Seminíferos/metabolismo , Síndrome de Células de Sertoli/metabolismo , Síndrome de Células de Sertoli/patologia , Células de Sertoli/citologia , Células de Sertoli/transplante , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/metabolismo , Fator Esteroidogênico 1/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante Heterólogo/métodos , Proteínas WT1/genética , Proteínas WT1/metabolismo
14.
In Vivo ; 33(6): 1785-1792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662503

RESUMO

BACKGROUND/AIM: Establishment of mouse xenograft models is necessary for oncological research and depends on the characteristics of the cell lines and the immune system of the host. In this study, we describe the development of mouse xenograft models using human gastric cancer (GC) cell lines. MATERIALS AND METHODS: MKN1 stably-expressing luciferase (MKN1-Luc), N87, KATO III, MKN45 stably-expressing luciferase (MKN45-Luc), NUGC4, and OCUM-1 human GC cell lines were injected intraperitoneally into mice to establish peritoneal metastasis models. MKN45-Luc were injected into subcutaneously implanted spleen, and MKN1-Luc and MKN45-Luc were injected directly into the portal veins of mice for the establishment of hepatic metastasis models. RESULTS: Peritoneal metastasis was formed after implantation of MKN1-Luc, N87, KATO III, MKN45-Luc, and NUGC4 in nude mice, but not formed in OCUM-1 even in NOD/SCID mice. After intrasplenic injection of MKN45-Luc, we found no hepatic metastasis formation. We identified hepatic metastasis formation after direct injection of MKN45-Luc and MKN1-Luc into the portal veins of NOD/SCID mice. CONCLUSION: Peritoneal and hepatic metastasis mouse xenograft models were successfully established using several human GC cell lines.


Assuntos
Xenoenxertos/patologia , Neoplasias Hepáticas/patologia , Neoplasias Gástricas/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias Peritoneais/patologia , Peritônio/patologia , Transplante Heterólogo/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
Nat Commun ; 10(1): 4491, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582751

RESUMO

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Células Epiteliais/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Organoides/transplante , Engenharia Tecidual/métodos , Âmnio/citologia , Animais , Sobrevivência Celular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Células Epiteliais/transplante , Sobrevivência de Enxerto , Xenoenxertos/irrigação sanguínea , Xenoenxertos/metabolismo , Xenoenxertos/transplante , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos SCID , Organoides/irrigação sanguínea , Organoides/metabolismo , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa/métodos , Esferoides Celulares , Estreptozocina , Técnicas de Cultura de Tecidos/métodos , Transplante Heterólogo/métodos
17.
Nat Commun ; 10(1): 4067, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492885

RESUMO

ARID1A inactivation causes mitotic defects. Paradoxically, cancers with high ARID1A mutation rates typically lack copy number alterations (CNAs). Here, we show that ARID1A inactivation causes defects in telomere cohesion, which selectively eliminates gross chromosome aberrations during mitosis. ARID1A promotes the expression of cohesin subunit STAG1 that is specifically required for telomere cohesion. ARID1A inactivation causes telomere damage that can be rescued by STAG1 expression. Colony formation capability of single cells in G2/M, but not G1 phase, is significantly reduced by ARID1A inactivation. This correlates with an increase in apoptosis and a reduction in tumor growth. Compared with ARID1A wild-type tumors, ARID1A-mutated tumors display significantly less CNAs across multiple cancer types. Together, these results show that ARID1A inactivation is selective against gross chromosome aberrations through causing defects in telomere cohesion, which reconciles the long-standing paradox between the role of ARID1A in maintaining mitotic integrity and the lack of genomic instability in ARID1A-mutated cancers.


Assuntos
Instabilidade Genômica , Mutação , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Telômero/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Variações do Número de Cópias de DNA , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Transplante Heterólogo/métodos , Carga Tumoral/genética
19.
Int J Surg ; 70: 84-91, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31445094

RESUMO

In pig-to-baboon organ xenotransplantation, coagulation dysfunction and inflammation have been suggested to be associated with acute humoral xenograft rejection. We have evaluated platelet counts, plasma fibrinogen, and parameters of inflammation as indicators of xenograft failure in baboons with kidney and heart grafts from genetically-engineered pigs. Blood chemistry, hematologic, immune, and inflammatory parameters were measured in recipient baboons (n = 16) with organs from α1,3-galactosyltransferase gene-knockout pigs expressing human complement- and coagulation-regulatory proteins. Thrombocytopenia and reduction of plasma fibrinogen level were observed in baboons developing graft failure, and these correlated with histopathologic findings of glomerular and interstitial thrombosis, and vasculitis in the graft. Not infrequently, in baboons with pig kidney grafts, a consumptive coagulopathy developed prior to a rise in serum creatinine. In contrast, when kidney graft survival was prolonged, no changes were observed in platelet count or fibrinogen. Indicators of the inflammatory response, particularly the serum amyloid A (SAA) assay, increased when graft failure was developing. There were no changes in cellular immune parameters, e.g., T or B cell counts or phenotypes that indicated graft failure. Therefore, in clinical xenotransplantation, noninvasive parameters (e.g., platelet count, fibrinogen level, SAA) might provide more reliable indicators of impending xenograft failure than measurements of immune parameters or even of serum creatinine.


Assuntos
Fibrinogênio/análise , Sobrevivência de Enxerto , Transplante de Coração/métodos , Transplante de Rim/métodos , Contagem de Plaquetas , Proteína Amiloide A Sérica/análise , Transplante Heterólogo/métodos , Animais , Animais Geneticamente Modificados , Humanos , Papio , Suínos
20.
Transplant Proc ; 51(6): 2043-2050, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31399182

RESUMO

BACKGROUND: Pigs are considered suitable animal donor models for xenotransplantation. For successful organ transplantation, immune rejection must be overcome. Xenotransplantation has recently been successfully performed using galactose-alpha1,3-galactose epitopes knockout (GalTKO) and a human membrane cofactor protein (hCD46) in a pig model. However, the growth and lifespan of the grafted organ have not been evaluated. Therefore, in the present study we evaluated aging and 84 senescence-related genes using the RT2 Profiler PCR array and whole blood samples from GalTKO/hCD46 Massachusetts General Hospital (MGH) pigs. METHODS: Experimental groups were double GalTKO/hCD46 (5-month-old), single GalTKO/hCD46 (2-year-old), and non-genetically modified (>3.5-year-old; control group within the same strain). Age-matched white hairless Yucatan (WHY) miniature pig groups were used as controls. RESULTS: Among the 19 senescence-related genes selected from the 84 genes for further evaluation, 13 were upregulated in the double GalTKO/hCD46 MGH pigs compared to control MGH pigs; however, in WHY pigs, only 4 genes were up- or down-regulated among the 19 genes. Moreover, in double GalTKO/hCD46 MGH and WHY pigs, the expression of the 19 genes changed only 1- to 2-fold, suggesting that there were no significant differences in senescence signals between the 2 pig lines. CONCLUSIONS: The present results indicate that the double GalTKO/hCD46 MGH pig might be a suitable model for human xenotransplantation studies. However, we used a limited number of experimental individuals, so further studies using larger experimental groups should be conducted to verify the present results.


Assuntos
Envelhecimento/genética , Animais Geneticamente Modificados , Galactose/deficiência , Proteína Cofatora de Membrana/genética , Transplante Heterólogo/métodos , Animais , Pré-Escolar , Modelos Animais de Doenças , Epitopos , Galactose/genética , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Pessoa de Meia-Idade , Suínos , Porco Miniatura
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