| Part 1:The investigation of Rat iPSCs derived Lung in Rat-Mouse Interspecific ChimeraBackgroudThe complexity of organogenesis restricts generation of organs derived from a patient's pluripotent stem cells(PSCs)in vitro,an ultimate goal of regenerative medicine.We hypothesized that if we injected rat iPSCs into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation.ObjectiveTo generate the rat-mouse chimeras by blastocyst complementation,we injected rat iPSCs into TTF-1-/-mouse blastocysts,thus confirming that rat iPSCs can contribute to xenogenic development between mouse and rat.The development of lung in rat-mouse chimeras was derived from rat iPSCs.This research will provide new ideas and ways for lung regeneration in vivo and make the foundation of regenerating a humanized lung organ in pigs.Methods(1)E19dTTF-1-/-mice were generated by intercross between TTF-1+/-male and female mice.H&E staining was done for lung phenotyping.(2)Allkaline phosphatase(AP)staining and immunofluorescent stainings using anti-C-myc,anti-Oct4,anti-SSEA-1and anti-Nanog antibody,were performed to evaluated rat iPSCs pluripotency.(3)To generate the rat-mouse chimeras by blastocyst complementation,we injected rat iPSCs into TTF-1-/-mouse blastocysts,and transfer the injected mouse blastocysts into foster mother.Thus H&E staining and IHC was applied for lung phenotyping using CCSP,SP-C and TTF-1 antibodies.(4)We designed 4 kinds of guide RNA which targeting TTF-1 gene,and generated TTF-1-/-knockout mouse embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs.We took phenotypic analysis of mouse blastocyst by gene sequencing.Results(1)Mouse TTF-1 gene can be completely knocked out in mouse embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs(2)Alkaline phosphatase(AP)staining and immunofluorescent stainings using anti-C-myc,anti-Oct4,anti-SSEA-land anti-Nanog antibody,were all positive in rat iPSCs.(3)TTF-1+/-mice developed normally,whereas TTF-1-/-mice were born dead caused by lacking of lung parenchyma.Instead,TTF-1-/-mice had a rudimentary bronchial tree which was associated with an abnormal epithelium in their pleural cavities.(4)After 12h or 24 h culture of rat iPSCs-injected embryos,rat iPSCs were enclosed within the inner cell mass in almost all blastocysts.Clara cells,pulmonary alveolar type ? cells and pulmonary progenitor cells were detected in the lung of rat-mouse chimera.ConclusionTo examine the potential for xenogenic approaches in blastocyst complementation,we have generated the rat-mouse chimeras by injecting rat iPSCs into TTF-1-/-mouse blastocysts,confirming that rat iPSCs can contribute to xenogenic development of lung.Meaningly,clara cells,pulmonary alveolar type ? cells and pulmonary progenitor cells were detected in the lung tissues of rat-mouse chimera.Part 2:Investigation of Immunogenicity of GGTA1/CMAH/?4GalNT2 Triple Gene Knockout Pig Organs and TissuesBackgroundPorcine Organs/tissues and red blood cells(RBCs)will resolve the increasing shortage of human donors demanded for allotransplantation,but xenoantigens recognized by human antibodies are an obstacle of clinical xenotransplantation.Simultaneous deletion of pig GGTA1,CMAH and ?4GalNT2 genes eliminates those xenoantigens,and may facilitate the clinical application.Human antibody binding to the heart valves or pericardium from GGTA1/CMAH/?4GalNT2 knockout(TKO)pigs was reduced in former analysis.But the immunogenicity of other porcine organs/tissues until keep unknown.ObjectiveTo analyse the immunogenicity of some porcine organs/tissues(heart,liver,spleen,lung,kidney,pancreas,cornea,and skin)and RBCs,it provides experimental basis and direction for the clinical application of porcine organs/tissues.Methods(1)For aGal,sd(a)and Neu5Gc detection RBCs from(wildtype)WT and TKO pigs as well as humans were respectively incubated with IB4,DBA and anti-Neu5Gc antibodies,with human serum and the level of IgG and IgM binding to these cells were compared using flow cytometry.(2)Corneal tissues from WT,GTKO/CD46 and TKO pigs were also applied by for aGal,sd(a)and Neu5Gc detection respectively immunofluorescence staining IB4,DBA and anti-Neu5Gc antibodies.Different corneas were incubated with human serum to determine immunoglobulin(Ig)M and IgG binding to corneal tissue by means of fluorescent microscopy.(3)Different organs/tissues from WT and TKO pigs,such as heart,liver,spleen,lung,kidney,pancreas and skin were used for aGal,sd(a)and Neu5Gc detection respectively by IB4,DBA and anti-Neu5Gc antibodies immunofluorescence staining,and WT porcine organs/tissues were set as positive control.These tissues were incubated with human serum to determine immunoglobulin(Ig)M and IgG binding to corneal tissue using fluorescent microscopy.Results(1)Expression of aGal,Neu5Gc and sd(a)antigen was detected in WT pig RBC,but TKO pig and human RBC were not detected.Human IgG/IgM binding to the human RBC and TKO pig RBC was significantly lower than to the WT pig RBC,and IgM binding to RBC kept no difference compared with human RBC(2)The corneal structure and cell morphology from TKO pigs and GTKO/CD46 pigs were not significantly different from those of WT pigs.The overall staining of aGal epitopes was low in the cornea with weak signals distributed in several keratocytes in the anterior-most part of WT pig corneal stroma,whereas GTKO/CD46 and TKO porcine keratocytes did not show any expression of the aGal epitopes.The expression of sd(a)and Neu5Gc antigens was detected in keratocytes of the anterior stroma of WT pig corneas with weak diffuse expression in the stroma.There was no aGal expression in TKO or GTKO pigs,nor were sd(a)antigen or Neu5Gc detected in TKO pigs(3)The tissue structure and cell morphology from TKO pigs and GTKO/CD46 pigs were not significantly different from those of WT pigs.The expression of aGal,Neu5Gc and sd(a)antigen was mostly found in WT porcine organs/tissues(heart,liver,lung,kidney,spleen,pancreas,and skin),but there was no aGal expression in TKO pigs,nor were sd(a)antigen or Neu5Gc detected in TKO pigs.Human serum IgG and IgM binding was decreased in some TKO porcine tissues,such as heart,lung,and kidney,but not in other organs/tissues.ConclusionsWhen aGal,Neu5Gc,and sd(a)antigens are eliminated in porcine red blood cells,human serum IgG and IgM binding was decreased significantly.Using relevant lectins or antibodies,we detected the expression of aGal,Neu5Gc,and sd(a)antigens in different organs and tissues,such as heart,liver,lung,kidney,spleen,and pancreas.Immunofluorescence staining indicated that these three carbohydrate antigens are mostly found in WT porcine vascular endothelial cells of the tested organs.Tissue-specific distribution of these antigens was observed as aGal was strongly expressed in the kidney,sd(a)was strongly expressed in the pancreas,and Neu5Gc was strongly expressed in the heart.As anticipated,the expression aGal,Neu5Gc,and sd(a)was absent in TKO pig organs/tissues(heart,liver,lung,kidney,spleen,and pancreas).Human serum IgG and IgM binding was decreased in some TKO porcine tissues,such as heart,lung,and kidney,showing that eliminating the reactivity of preformed human antibodies with those tissues can be achieved by gene targeting.However,comparable levels of IgG and IgM binding were observed in liver,spleen,and pancreas of TKO and WT pig,suggesting that other immunoreactive xenoanigens such as swine leukocyte antigens(SLA)maybe the dominant xenoantigens in those organs. |
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